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  • 99
    Millipore rabbit anti chip antibody
    Hsp70 is required for the association between <t>CHIP</t> and <t>BAG5.</t> (A) PDAs with GST fusion proteins using lysates of H4 cells transfected with the CHIP deletion constructs CHIP ΔU and CHIP ΔTPR are shown. The blots were probed with anti-CHIP (upper) and anti-Hsp70 (lower) antibodies. The inputs shown are 10% of total lysates used in each assay. Molecular weight markers are indicated. Results are representative of three independent experiments. (B) PDAs were performed using GST fusion proteins and purified recombinant CHIP with or without Hsp70 as indicated. Proteins that associated with GST alone, GST-BAG5, GST-BAG5 DARA, or GST-BAG1 were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of proteins used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining (lower). Molecular weight markers are indicated. Results are representative of three independent experiments.
    Rabbit Anti Chip Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher vps75 chip chip
    Changes to chromatin in cells lacking <t>Vps75.</t> (A) Steady-state histone levels are not affected in cells lacking Vps75. The relative levels of histones H2B and H3 in the wild-type (WT) or vps75Δ strain grown in YPD were determined by ChIP assays
    Vps75 Chip Chip, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam sp1 antibody chip grade
    Oxidative stress attenuated <t>Sp1</t> binding to its GC-Box elements present in hPrdx6 gene promoter. Table 1 . Evolutionary conserved Sp1 binding sequences in TATA-less Prdx6 promoters of mouse, rat and human cells. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 (GC-Box) binding sites showing primer location and sequences used in ChIP assay. ( B and C ) Oxidative stress (H 2 O 2 or UVB)-induced reduction in DNA binding activity of Sp1 to hPrdx6 gene promoter containing GC-Box (Sp1 sites) in SRA-hLECs. ChIP assay was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis kits (Active motif). Chromatin samples prepared from SRA-hLECs were exposed to varying concentrations of H 2 O 2 (0, 50, 75 and 100µM) or UVB (0, 40, 80 and 120J/m 2 ), and were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 gene promoter bearing GC-box (Sp1 sites). Histogram showed the amplified DNA by qPCR analysis: ( B ) Control (0) vs 50µM vs 75µM vs 100µM H 2 O 2 treatment. ( C ) Control (0) vs 40J/m 2 vs 80J/m 2 vs 120J/m 2 UVB exposure. The data represent mean ± SD from three independent experiments (** p
    Sp1 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam kdm1 lsd1 antibody chip grade
    Loss of <t>LSD1</t> or HDAC1 induces G 1 cell cycle arrest in F9 and PA-1 cells. (A and B) F9 and PA-1 cells were transfected with the indicated siRNAs, and the cell cycle was analyzed by FACS. F9 and PA-1 cells were arrested in the G 1 cell cycle by LSD1 or HDAC1
    Kdm1 Lsd1 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam kap1 antibody chip grade
    Interaction with HP1 is required for efficient tethering of <t>KAP1</t> to DNA. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged M2-KAP1 (this KAP1 construct harbors two amino acid substitutions
    Kap1 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam hdac3 antibody chip grade
    H3K9ac signal and H3K27ac signal at silenced genes and housekeeping genes in thymocytes. ( A–B ) Average H3K9ac and H3K27ac signal ±20 kb around the gene body among gene sets that are silenced in thymocytes ( A ) or housekeeping genes ( B ). Below are example H3K9ac ChIP-seq snapshots of genes within each of the genesets— Csf1r for macrophage, Pax5 for B cell, Cd34 for HSC/progenitor, Hprt for housekeeping, Atp5g1 for mitochondrial, and Rpl15 for ribosomal. Each image depicts an overlay of ChIP-seq tracks between OT-II (blue) and OT-II <t>HDAC3-cKO</t> (orange) mice. Refer to Figure 3—source data 2 for list of genes in these gene sets (macrophage, B cell, HSC/Progenitor, mitochondrial, ribosomal). TSS represents transcription start site; TES represents transcription end site. Scale bar in kb below ChIP-seq tracks identifies scale of snapshot.
    Hdac3 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam foxg1 antibody chip grade
    Mitochondrial membrane potential and cellular respiration in <t>Foxg1-transfected</t> HN9.10e cells. ( A ) Exemplificative pictures of untagged FL-Foxg1-expressing and mt-Foxg1-expressing (272–481) HN9.10e cells, loaded with TMRM. (Scale bar, 5 µm.)
    Foxg1 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Cell Signaling Technology Inc chip src 3
    PFKFB4 functions as a protein kinase by phosphorylating <t>SRC-3</t> at the S857 residue a , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, fructose-6-phosphate (F6P), ATP and increasing concentration of recombinant PFKFB4 enzyme followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of phosphorylated Ser/Thr-SRC-3 protein. b , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, PFKFB4 enzyme and varying concentrations of F6P and ATP followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of p-SRC-3 protein. c , Coomassie blue stain showing the levels of GST-fused SRC-3 fragments used in in vitro kinase reactions performed in Fig. 2b . d , Proteomics analysis of in vitro kinase assay using the GST-SRC-3-CID fragment in the presence of PFKFB4 enzyme and ATP followed by mass spectrometric analyses. Mass spectrum shows the green phosphorylation peak. e , Proteomics analysis of an in vitro kinase assay using a S857A-mutated GST-SRC-3-CID protein in the presence of PFKFB4 enzyme and ATP, followed by mass spectrometric analyses. Mass spectrum failed to detect phosphorylation peaks in the S857A mutated SRC-3-CID protein. f , Expression of PFKFB1, PFKFB2, PFKFB3 and PFKFB4 in MDA-MB-231 cells expressing shRNAs targeting PFKFB4 (sh#09 and sh#20). mRNA levels were normalized to internal housekeeping gene actin. [Mean ± s.d., n =3, biological replicates, two-way ANOVA with Tukey’s Multiple comparisons test, * P
    Chip Src 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Santa Cruz Biotechnology asxl1 chip seq
    Identification of genes significantly dysregulated with deletion of <t>Asxl1</t> alone and in concert with deletion of Tet2 and their functional impact. (A) Volcano plot of differentially expressed transcripts from RNA-Seq data of 1-yr-old control versus littermate Asxl1 KO ( Mx1-cre Asxl1 fl/fl ) LSK and MP (lineage − Sca-1 − c-Kit + ) cells (experiment included cells from two individual mice per genotype). (B) Venn diagrams of genes significantly up- and down-regulated with Asxl1 loss in LSK and MP (lineage − , Sca-1 − , cKit + ) cells from 1-yr-old Mx1-cre Asxl1 fl/fl mice and littermate Cre − controls as identified in A. (C) qRT-PCR analysis of HoxA and Hox -associated transcription factor genes in LSK cells of 1-yr-old Cre − Asxl1 fl/fl control versus littermate Vav-cre Asxl1 fl/fl . (D) qRT-PCR analysis of p16 INK4a in LT-HSCs (lineage − , Sca-1 + , c-Kit + , CD150 + , CD48 − ) and MPP cells (lineage − , Sca-1 + , c-Kit + , CD150 − , CD48 + ) from 6-wk- and 6-mo-old control (C) versus littermate Vav-cre Asxl1 fl/fl (KO) mice. (E) Cell cycle analysis of MPPs from 72-wk-old Vav-cre Asxl1 fl/fl or littermate Cre − Asxl1 fl/fl control mice with in vivo BrdU administration. Representative FACS plot analysis showing gating on MPP cells followed by BrdU versus DAPI stain is shown on the left (parent gate is LSK cells). Relative quantification of the percentage of MPP cells in S, G2/M, and G0/1 phase is shown on the right ( n = 5 mice per group). (F) Assessment of the proportion of HSPCs undergoing apoptosis was performed by Annexin V/DAPI stain of LSK cells from 72-wk-old Vav-cre Asxl1 fl/fl mice or Cre − Asxl1 fl/fl littermate controls. Representative FACS plot analysis showing gating on LSK cells followed by Annexin V versus DAPI stain is shown on the left (parent gate is lineage − cells). Relative quantification of the percentage of Annexin V + /DAPI − and Annexin V + /DAPI + LSK cells is shown on the right ( n = 5 mice per group). (G) Comparison of significant differentially expressed genes in LSK cells from 6-wk-old Mx1-cre Asxl1 fl/fl , Mx1-cre Tet2 fl/fl , or Mx1-cre Asxl1 fl/fl Tet2 fl/fl relative to controls (or Mx1-cre Asxl1 WT Tet2 WT). 99 genes are uniquely down-regulated in Asxl1 / Tet2 DKO mice relative to all other genotypes (left), whereas 49 genes are significantly up-regulated (right). (H) GSEA of overlapping and statistically significant gene sets enriched in the LSK cells of mice with deletion of Asxl1 alone or with combined Asxl1 and Tet2 deletion. (I) Gene sets uniquely enriched in mice with concomitant deletion of Asxl1 and Tet2 relative to all other genotypes as determined by GSEA. Error bars represent ±SD; *, P
    Asxl1 Chip Seq, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Abcam chip anti h3k9me2
    R-loops and RNAi promote <t>H3K9me2</t> mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.
    Chip Anti H3k9me2, supplied by Abcam, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher znf217 knockdown chip assays
    Schematic illustrating three models for <t>ZNF217</t> and CtBP2 interaction
    Znf217 Knockdown Chip Assays, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Abcam brf2 antibody chip grade
    The <t>Brf2</t> Molecular Pin (A) The Brf2 TBP anchor domain but not the molecular pin is essential for Brf2-TBP interaction in absence of the DNA, as shown by a pull-down assay. (B) The Brf2 TBP anchor domain and the molecular pin are essential for the formation of a Brf2-TBP/DNA complex, as shown in an EMSA. (C) Close-up view of the Brf2 molecular pin at the interface between the Brf2 C-cyclin repeat, TBP, and the DNA. See also Figures S3 and S7 .
    Brf2 Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Santa Cruz Biotechnology chip grade nurr1 antibodies
    Microarray analysis of <t>Nurr1-overexpressing</t> MN9D cells. ( A ) Western blot revealing high levels of Nurr1 protein upon treatment of MN9D- Nurr1 Tet On 13N cells with doxycyclin (Dox). ( B ) The relative percentage increase or decrease of a selection of transcripts
    Chip Grade Nurr1 Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Hsp70 is required for the association between CHIP and BAG5. (A) PDAs with GST fusion proteins using lysates of H4 cells transfected with the CHIP deletion constructs CHIP ΔU and CHIP ΔTPR are shown. The blots were probed with anti-CHIP (upper) and anti-Hsp70 (lower) antibodies. The inputs shown are 10% of total lysates used in each assay. Molecular weight markers are indicated. Results are representative of three independent experiments. (B) PDAs were performed using GST fusion proteins and purified recombinant CHIP with or without Hsp70 as indicated. Proteins that associated with GST alone, GST-BAG5, GST-BAG5 DARA, or GST-BAG1 were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of proteins used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining (lower). Molecular weight markers are indicated. Results are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: Hsp70 is required for the association between CHIP and BAG5. (A) PDAs with GST fusion proteins using lysates of H4 cells transfected with the CHIP deletion constructs CHIP ΔU and CHIP ΔTPR are shown. The blots were probed with anti-CHIP (upper) and anti-Hsp70 (lower) antibodies. The inputs shown are 10% of total lysates used in each assay. Molecular weight markers are indicated. Results are representative of three independent experiments. (B) PDAs were performed using GST fusion proteins and purified recombinant CHIP with or without Hsp70 as indicated. Proteins that associated with GST alone, GST-BAG5, GST-BAG5 DARA, or GST-BAG1 were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of proteins used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining (lower). Molecular weight markers are indicated. Results are representative of three independent experiments.

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Transfection, Construct, Molecular Weight, Purification, Recombinant, Staining

    CHIP forms a protein complex with BAG5. (A) Immunoprecipitations with anti-FLAG antibodies were performed from lysates of H4 cells transfected with CHIP with or without FLAG-BAG5 as indicated. Immunoprecipitates were sequentially probed with anti-CHIP (upper), anti-Hsp70 (middle), and anti-FLAG (lower) antibodies. Ten percent of lysates used for immunoprecipitation was loaded as input. The upper CHIP band corresponds to monoubiquitinylated CHIP [62] . Molecular weight markers are shown on right. Similar results were found in three separate experiments. (B) PDAs were performed using lysates of H4 cells transfected with CHIP. Proteins that associated with GST alone, GST-BAG5, or GST-BAG5 DARA were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of lysates used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining of the membranes (lower). Molecular weight markers are indicated on right. Results are representative of four independent experiments.

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: CHIP forms a protein complex with BAG5. (A) Immunoprecipitations with anti-FLAG antibodies were performed from lysates of H4 cells transfected with CHIP with or without FLAG-BAG5 as indicated. Immunoprecipitates were sequentially probed with anti-CHIP (upper), anti-Hsp70 (middle), and anti-FLAG (lower) antibodies. Ten percent of lysates used for immunoprecipitation was loaded as input. The upper CHIP band corresponds to monoubiquitinylated CHIP [62] . Molecular weight markers are shown on right. Similar results were found in three separate experiments. (B) PDAs were performed using lysates of H4 cells transfected with CHIP. Proteins that associated with GST alone, GST-BAG5, or GST-BAG5 DARA were probed with anti-CHIP (upper) and anti-Hsp70 (middle) antibodies. Input was 10% of lysates used for PDAs. The presence of equal amounts of GST fusion proteins was confirmed by Ponceau S staining of the membranes (lower). Molecular weight markers are indicated on right. Results are representative of four independent experiments.

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Molecular Weight, Staining

    BAG5 negatively regulates CHIP-mediated reduction of α-syn oligomer levels. (A) PDAs were performed using GST fusion proteins and lysates from H4 cells transfected with syn-luc1, syn-luc2, CHIP, and Hsp70. Proteins that associated with GST alone or GST-BAG5 were probed with anti-α-syn (upper), anti-CHIP (middle), and anti-Hsp70 (lower) antibodies. Input was 10% of total lysates used for PDAs. Results are representative of three independent experiments. (B) Luciferase activity was measured from H4 cells transfected with syn-luc1 and syn-luc2 plus the vectors indicated on the horizontal axis of the graph. The total DNA used in each condition was equalized using the empty vector pcDNA. Bars correspond to mean (±s.e.m.) luciferase activity normalized to measures obtained for the co-transfection of syn-luc1 and syn-luc2 with pcDNA as control (*P

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: BAG5 negatively regulates CHIP-mediated reduction of α-syn oligomer levels. (A) PDAs were performed using GST fusion proteins and lysates from H4 cells transfected with syn-luc1, syn-luc2, CHIP, and Hsp70. Proteins that associated with GST alone or GST-BAG5 were probed with anti-α-syn (upper), anti-CHIP (middle), and anti-Hsp70 (lower) antibodies. Input was 10% of total lysates used for PDAs. Results are representative of three independent experiments. (B) Luciferase activity was measured from H4 cells transfected with syn-luc1 and syn-luc2 plus the vectors indicated on the horizontal axis of the graph. The total DNA used in each condition was equalized using the empty vector pcDNA. Bars correspond to mean (±s.e.m.) luciferase activity normalized to measures obtained for the co-transfection of syn-luc1 and syn-luc2 with pcDNA as control (*P

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Cotransfection

    BAG5 inhibits CHIP-mediated ubiquitinylation of α-syn in vitro and in cells. (A) In vitro ubiquitinylation assays were performed with increasing amounts of GST or GST-BAG5 as indicated. Ubiquitinylation was determined by Western blot using anti-α-syn, anti-CHIP, or anti-Hsp70 antibodies. GST fusion proteins were stained with Ponceau S. Similar results were found in three experiments. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, myc-CHIP, HA-Ub, and FLAG-BAG5 as shown. Ubiquitinylation of α-syn was detected using anti-HA antibodies (upper). The arrow indicates the monoubiquitinylated form of α-syn (UB-α-syn). Immunoprecipitation of equivalent amounts of α-syn was confirmed by probing with anti-α-syn antibodies (lower). The asterisk (*) indicates cross-reactivity of the secondary antibody with the immunoglobulin light chain. (C) Quantification by densitometric analysis of the UB-α-syn band with co-transfection of a control vector or FLAG-BAG5 from three independent experiments, one of which is represented in (B). Bars shown correspond to mean (±S.D.) gray value normalized to measures obtained for the control vector. *P

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: BAG5 inhibits CHIP-mediated ubiquitinylation of α-syn in vitro and in cells. (A) In vitro ubiquitinylation assays were performed with increasing amounts of GST or GST-BAG5 as indicated. Ubiquitinylation was determined by Western blot using anti-α-syn, anti-CHIP, or anti-Hsp70 antibodies. GST fusion proteins were stained with Ponceau S. Similar results were found in three experiments. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, myc-CHIP, HA-Ub, and FLAG-BAG5 as shown. Ubiquitinylation of α-syn was detected using anti-HA antibodies (upper). The arrow indicates the monoubiquitinylated form of α-syn (UB-α-syn). Immunoprecipitation of equivalent amounts of α-syn was confirmed by probing with anti-α-syn antibodies (lower). The asterisk (*) indicates cross-reactivity of the secondary antibody with the immunoglobulin light chain. (C) Quantification by densitometric analysis of the UB-α-syn band with co-transfection of a control vector or FLAG-BAG5 from three independent experiments, one of which is represented in (B). Bars shown correspond to mean (±S.D.) gray value normalized to measures obtained for the control vector. *P

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, In Vitro, Western Blot, Staining, Transfection, Hemagglutination Assay, Immunoprecipitation, Cotransfection, Plasmid Preparation

    Targeted knockdown of CHIP or absence of α-syn ubiquitinylation increases oligomerization of α-syn. (A) Knockdown of CHIP protein expression in the CHIP shRNA stable cell line versus a control shRNA cell line was confirmed by Western blot. The membrane was sequentially probed with anti-CHIP, anti-Hsp70, and anti-α-syn antibodies. GAPDH was used as a loading control. (B) Luciferase activity was measured from CHIP shRNA or control shRNA stable cell lines transiently co-transfected with syn-luc1 and syn-luc2. Bars correspond to mean (± S.D.) luciferase activity normalized to measures obtained for control shRNA cells co-transfected with syn-luc1 and syn-luc2. *P

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: Targeted knockdown of CHIP or absence of α-syn ubiquitinylation increases oligomerization of α-syn. (A) Knockdown of CHIP protein expression in the CHIP shRNA stable cell line versus a control shRNA cell line was confirmed by Western blot. The membrane was sequentially probed with anti-CHIP, anti-Hsp70, and anti-α-syn antibodies. GAPDH was used as a loading control. (B) Luciferase activity was measured from CHIP shRNA or control shRNA stable cell lines transiently co-transfected with syn-luc1 and syn-luc2. Bars correspond to mean (± S.D.) luciferase activity normalized to measures obtained for control shRNA cells co-transfected with syn-luc1 and syn-luc2. *P

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Expressing, shRNA, Stable Transfection, Western Blot, Luciferase, Activity Assay, Transfection

    Wild-type α-syn is a substrate of CHIP E3 ubiquitin ligase activity in vitro . (A) In vitro ubiquitinylation assays were performed by incubating purified recombinant proteins of the E1 Ube1, the E2 UbcH5b, and the CHIP substrate Hsp70 with and without purified ubiquitin or CHIP. Ubiquitinylation of CHIP and Hsp70 was determined by Western blot using anti-CHIP (upper) and anti-Hsp70 (middle) antibodies, respectively. The high molecular weight smears represented ubiquitinylated ((UB) n ) forms of CHIP and Hsp70. The purified recombinant proteins used in the assay were stained with Ponceau S (lower). Similar results were found in more than five experiments. Molecular weight markers are shown on left in kDa. (B) In vitro ubiquitinylation assays were performed by incubating purified recombinant human untagged wild-type α-syn with ubiquitin, Ube1, UbcH5b, CHIP, and Mg-ATP as indicated. Ubiquitinylation of α-syn (UB-α-syn and (UB) n -α-syn) was determined by Western blot using anti-α-syn (upper) and anti-ubiquitin (middle) antibodies. The asterisk (*) indicates a non-specific band. Ubiquitinylation of CHIP was detected with anti-CHIP antibodies (lower). Results are representative of more than five experiments. Molecular weight markers are shown in kDa. (C) In vitro ubiquitinylation assays were performed with α-syn, Ube1, and either the E2 UbcH5a or UbcH5b plus His-tagged CHIP, HDM2, or MuRF1 or in the absence of an E3 ubiquitin ligase. Experiments were performed without and with ubiquitin as indicated. Ubiquitinylation of α-syn and the E3 ubiquitin ligases was detected with anti-α-syn and anti-His antibodies, respectively. The asterisk (*) indicates a non-specific band. Molecular weight markers in kDa are indicated.

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: Wild-type α-syn is a substrate of CHIP E3 ubiquitin ligase activity in vitro . (A) In vitro ubiquitinylation assays were performed by incubating purified recombinant proteins of the E1 Ube1, the E2 UbcH5b, and the CHIP substrate Hsp70 with and without purified ubiquitin or CHIP. Ubiquitinylation of CHIP and Hsp70 was determined by Western blot using anti-CHIP (upper) and anti-Hsp70 (middle) antibodies, respectively. The high molecular weight smears represented ubiquitinylated ((UB) n ) forms of CHIP and Hsp70. The purified recombinant proteins used in the assay were stained with Ponceau S (lower). Similar results were found in more than five experiments. Molecular weight markers are shown on left in kDa. (B) In vitro ubiquitinylation assays were performed by incubating purified recombinant human untagged wild-type α-syn with ubiquitin, Ube1, UbcH5b, CHIP, and Mg-ATP as indicated. Ubiquitinylation of α-syn (UB-α-syn and (UB) n -α-syn) was determined by Western blot using anti-α-syn (upper) and anti-ubiquitin (middle) antibodies. The asterisk (*) indicates a non-specific band. Ubiquitinylation of CHIP was detected with anti-CHIP antibodies (lower). Results are representative of more than five experiments. Molecular weight markers are shown in kDa. (C) In vitro ubiquitinylation assays were performed with α-syn, Ube1, and either the E2 UbcH5a or UbcH5b plus His-tagged CHIP, HDM2, or MuRF1 or in the absence of an E3 ubiquitin ligase. Experiments were performed without and with ubiquitin as indicated. Ubiquitinylation of α-syn and the E3 ubiquitin ligases was detected with anti-α-syn and anti-His antibodies, respectively. The asterisk (*) indicates a non-specific band. Molecular weight markers in kDa are indicated.

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Activity Assay, In Vitro, Purification, Recombinant, Western Blot, Molecular Weight, Staining

    CHIP mediates ubiquitinylation of α-syn in cells. (A) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with HA-Ub, syn-luc1, and control vector or myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (middle) antibodies. Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-myc or anti-α-syn antibodies (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) indicate immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence (see (B)). Molecular weight markers are indicated on left in kDa. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub, and myc-CHIP as indicated. Immunoprecipitated proteins were probed with anti-α-syn antibodies (H3C) which recognizes both syn-luc1 and synKR-luc1. The asterisks (*) correspond with the same bands indicated as such in (A). (C) Densitometric quantification of the band representing monoubiquitinylated α-syn when co-transfected with a control vector or myc-CHIP was performed from three independent experiments, one of which is represented in (A). Bars correspond to mean (± S.D.) gray value normalized to measures obtained for co-transfection of syn-luc1 and HA-Ub with control vector. *P

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: CHIP mediates ubiquitinylation of α-syn in cells. (A) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with HA-Ub, syn-luc1, and control vector or myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (middle) antibodies. Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-myc or anti-α-syn antibodies (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) indicate immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence (see (B)). Molecular weight markers are indicated on left in kDa. (B) Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub, and myc-CHIP as indicated. Immunoprecipitated proteins were probed with anti-α-syn antibodies (H3C) which recognizes both syn-luc1 and synKR-luc1. The asterisks (*) correspond with the same bands indicated as such in (A). (C) Densitometric quantification of the band representing monoubiquitinylated α-syn when co-transfected with a control vector or myc-CHIP was performed from three independent experiments, one of which is represented in (A). Bars correspond to mean (± S.D.) gray value normalized to measures obtained for co-transfection of syn-luc1 and HA-Ub with control vector. *P

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Transfection, Hemagglutination Assay, Plasmid Preparation, Immunoprecipitation, Sequencing, Molecular Weight, Cotransfection

    CHIP monoubiquitinylates α-syn. Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub WT, HA-Ub KO, and myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (Syn-1) (middle) antibodies. For the anti-α-syn blots, short exposure times were used to allow for the comparison of amount of α-syn immunoprecipitated in each of the conditions. Ubiquitinylated forms of α-syn are not detectable by Syn-1 at these shorter exposure times (see Figure 2D ). Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-HA antibodies which recognize HA-ubiquitin monomers (UB) and proteins with covalently attached HA-ubiquitin (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) correspond with immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence. These are the same bands seen in Figure 2 . Similar results were found in each of three experiments.

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: CHIP monoubiquitinylates α-syn. Immunoprecipitations with anti-luc were performed from lysates of H4 cells transfected with syn-luc1, synKR-luc1, HA-Ub WT, HA-Ub KO, and myc-CHIP as indicated. Immunoprecipitates were sequentially probed with anti-HA (upper) and anti-α-syn (Syn-1) (middle) antibodies. For the anti-α-syn blots, short exposure times were used to allow for the comparison of amount of α-syn immunoprecipitated in each of the conditions. Ubiquitinylated forms of α-syn are not detectable by Syn-1 at these shorter exposure times (see Figure 2D ). Five percent of lysates used for immunoprecipitation was loaded as input and probed with anti-HA antibodies which recognize HA-ubiquitin monomers (UB) and proteins with covalently attached HA-ubiquitin (lower). The middle band represents monoubiquitinylated α-syn (UB-α-syn). The asterisks (*) correspond with immunoprecipitated bands that remain detectable by the anti-HA antibody following the substitution of all lysines within the α-syn sequence. These are the same bands seen in Figure 2 . Similar results were found in each of three experiments.

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Transfection, Hemagglutination Assay, Gene Knockout, Immunoprecipitation, Sequencing

    CHIP overexpression decreases α-syn oligomer levels. (A) Schematic of the constructs used in the bioluminescent PCA with Gaussia princeps luciferase (luc) to assay for α-syn oligomerization. Fragments of luc were cloned downstream of full-length human α-syn to generate the expression constructs referred to as syn-luc1 and syn-luc2. Reconstitution of luc from the fragments can occur upon α-syn-α-syn interactions in cells and thus luciferase activity provides a surrogate measure of α-syn oligomerization [16] , [19] . Control experiments were performed with a luc fragment without α-syn (linker-luc1) and with full-length luciferase. (B) Luciferase activity was measured from H4 cells transfected with pcDNA control, syn-luc1 or syn-luc2 alone, or co-transfected with syn-luc1 and syn-luc2. Bars correspond to mean (± S.D.) luciferase activity measured in relative luciferase units (RLU). *P

    Journal: PLoS ONE

    Article Title: Ubiquitinylation of ?-Synuclein by Carboxyl Terminus Hsp70-Interacting Protein (CHIP) Is Regulated by Bcl-2-Associated Athanogene 5 (BAG5)

    doi: 10.1371/journal.pone.0014695

    Figure Lengend Snippet: CHIP overexpression decreases α-syn oligomer levels. (A) Schematic of the constructs used in the bioluminescent PCA with Gaussia princeps luciferase (luc) to assay for α-syn oligomerization. Fragments of luc were cloned downstream of full-length human α-syn to generate the expression constructs referred to as syn-luc1 and syn-luc2. Reconstitution of luc from the fragments can occur upon α-syn-α-syn interactions in cells and thus luciferase activity provides a surrogate measure of α-syn oligomerization [16] , [19] . Control experiments were performed with a luc fragment without α-syn (linker-luc1) and with full-length luciferase. (B) Luciferase activity was measured from H4 cells transfected with pcDNA control, syn-luc1 or syn-luc2 alone, or co-transfected with syn-luc1 and syn-luc2. Bars correspond to mean (± S.D.) luciferase activity measured in relative luciferase units (RLU). *P

    Article Snippet: Sections were incubated in blocking solution at 4°C overnight with a combination of mouse anti-α-syn antibody (dilution 1∶250; BD Bioscience) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem), or with a combination of mouse anti-BAG5 antibody (dilution 1∶250; Santa Cruz) and rabbit anti-CHIP antibody (dilution 1∶200; Calbiochem).

    Techniques: Chromatin Immunoprecipitation, Over Expression, Construct, Luciferase, Clone Assay, Expressing, Activity Assay, Transfection

    Changes to chromatin in cells lacking Vps75. (A) Steady-state histone levels are not affected in cells lacking Vps75. The relative levels of histones H2B and H3 in the wild-type (WT) or vps75Δ strain grown in YPD were determined by ChIP assays

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Changes to chromatin in cells lacking Vps75. (A) Steady-state histone levels are not affected in cells lacking Vps75. The relative levels of histones H2B and H3 in the wild-type (WT) or vps75Δ strain grown in YPD were determined by ChIP assays

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques: Chromatin Immunoprecipitation

    Genes affected by deletion of VPS75 belong to an acid-responsive regulon and cluster together. (A) Deletion of VPS75 confers resistance to acetic acid. The indicated strains were grown overnight in YPD, spotted as 10-fold serial dilutions on YPD or YPD

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Genes affected by deletion of VPS75 belong to an acid-responsive regulon and cluster together. (A) Deletion of VPS75 confers resistance to acetic acid. The indicated strains were grown overnight in YPD, spotted as 10-fold serial dilutions on YPD or YPD

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Vps75 is physically associated with sites of active transcription, and its expression remains constant throughout the cell cycle. (A) Vps75 is recruited to GAL10 following galactose induction. Cells expressing a tagged form of Vps75 were grown in medium

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Vps75 is physically associated with sites of active transcription, and its expression remains constant throughout the cell cycle. (A) Vps75 is recruited to GAL10 following galactose induction. Cells expressing a tagged form of Vps75 were grown in medium

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques: Expressing

    Transcriptional defects in the absence of Vps75.

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Transcriptional defects in the absence of Vps75.

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Transcriptional defects in the absence of Vps75.

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Transcriptional defects in the absence of Vps75.

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Vps75 promotes loss of H2A/H2B dimers from mononucleosomes in the presence of RSC and ATP. (A) Nucleosomes were treated for 2 h at 30°C with the indicated proteins and then fractionated by gel electrophoresis. An autoradiograph of the gel is shown.

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Vps75 promotes loss of H2A/H2B dimers from mononucleosomes in the presence of RSC and ATP. (A) Nucleosomes were treated for 2 h at 30°C with the indicated proteins and then fractionated by gel electrophoresis. An autoradiograph of the gel is shown.

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques: Nucleic Acid Electrophoresis, Autoradiography

    VPS75 interacts genetically with genes involved in transcription. (A) Scatter plot of the CCs of vps75 Δ, asf1Δ , and rtt109 ). The profile of RTT109 correlates with

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: VPS75 interacts genetically with genes involved in transcription. (A) Scatter plot of the CCs of vps75 Δ, asf1Δ , and rtt109 ). The profile of RTT109 correlates with

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    VPS75 genetically interacts with RNAPII and other factors involved in transcription and chromatin function.

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: VPS75 genetically interacts with RNAPII and other factors involved in transcription and chromatin function.

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Vps75 regulates transcription-dependent accumulation of histone H3 acetylated at lysine 56 (H3K56ac) at GAL10 . (A) Loss of histone H3 at GAL10 following induction. Wild-type (WT) or vps75Δ cells were grown in raffinose and arrested in G 1 phase

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Vps75 regulates transcription-dependent accumulation of histone H3 acetylated at lysine 56 (H3K56ac) at GAL10 . (A) Loss of histone H3 at GAL10 following induction. Wild-type (WT) or vps75Δ cells were grown in raffinose and arrested in G 1 phase

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Transcriptional defects in the absence of Vps75.

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Transcriptional defects in the absence of Vps75.

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Genome-wide analysis of Vps75 binding sites reveals an association with transcription and histone acetylation. (A) Composite profile of Vps75 occupancy (detected by ChIP) across the average gene. The ends of genes were defined at fixed points according

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Genome-wide analysis of Vps75 binding sites reveals an association with transcription and histone acetylation. (A) Composite profile of Vps75 occupancy (detected by ChIP) across the average gene. The ends of genes were defined at fixed points according

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques: Genome Wide, Binding Assay, Chromatin Immunoprecipitation

    Induction of GAL-VPS13 is affected by Vps75. Recruitment of RNAPII to the promoter and through the coding region (2.2 kb, 5.5 kb, and 8.3 kb) of GAL-VPS13 was measured by ChIP assays followed by quantitative PCR in wild-type (WT) and vps75Δ cells

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Induction of GAL-VPS13 is affected by Vps75. Recruitment of RNAPII to the promoter and through the coding region (2.2 kb, 5.5 kb, and 8.3 kb) of GAL-VPS13 was measured by ChIP assays followed by quantitative PCR in wild-type (WT) and vps75Δ cells

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Effect of deletion of VPS75 and RTT109 on intragenic transcription. (A) Vps75 and Rtt109 do not regulate intragenic transcription from the cryptic promoter of the GAL1 :: FLO8-HIS3 reporter gene. The indicated wild-type (WT) and deletion strains containing

    Journal:

    Article Title: An Rtt109-Independent Role for Vps75 in Transcription-Associated Nucleosome Dynamics

    doi: 10.1128/MCB.01882-08

    Figure Lengend Snippet: Effect of deletion of VPS75 and RTT109 on intragenic transcription. (A) Vps75 and Rtt109 do not regulate intragenic transcription from the cryptic promoter of the GAL1 :: FLO8-HIS3 reporter gene. The indicated wild-type (WT) and deletion strains containing

    Article Snippet: To determine the genome-wide distribution of Vps75 (ChIP-chip), chromatin immunoprecipitates were prepared and hybridized to Affymetrix S. cerevisiae whole-genome forward tiling arrays (Tiling 1.0F array; P/N 520286) as described previously ( ).

    Techniques:

    Oxidative stress attenuated Sp1 binding to its GC-Box elements present in hPrdx6 gene promoter. Table 1 . Evolutionary conserved Sp1 binding sequences in TATA-less Prdx6 promoters of mouse, rat and human cells. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 (GC-Box) binding sites showing primer location and sequences used in ChIP assay. ( B and C ) Oxidative stress (H 2 O 2 or UVB)-induced reduction in DNA binding activity of Sp1 to hPrdx6 gene promoter containing GC-Box (Sp1 sites) in SRA-hLECs. ChIP assay was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis kits (Active motif). Chromatin samples prepared from SRA-hLECs were exposed to varying concentrations of H 2 O 2 (0, 50, 75 and 100µM) or UVB (0, 40, 80 and 120J/m 2 ), and were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 gene promoter bearing GC-box (Sp1 sites). Histogram showed the amplified DNA by qPCR analysis: ( B ) Control (0) vs 50µM vs 75µM vs 100µM H 2 O 2 treatment. ( C ) Control (0) vs 40J/m 2 vs 80J/m 2 vs 120J/m 2 UVB exposure. The data represent mean ± SD from three independent experiments (** p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Oxidative stress attenuated Sp1 binding to its GC-Box elements present in hPrdx6 gene promoter. Table 1 . Evolutionary conserved Sp1 binding sequences in TATA-less Prdx6 promoters of mouse, rat and human cells. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 (GC-Box) binding sites showing primer location and sequences used in ChIP assay. ( B and C ) Oxidative stress (H 2 O 2 or UVB)-induced reduction in DNA binding activity of Sp1 to hPrdx6 gene promoter containing GC-Box (Sp1 sites) in SRA-hLECs. ChIP assay was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis kits (Active motif). Chromatin samples prepared from SRA-hLECs were exposed to varying concentrations of H 2 O 2 (0, 50, 75 and 100µM) or UVB (0, 40, 80 and 120J/m 2 ), and were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 gene promoter bearing GC-box (Sp1 sites). Histogram showed the amplified DNA by qPCR analysis: ( B ) Control (0) vs 50µM vs 75µM vs 100µM H 2 O 2 treatment. ( C ) Control (0) vs 40J/m 2 vs 80J/m 2 vs 120J/m 2 UVB exposure. The data represent mean ± SD from three independent experiments (** p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Binding Assay, Gas Chromatography, Chromatin Immunoprecipitation, Activity Assay, Real-time Polymerase Chain Reaction, Amplification

    Mutagenesis and in vivo DNA binding assay revealed increased binding of Sumoylation-deficient Sp1K16R to Sp1 site in Prdx6 promoter by skipping aberrant Sumoylation effect. ( A ) Schematic representation of the regulatory region of proximal promoter of Prdx6 gene containing GC-box (Sp1 binding sites) showing primer location used in ChIP assay. ( B ) Sumo1 failed to affect Sp1-DNA binding activity in Sumoylation-deficient Sp1K16R transfected LECs. SRA-hLECs were transfected with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R alone or cotransfected with different concentrations of pEGFP-Sumo1. ChIP experiment was carried out as described in Materials and Methods. Chromatin samples prepared from LECs cotransfected with pEGFP-Sumo1 with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R were subjected to ChIP assay with a ChIP grade antibody, anti-HA (gray and black bars) and control IgG (open bars). The DNA fragments were used as templates for RT-qPCR by using primers designed to amplify −342 to +30 region of the Prdx6 gene promoter bearing Sp1 binding sites as shown. Histogram shows the amplified DNA through real-time qPCR analysis. 0 µg vs 2 µg and 4µg pEGFP-Sumo1, pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (* p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Mutagenesis and in vivo DNA binding assay revealed increased binding of Sumoylation-deficient Sp1K16R to Sp1 site in Prdx6 promoter by skipping aberrant Sumoylation effect. ( A ) Schematic representation of the regulatory region of proximal promoter of Prdx6 gene containing GC-box (Sp1 binding sites) showing primer location used in ChIP assay. ( B ) Sumo1 failed to affect Sp1-DNA binding activity in Sumoylation-deficient Sp1K16R transfected LECs. SRA-hLECs were transfected with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R alone or cotransfected with different concentrations of pEGFP-Sumo1. ChIP experiment was carried out as described in Materials and Methods. Chromatin samples prepared from LECs cotransfected with pEGFP-Sumo1 with either pCl-neo-HA-Sp1 or its mutant pCl-neo-HA-Sp1K16R were subjected to ChIP assay with a ChIP grade antibody, anti-HA (gray and black bars) and control IgG (open bars). The DNA fragments were used as templates for RT-qPCR by using primers designed to amplify −342 to +30 region of the Prdx6 gene promoter bearing Sp1 binding sites as shown. Histogram shows the amplified DNA through real-time qPCR analysis. 0 µg vs 2 µg and 4µg pEGFP-Sumo1, pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (* p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Mutagenesis, In Vivo, DNA Binding Assay, Binding Assay, Gas Chromatography, Chromatin Immunoprecipitation, Activity Assay, Transfection, Quantitative RT-PCR, Amplification, Real-time Polymerase Chain Reaction

    Sumoylation-deficient Prdx6K122/142R fused to transduction protein domain (TAT) internalized in cells and blunted oxidative stress-induced aberrant Sumoylation. ( A and B ) Prdx6 -/- mLECs were transduced with Sumoylation-deficient protein, TAT-HA-Prdx6K122/142R conferred higher resistance to oxidative stress-induced Sumoylation than did Prdx6WT. Prdx6 -/- LECs were pretreated with TAT-HA-Prdx6 WT or TAT-HA-Prdx6K122/142R and then exposed to different concentrations of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ). 48h later, nuclear extracts containing equal amounts of proteins were processed for Sumo1-ELISA assay to assess the relative levels of Sp1 Sumoylation in Prdx6 WT (gray bars) and its mutant Prdx6K122/142R (black bars) transduced in cells as shown. Data represent the mean ± SD from three independent experiments (** p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Sumoylation-deficient Prdx6K122/142R fused to transduction protein domain (TAT) internalized in cells and blunted oxidative stress-induced aberrant Sumoylation. ( A and B ) Prdx6 -/- mLECs were transduced with Sumoylation-deficient protein, TAT-HA-Prdx6K122/142R conferred higher resistance to oxidative stress-induced Sumoylation than did Prdx6WT. Prdx6 -/- LECs were pretreated with TAT-HA-Prdx6 WT or TAT-HA-Prdx6K122/142R and then exposed to different concentrations of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ). 48h later, nuclear extracts containing equal amounts of proteins were processed for Sumo1-ELISA assay to assess the relative levels of Sp1 Sumoylation in Prdx6 WT (gray bars) and its mutant Prdx6K122/142R (black bars) transduced in cells as shown. Data represent the mean ± SD from three independent experiments (** p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Transduction, Enzyme-linked Immunosorbent Assay, Mutagenesis

    Sp1 (K16R) mutated at Sumoylation site enhanced its transcription potential by increasing steady state of Sp1 in cells. ( A ) SRA-hLECs were cotransfected with wild type Prdx6 promoter linked to CAT along with either pCl-neo-HA-Sp1 or pCl-neo-HA-Sp1K16R as shown. After 72h cell lysates were analyzed for CAT activity. Histograms represent values derived from three independent experiments. * p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Sp1 (K16R) mutated at Sumoylation site enhanced its transcription potential by increasing steady state of Sp1 in cells. ( A ) SRA-hLECs were cotransfected with wild type Prdx6 promoter linked to CAT along with either pCl-neo-HA-Sp1 or pCl-neo-HA-Sp1K16R as shown. After 72h cell lysates were analyzed for CAT activity. Histograms represent values derived from three independent experiments. * p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Activity Assay, Derivative Assay

    Enhanced Sp1 binding to Prdx6 promoter in cells transduced with Sumoylation–deficient Prdx6 against oxidative stress. ( A and B ) SRA-hLECs were transduced with TAT-HA-Prdx6WT and its mutant TAT-HA-Prdx6K122/142R mutated at Sumoylation sites recombinant protein followed by different concentrations of H 2 O 2 ( A ) or UVB ( B ) exposure as indicated. ChIP assay was carried out using ChIP grade anti-Sp1 antibody. The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 promoter bearing GC-box (Sp1 sites). Histogram shows the amplified DNA with real-time PCR analysis; open bars vs gray bars vs black bars. The data represent mean ± SD from two independent experiments (** p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Enhanced Sp1 binding to Prdx6 promoter in cells transduced with Sumoylation–deficient Prdx6 against oxidative stress. ( A and B ) SRA-hLECs were transduced with TAT-HA-Prdx6WT and its mutant TAT-HA-Prdx6K122/142R mutated at Sumoylation sites recombinant protein followed by different concentrations of H 2 O 2 ( A ) or UVB ( B ) exposure as indicated. ChIP assay was carried out using ChIP grade anti-Sp1 antibody. The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 promoter bearing GC-box (Sp1 sites). Histogram shows the amplified DNA with real-time PCR analysis; open bars vs gray bars vs black bars. The data represent mean ± SD from two independent experiments (** p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Binding Assay, Transduction, Mutagenesis, Recombinant, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Gas Chromatography, Amplification

    ChIP analysis of genomic DNA from LECs facing oxidative stress disclosed a significant loss in Sp1-DNA binding to Prdx6 gene promoter. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 binding sites showing primer location and sequences used in ChIP assay. ( B and C ) ChIP assay showing Sp1 binding to Prdx6 promoter in vivo . Chromatin samples were prepared from Prdx6 +/+ LECs (mLECs) exposed to different doses of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ) as indicated. 72h later samples were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 or IgG control. The DNA fragments were amplified by using primers designed to amplify −208 to +27 region of the Prdx6 promoter bearing Sp1 sites (**) and contiguous sequence (−2229 to −2356) to which Sp1 does not bind (*) as indicated. PCR products were resolved onto agarose gel and visualized with ethidium bromide staining. Photographs are representative of three experiments. ( D and E ) Expression assays showing H 2 O 2 - and UVB- induced declined expression of Sp1 in mLECs. mLECs cells were treated with different concentrations of H 2 O 2 ( D ) and/or UVB ( E ) multiple time for 3 days as indicated. Total RNA and protein were isolated and subjected to real-time PCR and Western analysis with Sp1 specific probes, respectively. Data revealed a concentration –dependent reduced expression of Sp1 mRNA ( Da and Ea ; Gray vs black bars; * p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: ChIP analysis of genomic DNA from LECs facing oxidative stress disclosed a significant loss in Sp1-DNA binding to Prdx6 gene promoter. ( A ) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 binding sites showing primer location and sequences used in ChIP assay. ( B and C ) ChIP assay showing Sp1 binding to Prdx6 promoter in vivo . Chromatin samples were prepared from Prdx6 +/+ LECs (mLECs) exposed to different doses of H 2 O 2 (0, 25, 50 and 75µM) and/or UVB (0, 30, 60 and 90J/m 2 ) as indicated. 72h later samples were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 or IgG control. The DNA fragments were amplified by using primers designed to amplify −208 to +27 region of the Prdx6 promoter bearing Sp1 sites (**) and contiguous sequence (−2229 to −2356) to which Sp1 does not bind (*) as indicated. PCR products were resolved onto agarose gel and visualized with ethidium bromide staining. Photographs are representative of three experiments. ( D and E ) Expression assays showing H 2 O 2 - and UVB- induced declined expression of Sp1 in mLECs. mLECs cells were treated with different concentrations of H 2 O 2 ( D ) and/or UVB ( E ) multiple time for 3 days as indicated. Total RNA and protein were isolated and subjected to real-time PCR and Western analysis with Sp1 specific probes, respectively. Data revealed a concentration –dependent reduced expression of Sp1 mRNA ( Da and Ea ; Gray vs black bars; * p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Amplification, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

    Aging human LECs/lenses showed age-dependent decline in Senp1 expression and increased Sumo1 expression, which were directly related to increase Sp1 Sumoylation. ( A ) Aging hLECs/lenses displayed a progressive decline in the deSumoylating agent Senp1 and an increase in Sumo1 levels. Total RNA was isolated from human lenses/LECs of different ages as indicated, and was processed for real-time qPCR analysis as stated in Materials and Methods. The data represent the mean ± S.D. values from three independent experiments. P values were determined for younger (18y) vs aging samples. ** p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Aging human LECs/lenses showed age-dependent decline in Senp1 expression and increased Sumo1 expression, which were directly related to increase Sp1 Sumoylation. ( A ) Aging hLECs/lenses displayed a progressive decline in the deSumoylating agent Senp1 and an increase in Sumo1 levels. Total RNA was isolated from human lenses/LECs of different ages as indicated, and was processed for real-time qPCR analysis as stated in Materials and Methods. The data represent the mean ± S.D. values from three independent experiments. P values were determined for younger (18y) vs aging samples. ** p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction

    Prdx6 -/- LECs, a model for aging, bore an enhanced Sumoylated form of Sp1, and levels were further increased with exposure to oxidative stress. ( A ) Nuclear extracts were prepared from Prdx6 +/+ and Prdx6 −/− mLECs and submitted to Sp1 Sandwich/Sumo1-ELISA assays to examine the total and Sumoylated forms of Sp1 protein. Sumoylated Sp1 protein was subtracted from total Sp1 protein, and results are presented as deSumoylated (gray bars) and Sumoylated (black bars) forms of Sp1. The data represent mean ± SD from three independent experiments. Prdx6 +/+ vs Prdx6 −/− ; * p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Prdx6 -/- LECs, a model for aging, bore an enhanced Sumoylated form of Sp1, and levels were further increased with exposure to oxidative stress. ( A ) Nuclear extracts were prepared from Prdx6 +/+ and Prdx6 −/− mLECs and submitted to Sp1 Sandwich/Sumo1-ELISA assays to examine the total and Sumoylated forms of Sp1 protein. Sumoylated Sp1 protein was subtracted from total Sp1 protein, and results are presented as deSumoylated (gray bars) and Sumoylated (black bars) forms of Sp1. The data represent mean ± SD from three independent experiments. Prdx6 +/+ vs Prdx6 −/− ; * p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Enzyme-linked Immunosorbent Assay

    Cells overexpressing Sumo1 showed reduced Sp1 binding to its responsive elements in Prdx6 promoter. ( A ) Schematic illustration of Prdx6 gene promoter. ( B ) Gel-shift mobility assay showing that Sumo1 reduced the Sp1 DNA–binding activity of Prdx6 gene promoter. Gel-shift mobility assay was carried out using nuclear extracts isolated from mLECs transfected with pEGFP-Vector (Lanes 1 and 2) or pEGFP-Sumo1 (Lanes 3 and 4) incubated with 32p-labeled wild type probe (Lanes 1 and 3) or its mutant (Lanes 2 and 4). A diminished Cm1 band was observed in cells overexpressing Sumo1 (Lane 3) in comparison to vector control (Lane 1). No binding occurred in mutant probes (Lanes 2 and 4). ( C ) Histogram represents densitometry analysis of DNA-protein complex formed in gel-shift assay. Lane 1 vs lane 3, * p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Cells overexpressing Sumo1 showed reduced Sp1 binding to its responsive elements in Prdx6 promoter. ( A ) Schematic illustration of Prdx6 gene promoter. ( B ) Gel-shift mobility assay showing that Sumo1 reduced the Sp1 DNA–binding activity of Prdx6 gene promoter. Gel-shift mobility assay was carried out using nuclear extracts isolated from mLECs transfected with pEGFP-Vector (Lanes 1 and 2) or pEGFP-Sumo1 (Lanes 3 and 4) incubated with 32p-labeled wild type probe (Lanes 1 and 3) or its mutant (Lanes 2 and 4). A diminished Cm1 band was observed in cells overexpressing Sumo1 (Lane 3) in comparison to vector control (Lane 1). No binding occurred in mutant probes (Lanes 2 and 4). ( C ) Histogram represents densitometry analysis of DNA-protein complex formed in gel-shift assay. Lane 1 vs lane 3, * p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Activity Assay, Isolation, Transfection, Plasmid Preparation, Incubation, Labeling, Mutagenesis

    Aging human LECs/lenses showing elevated levels of ROS and progressive decline in Sp1 and Prdx6 expression connected to reduction in Sp1 activity. ( A ) ROS levels increased progressively in aging hLECs. Cells were cultured in 96 well plate (5000/well), and ROS were quantified using H2-DCF-DA dye assay as shown. Data represent the mean ± S.D. of two independent experiments. Younger (18y) vs aging samples; * p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Aging human LECs/lenses showing elevated levels of ROS and progressive decline in Sp1 and Prdx6 expression connected to reduction in Sp1 activity. ( A ) ROS levels increased progressively in aging hLECs. Cells were cultured in 96 well plate (5000/well), and ROS were quantified using H2-DCF-DA dye assay as shown. Data represent the mean ± S.D. of two independent experiments. Younger (18y) vs aging samples; * p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Expressing, Activity Assay, Cell Culture

    Sp1K16R mutated at Sumo1 binding site provided enhanced cytoprotection against oxidative stress. ( A and B ) SRA-hLECs were transfected with either pEGFP-Vector, pCl-neo-HA-Sp1, or pCl-neo-HA-Sp1K16R and then exposed to different concentrations of H 2 O 2 as indicated. After 8h of H 2 O 2 exposure, ROS intensity was quantified with CellROX deep red reagent ( A ). 24h later viability of cells was analyzed by MTS assay ( B ) as shown. Histogram values represent mean ± SD of three independent experiments. 0 vs 100 vs 150µM H 2 O 2 and pEGFP-Vector vs pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (** p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Sp1K16R mutated at Sumo1 binding site provided enhanced cytoprotection against oxidative stress. ( A and B ) SRA-hLECs were transfected with either pEGFP-Vector, pCl-neo-HA-Sp1, or pCl-neo-HA-Sp1K16R and then exposed to different concentrations of H 2 O 2 as indicated. After 8h of H 2 O 2 exposure, ROS intensity was quantified with CellROX deep red reagent ( A ). 24h later viability of cells was analyzed by MTS assay ( B ) as shown. Histogram values represent mean ± SD of three independent experiments. 0 vs 100 vs 150µM H 2 O 2 and pEGFP-Vector vs pCl-neo-HA-Sp1 WT vs pCl-neo-HA-Sp1K16R (** p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, MTS Assay

    Sumo1 modified Sp1 at K16 residue in vivo . ( A ) SRA-hLECs (1.2X10 6 ) were transfected with pEGFP-Sumo1 (3µg) along with Sp1 WT (3µg) or its mutant Sp1K16R (mutated at Sumoylation sites) using (3µg) plasmid as indicated. Exogenous Sp1 was immunoprecipitated (IP) from cell lysates containing equal amounts of proteins, and its Sumoylated form was detected with anti-HA ( Ba ) and anti-Sumo1 ( Bb ) rabbit polyclonal antibodies as indicated. Cell lysates were prepared and subjected to IP using anti-HA monoclonal antibody. IP with anti-HA monoclonal antibody shows a single-exogenous Sumoylated band at ~145 kDa (lane 2, pEGFP-Sumo1+pCl-neo-HA-Sp1WT). No Sumoylation band could be detected in cell extracts of pEGFP-Vector+pCl-neo-HA-Sp1WT or pEGFP-Sumo1+pCl-neo-HA-Sp1-K16R transfected cells ( B , a and b ; lanes 1 and 3). ( B ) SRA-hLECs were transfected with pCl-neo-HA-Sp1WT plus pEGFP-Vector, or pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, or pCl-neo-HA-Sp1-K16R plus pEGFP-Sumo1. 48h later, total cell lysates were prepared and processed for Sumo1-ELISA assay according to the manufacturer’s protocol (EpiQuik TM ) to measure the Sumoylated form of Sp1. Data represent mean ± SD from three independent experiments: pCl-neo-HA-Sp1WT plus pEGFP-Vector, vs pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, vs pCl-neo-HA-Sp1 K16R plus pEGFP-Sumo1 (* p

    Journal: Aging (Albany NY)

    Article Title: Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

    doi: 10.18632/aging.101547

    Figure Lengend Snippet: Sumo1 modified Sp1 at K16 residue in vivo . ( A ) SRA-hLECs (1.2X10 6 ) were transfected with pEGFP-Sumo1 (3µg) along with Sp1 WT (3µg) or its mutant Sp1K16R (mutated at Sumoylation sites) using (3µg) plasmid as indicated. Exogenous Sp1 was immunoprecipitated (IP) from cell lysates containing equal amounts of proteins, and its Sumoylated form was detected with anti-HA ( Ba ) and anti-Sumo1 ( Bb ) rabbit polyclonal antibodies as indicated. Cell lysates were prepared and subjected to IP using anti-HA monoclonal antibody. IP with anti-HA monoclonal antibody shows a single-exogenous Sumoylated band at ~145 kDa (lane 2, pEGFP-Sumo1+pCl-neo-HA-Sp1WT). No Sumoylation band could be detected in cell extracts of pEGFP-Vector+pCl-neo-HA-Sp1WT or pEGFP-Sumo1+pCl-neo-HA-Sp1-K16R transfected cells ( B , a and b ; lanes 1 and 3). ( B ) SRA-hLECs were transfected with pCl-neo-HA-Sp1WT plus pEGFP-Vector, or pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, or pCl-neo-HA-Sp1-K16R plus pEGFP-Sumo1. 48h later, total cell lysates were prepared and processed for Sumo1-ELISA assay according to the manufacturer’s protocol (EpiQuik TM ) to measure the Sumoylated form of Sp1. Data represent mean ± SD from three independent experiments: pCl-neo-HA-Sp1WT plus pEGFP-Vector, vs pCl-neo-HA-Sp1WT plus pEGFP-Sumo1, vs pCl-neo-HA-Sp1 K16R plus pEGFP-Sumo1 (* p

    Article Snippet: The following antibodies were used: control IgG and antibody specific to Sp1 (ab13370, Abcam) and/or HA (ab9110 and ab18181, Abcam).

    Techniques: Modification, In Vivo, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

    Loss of LSD1 or HDAC1 induces G 1 cell cycle arrest in F9 and PA-1 cells. (A and B) F9 and PA-1 cells were transfected with the indicated siRNAs, and the cell cycle was analyzed by FACS. F9 and PA-1 cells were arrested in the G 1 cell cycle by LSD1 or HDAC1

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Loss of LSD1 or HDAC1 induces G 1 cell cycle arrest in F9 and PA-1 cells. (A and B) F9 and PA-1 cells were transfected with the indicated siRNAs, and the cell cycle was analyzed by FACS. F9 and PA-1 cells were arrested in the G 1 cell cycle by LSD1 or HDAC1

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Transfection, FACS

    Regulation of H4K16 acetylation by elevated HDAC1 and LSD1 levels in ES/EC cells. (A) The acetylation of H4K16 is sensitive to pan-HDAC inhibitors in HeLa cells. F9 and HeLa cells were treated with the HDAC inhibitor VPA (1 μM) or MS-275 (2 μM)

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Regulation of H4K16 acetylation by elevated HDAC1 and LSD1 levels in ES/EC cells. (A) The acetylation of H4K16 is sensitive to pan-HDAC inhibitors in HeLa cells. F9 and HeLa cells were treated with the HDAC inhibitor VPA (1 μM) or MS-275 (2 μM)

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Mass Spectrometry

    Schematic summarizing LSD1- or HDAC1-regulated pluripotency of mES or EC cells through HDAC1-mediated H4K16 acetylation. LSD1 and HDAC1 form a complex with CoREST, and the activities of LSD1 and HDAC1 in the CoREST complex are mutually dependent. Acetylated

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Schematic summarizing LSD1- or HDAC1-regulated pluripotency of mES or EC cells through HDAC1-mediated H4K16 acetylation. LSD1 and HDAC1 form a complex with CoREST, and the activities of LSD1 and HDAC1 in the CoREST complex are mutually dependent. Acetylated

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques:

    Regulation of LSD1 and HDAC1 by mutual activities in the LSD1-CoREST-HDAC1 complex. (A) Purified recombinant GST-LSD1, 6-histidine-tagged LSD1 (His-LSD1), GST-HDAC1, and GST-CoREST proteins. Lanes M.W., molecular weight markers (in thousands). (B) Analysis

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Regulation of LSD1 and HDAC1 by mutual activities in the LSD1-CoREST-HDAC1 complex. (A) Purified recombinant GST-LSD1, 6-histidine-tagged LSD1 (His-LSD1), GST-HDAC1, and GST-CoREST proteins. Lanes M.W., molecular weight markers (in thousands). (B) Analysis

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Purification, Recombinant, Molecular Weight

    Inactivation of MOF reverses the effects of LSD1 inactivation on increased acetylation of H4K16 and gene expression in ES/EC cells. (A) F9, mES, and PA-1 cells were transfected with the indicated siRNAs. Histone modifications of H3 and H4 were monitored

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Inactivation of MOF reverses the effects of LSD1 inactivation on increased acetylation of H4K16 and gene expression in ES/EC cells. (A) F9, mES, and PA-1 cells were transfected with the indicated siRNAs. Histone modifications of H3 and H4 were monitored

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Expressing, Transfection

    LSD1 forms a complex with HDAC1, and loss of HDAC1 phenocopies the selective growth-inhibitory effects of LSD1 inactivation in ES/EC cells.

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: LSD1 forms a complex with HDAC1, and loss of HDAC1 phenocopies the selective growth-inhibitory effects of LSD1 inactivation in ES/EC cells.

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques:

    Loss of HDAC1 or LSD1 induces the expression of genes for differentiation. (A) PA-1 and F9 cells were transfected with the indicated siRNAs. The mRNAs were isolated, and the levels of the differentiation genes FOXA2 , HNF4A , SOX17 , BMP2 , and EOMES were

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Loss of HDAC1 or LSD1 induces the expression of genes for differentiation. (A) PA-1 and F9 cells were transfected with the indicated siRNAs. The mRNAs were isolated, and the levels of the differentiation genes FOXA2 , HNF4A , SOX17 , BMP2 , and EOMES were

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Expressing, Transfection, Isolation

    Regulation of LSD1 and HDAC1 activities by substrate modification. (A) Schematic outline for the experimental design. The hyperacetylated or hypermethylated nucleosomes were isolated from cells treated with either MS-275 or LSD1 inhibitors. The hyperacetylated

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Regulation of LSD1 and HDAC1 activities by substrate modification. (A) Schematic outline for the experimental design. The hyperacetylated or hypermethylated nucleosomes were isolated from cells treated with either MS-275 or LSD1 inhibitors. The hyperacetylated

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Modification, Isolation, Mass Spectrometry

    Loss of the acetyltransfe rase MOF rescues the growth-inhibitory effects of LSD1 inactivation in ES/EC cells. (A to C) F9, PA-1, and mES cells were transfected with siRNAs specific for luciferase, LSD1, LSD1 plus MOF, and MOF for 48 h. Cell growth was

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Loss of the acetyltransfe rase MOF rescues the growth-inhibitory effects of LSD1 inactivation in ES/EC cells. (A to C) F9, PA-1, and mES cells were transfected with siRNAs specific for luciferase, LSD1, LSD1 plus MOF, and MOF for 48 h. Cell growth was

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Transfection, Luciferase

    Inactivation of LSD1 or HDAC1 causes similar changes of histone methylation and acetylation in ES/EC cells. (A) F9 and NIH 3T3 cells were treated with the LSD1 inhibitors CBB1003 and CBB1007 for 24 h, and the acetylation and methylation of histones H3

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Inactivation of LSD1 or HDAC1 causes similar changes of histone methylation and acetylation in ES/EC cells. (A) F9 and NIH 3T3 cells were treated with the LSD1 inhibitors CBB1003 and CBB1007 for 24 h, and the acetylation and methylation of histones H3

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Methylation

    LSD1 or HDAC1 inactivation induces elevated levels of H3K4me2 and H4K16ac on the regulatory regions of differentiation genes. (A and B) F9 and PA-1 cells were transfected with siRNAs for 48 h (A) or treated with CBB1007 or MS-275 for 30 h (B), as indicated.

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: LSD1 or HDAC1 inactivation induces elevated levels of H3K4me2 and H4K16ac on the regulatory regions of differentiation genes. (A and B) F9 and PA-1 cells were transfected with siRNAs for 48 h (A) or treated with CBB1007 or MS-275 for 30 h (B), as indicated.

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Transfection, Mass Spectrometry

    The effects of inactivation of HDAC1 resemble the selective growth-inhibitory effects of LSD1 deficiency in ES/EC cells. (A) LSD1 formed a protein complex with CoREST and HDAC1. The 3× Flag/HA-tagged LSD1 protein complex was isolated by immunoprecipitation

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: The effects of inactivation of HDAC1 resemble the selective growth-inhibitory effects of LSD1 deficiency in ES/EC cells. (A) LSD1 formed a protein complex with CoREST and HDAC1. The 3× Flag/HA-tagged LSD1 protein complex was isolated by immunoprecipitation

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Hemagglutination Assay, Isolation, Immunoprecipitation

    Restoration of LSD1, HDAC1, and MOF siRNA ablation effects by reexpression of cognate cDNAs. (A to C) After they were transfected with siRNA specific for the LSD1 and HDAC1 5′ or 3′ UTR for 24 h, F9 and PA-1 cells were transfected with

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: Restoration of LSD1, HDAC1, and MOF siRNA ablation effects by reexpression of cognate cDNAs. (A to C) After they were transfected with siRNA specific for the LSD1 and HDAC1 5′ or 3′ UTR for 24 h, F9 and PA-1 cells were transfected with

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Transfection

    HDAC1 is required for the expression of Oct4 and Sox2 by directly binding to the regulatory regions. (A and B) Inactivation of HDAC1 or LSD1 suppresses the expression of pluripotent stem cell proteins Oct4 and Sox2. F9 and PA-1 cells were transfected

    Journal:

    Article Title: LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

    doi: 10.1128/MCB.00631-13

    Figure Lengend Snippet: HDAC1 is required for the expression of Oct4 and Sox2 by directly binding to the regulatory regions. (A and B) Inactivation of HDAC1 or LSD1 suppresses the expression of pluripotent stem cell proteins Oct4 and Sox2. F9 and PA-1 cells were transfected

    Article Snippet: Antibodies against HDAC1, HDAC2, HDAC3, HDAC6, Sox2, and CoREST were obtained from Bethyl Laboratories; anti-histone H4 peptide with acetylated lysine 16 (anti-H4K16ac; catalog no. 07-329), anti-histone H4 peptide with acetylated lysine 12 (anti-H4K12ac; catalog no. 04719), and histone H3 peptide with trimethylated lysine 4 (anti-H3K4me3) antibodies were from Millipore; anti-histone H3 peptide with acetylated lysine 56 (H3K56ac) antibodies (catalog no. 39281) were from Active Motif; anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), histone H3 peptide with methylated lysine 4 (H3K4me1; ab8895), histone H3 (ab1791), histone H3 peptide with acetylated lysine 14 (H3K14ac; ab52946), histone H3 peptide with acetylated lysine 9 (H3K9ac; ab4441), histone H3 peptide with acetylated lysine 27 (H3K27ac; ab4279), Sall4, Nanog, and LSD1 (ab17721) antibodies were from Abcam; and Lin28 and Klf4 antibodies were from Proteintech Group.

    Techniques: Expressing, Binding Assay, Transfection

    Abnormal behaviors in individual Kdm1a Vasa M-Z+ adults. ( A ) Behavioral ethogram of M. castaneus (CAST) controls versus Kdm1a Vasa M-Z+ adults. ( B ) Quantification of change in weight of food in the hopper of parents of Kdm1a Vasa M-Z+ adults and CAST controls versus Kdm1a Vasa M-Z+ adults. ( C ) Quantification of change in weight of food in the hopper of B6/CAST M+Z+ controls versus Kdm1a Vasa M-Z+ adults. ( D ) Quantification of change in weight of food in the hopper of F2 intercrossed M+Z+ adults versus Kdm1a Vasa M-Z+ adults. ( E ) Quantification of change in bedding height of parents of Kdm1a Vasa M-Z+ adults and CAST controls versus Kdm1a Vasa M-Z+ adults. ( F ) Quantification of change in bedding height of B6/CAST M+Z+ controls versus Kdm1a Vasa M-Z+ adults. ( G ) Quantification of change in bedding height of F2 intercrossed M+Z+ adults versus Kdm1a Vasa M-Z+ adults. The measurements for each individual animal ( B–D ) and ( E–G ) correspond to the averages shown in Figure 5 (D,E ). Yellow arrowheads represent animals heterozygous for Kdm1a . Data shown as mean for each day. p-values calculated using an unpaired t-test with * = p

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Abnormal behaviors in individual Kdm1a Vasa M-Z+ adults. ( A ) Behavioral ethogram of M. castaneus (CAST) controls versus Kdm1a Vasa M-Z+ adults. ( B ) Quantification of change in weight of food in the hopper of parents of Kdm1a Vasa M-Z+ adults and CAST controls versus Kdm1a Vasa M-Z+ adults. ( C ) Quantification of change in weight of food in the hopper of B6/CAST M+Z+ controls versus Kdm1a Vasa M-Z+ adults. ( D ) Quantification of change in weight of food in the hopper of F2 intercrossed M+Z+ adults versus Kdm1a Vasa M-Z+ adults. ( E ) Quantification of change in bedding height of parents of Kdm1a Vasa M-Z+ adults and CAST controls versus Kdm1a Vasa M-Z+ adults. ( F ) Quantification of change in bedding height of B6/CAST M+Z+ controls versus Kdm1a Vasa M-Z+ adults. ( G ) Quantification of change in bedding height of F2 intercrossed M+Z+ adults versus Kdm1a Vasa M-Z+ adults. The measurements for each individual animal ( B–D ) and ( E–G ) correspond to the averages shown in Figure 5 (D,E ). Yellow arrowheads represent animals heterozygous for Kdm1a . Data shown as mean for each day. p-values calculated using an unpaired t-test with * = p

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques:

    Relative expression of epigenetic regulators in Kdm1a Zp3 2C embryos. Quantitative RT-PCR analysis of epigenetic regulators including Trim28 ( A ), Zfp57 ( B ) Dppa3/stella ( C ), and Dnmt1 ( D ) in Kdm1a Zp3 M+Z+ 2C embryos compared to Kdm1a Zp3 M-Z+ 2C embryos. Y-axis represents average fold change. All gene expression was normalized to Hprt expression. DOI: http://dx.doi.org/10.7554/eLife.08848.016

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Relative expression of epigenetic regulators in Kdm1a Zp3 2C embryos. Quantitative RT-PCR analysis of epigenetic regulators including Trim28 ( A ), Zfp57 ( B ) Dppa3/stella ( C ), and Dnmt1 ( D ) in Kdm1a Zp3 M+Z+ 2C embryos compared to Kdm1a Zp3 M-Z+ 2C embryos. Y-axis represents average fold change. All gene expression was normalized to Hprt expression. DOI: http://dx.doi.org/10.7554/eLife.08848.016

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Expressing, Quantitative RT-PCR

    Expression of epigenetic regulators in Kdm1a fl/fl and Kdm1a Zp3 oocytes. Sequenced RNA-seq reads showing relative expression from Kdm1a fl/fl oocytes and Kdm1a Zp3 mutant oocytes aligned to the genome for Lsd1/Kdm1a ( A ), Tet1 ( B ), Trim28 ( C ), Zfp57 ( D ), Dppa3/stella ( E ), Dnmt1 ( F ) and Uhrf1 ( G ). Gene tracks visualized using Integrative Genomics Viewer. DOI: http://dx.doi.org/10.7554/eLife.08848.017

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Expression of epigenetic regulators in Kdm1a fl/fl and Kdm1a Zp3 oocytes. Sequenced RNA-seq reads showing relative expression from Kdm1a fl/fl oocytes and Kdm1a Zp3 mutant oocytes aligned to the genome for Lsd1/Kdm1a ( A ), Tet1 ( B ), Trim28 ( C ), Zfp57 ( D ), Dppa3/stella ( E ), Dnmt1 ( F ) and Uhrf1 ( G ). Gene tracks visualized using Integrative Genomics Viewer. DOI: http://dx.doi.org/10.7554/eLife.08848.017

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Expressing, RNA Sequencing Assay, Mutagenesis

    Imprinting analysis of Kdm1a Vasa progeny. ( A , C , E ) Allele-specific bisulfite analysis of, Igf2r ( A ), Mest ( C ), and Snrpn ( E ). Each line represents the clone of an allele. Each circle represents a CpG dinucleotide where closed circles indicate methylation and open circles indicate no methylation. Maternal and paternal alleles are indicated. ( B , D , F ) Relative expression analysis of Igf2r ( B ), Mest ( D ), and Snrpn ( F ). Expression normalized to β-actin . Error bars indicate ± S.E.M. p-values calculated using an unpaired t-test with n.s. indicating p > 0.05, * = p

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Imprinting analysis of Kdm1a Vasa progeny. ( A , C , E ) Allele-specific bisulfite analysis of, Igf2r ( A ), Mest ( C ), and Snrpn ( E ). Each line represents the clone of an allele. Each circle represents a CpG dinucleotide where closed circles indicate methylation and open circles indicate no methylation. Maternal and paternal alleles are indicated. ( B , D , F ) Relative expression analysis of Igf2r ( B ), Mest ( D ), and Snrpn ( F ). Expression normalized to β-actin . Error bars indicate ± S.E.M. p-values calculated using an unpaired t-test with n.s. indicating p > 0.05, * = p

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Methylation, Expressing

    Model. Loss of maternal LSD1 results in defects later in development in wild-type oocytes, after fertilization (denoted by blue sperm encircling oocyte) the fertilized egg undergoes the maternal to zygotic transition (MZT; green to blue/purple) at the 1–2 cell stage. These M+Z+ embryos proceed normally through development (indicated by blastocyst, perinatal stage pup, and adult mouse). In contrast, when Lsd1 is deleted with either Gdf9- or Zp3-Cre, the resulting Lsd1Gdf9 and Lsd1Zp3 progeny become arrest at the 1–2 cell stage and never undergo the MZT (green). When Lsd1 is deleted with Vasa-Cre, we observe 3 hypomorphic outcomes in resulting Lsd1Vasa progeny: (1) developmental arrest at the 1–2 cell stage, (2) perinatal lethality and (3) abnormal behavior in surviving adult animals. These outcomes are due to reduced LSD1 in the mothers oocyte, suggesting that lowered maternal LSD1 can result in defects much later in development. These long-range outcomes are associated with imprinting defects (depicted as wild-type versus mutant changes in DNA methylation within the yellow region). DOI: http://dx.doi.org/10.7554/eLife.08848.027

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Model. Loss of maternal LSD1 results in defects later in development in wild-type oocytes, after fertilization (denoted by blue sperm encircling oocyte) the fertilized egg undergoes the maternal to zygotic transition (MZT; green to blue/purple) at the 1–2 cell stage. These M+Z+ embryos proceed normally through development (indicated by blastocyst, perinatal stage pup, and adult mouse). In contrast, when Lsd1 is deleted with either Gdf9- or Zp3-Cre, the resulting Lsd1Gdf9 and Lsd1Zp3 progeny become arrest at the 1–2 cell stage and never undergo the MZT (green). When Lsd1 is deleted with Vasa-Cre, we observe 3 hypomorphic outcomes in resulting Lsd1Vasa progeny: (1) developmental arrest at the 1–2 cell stage, (2) perinatal lethality and (3) abnormal behavior in surviving adult animals. These outcomes are due to reduced LSD1 in the mothers oocyte, suggesting that lowered maternal LSD1 can result in defects much later in development. These long-range outcomes are associated with imprinting defects (depicted as wild-type versus mutant changes in DNA methylation within the yellow region). DOI: http://dx.doi.org/10.7554/eLife.08848.027

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Mutagenesis, DNA Methylation Assay

    KDM1A expression in stged oocytes. ( A–L ) Immunohistochemistry (IHC) of primordial follicles ( A–C ), primary follicles ( D–F ), secondary follicles ( G-I ) and pre-antral and antral follicles ( J–L ) stained with anti-KDM1A(brown) antibody and hematoxylin (blue). The oocyte nucleus is indicated with black arrowheads. Scale bars represent 50 μm. DOI: http://dx.doi.org/10.7554/eLife.08848.004

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: KDM1A expression in stged oocytes. ( A–L ) Immunohistochemistry (IHC) of primordial follicles ( A–C ), primary follicles ( D–F ), secondary follicles ( G-I ) and pre-antral and antral follicles ( J–L ) stained with anti-KDM1A(brown) antibody and hematoxylin (blue). The oocyte nucleus is indicated with black arrowheads. Scale bars represent 50 μm. DOI: http://dx.doi.org/10.7554/eLife.08848.004

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Expressing, Immunohistochemistry, Staining

    Lack of normal Kdm1a Gdf9 and Kdm1a Zp3 embryos at embryonic day 1.5 and 2.5. ( A , B , D , E , F ) Brightfield images of embryonic day 1.5 (e1.5) M+Z+ 1-cell ( A ) and 2-cell ( B ) embryos and M-Z+ 1-cell ( D ), 2-cell ( E ), and fragmented ( F ) embryos derived from Kdm1a Gdf9 control and mutant mothers. ( C , G , H ) Brightfield images of e2.5 M+Z+ 8-cell ( C ) embryo and M-Z+ abnormal 1-cell ( G ), and fragmented ( H ) embryos derived from Kdm1a Gdf9 control and mutant mothers. ( I ) Percentage of fragmented (purple), unfertilized oocyte or 1C (green), and 2C (yellow) embryos from Kdm1a Gdf9 control and mutant mothers. n = 123 for Kdm1a Gdf9 M+Z+ control embryos from 8 litters. n = 104 for Kdm1a Gdf9 M-Z+ embryos from 8 litters. ( J ) Brightfield image of 3-cell M-Z+ embryo derived from a Kdm1a Zp3 mutant mother. ( K ) Brightfield image of 4-cell M-Z+ embryo derived from a Kdm1a Zp3 mutant mother. DOI: http://dx.doi.org/10.7554/eLife.08848.007

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Lack of normal Kdm1a Gdf9 and Kdm1a Zp3 embryos at embryonic day 1.5 and 2.5. ( A , B , D , E , F ) Brightfield images of embryonic day 1.5 (e1.5) M+Z+ 1-cell ( A ) and 2-cell ( B ) embryos and M-Z+ 1-cell ( D ), 2-cell ( E ), and fragmented ( F ) embryos derived from Kdm1a Gdf9 control and mutant mothers. ( C , G , H ) Brightfield images of e2.5 M+Z+ 8-cell ( C ) embryo and M-Z+ abnormal 1-cell ( G ), and fragmented ( H ) embryos derived from Kdm1a Gdf9 control and mutant mothers. ( I ) Percentage of fragmented (purple), unfertilized oocyte or 1C (green), and 2C (yellow) embryos from Kdm1a Gdf9 control and mutant mothers. n = 123 for Kdm1a Gdf9 M+Z+ control embryos from 8 litters. n = 104 for Kdm1a Gdf9 M-Z+ embryos from 8 litters. ( J ) Brightfield image of 3-cell M-Z+ embryo derived from a Kdm1a Zp3 mutant mother. ( K ) Brightfield image of 4-cell M-Z+ embryo derived from a Kdm1a Zp3 mutant mother. DOI: http://dx.doi.org/10.7554/eLife.08848.007

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Derivative Assay, Mutagenesis

    Expression of epigenetic regulators in Kdm1a Zp3 2C embryos. Sequenced RNA-seq reads showing relative expression from Kdm1a fl/fl M+Z+ 2C embryos and Kdm1a Zp3 M-Z+ 2C embryos aligned to the genome for Lsd1/Kdm1a ( A ), Tet1 ( B ), Trim28 ( C ), Zfp57 ( D ), Dppa3/stella ( E ), Dnmt1 ( F ) and Uhrf1 ( G ). Gene tracks visualized using Integrative Genomics Viewer. DOI: http://dx.doi.org/10.7554/eLife.08848.015

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Expression of epigenetic regulators in Kdm1a Zp3 2C embryos. Sequenced RNA-seq reads showing relative expression from Kdm1a fl/fl M+Z+ 2C embryos and Kdm1a Zp3 M-Z+ 2C embryos aligned to the genome for Lsd1/Kdm1a ( A ), Tet1 ( B ), Trim28 ( C ), Zfp57 ( D ), Dppa3/stella ( E ), Dnmt1 ( F ) and Uhrf1 ( G ). Gene tracks visualized using Integrative Genomics Viewer. DOI: http://dx.doi.org/10.7554/eLife.08848.015

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Expressing, RNA Sequencing Assay

    Hypomorphic phenotype in Kdm1a Vasa progeny. ( A–D ) Brightfield images of M+Z+. ( A ) and M-Z+ ( B–D ) embryos derived from Kdm1a Vasa control and mutant mothers at embryonic day 3.5 (e3.5). Panels show blastocysts ( A , B ), a multicellular embryo ( C ) and a fragmented embryo ( D ). ( E ) Percentage of fragmented (purple), 1-cell (green), multi-cellular (blue) and blastocyst (yellow) embryos from Kdm1a Vasa control and mutant mothers at e3.5. n = 58 for Kdm1a Vasa M+Z+ embryos from 7 litters. n = 79 for Kdm1a Vasa M-Z+ embryos from 10 litters. ( F ) Litter sizes of Kdm1a Vasa control and mutant mothers. Average litter size for each indicated by red line. Each circle indicates one litter and n=number of litters analyzed. p-values calculated using an unpaired t-test with **** = p

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Hypomorphic phenotype in Kdm1a Vasa progeny. ( A–D ) Brightfield images of M+Z+. ( A ) and M-Z+ ( B–D ) embryos derived from Kdm1a Vasa control and mutant mothers at embryonic day 3.5 (e3.5). Panels show blastocysts ( A , B ), a multicellular embryo ( C ) and a fragmented embryo ( D ). ( E ) Percentage of fragmented (purple), 1-cell (green), multi-cellular (blue) and blastocyst (yellow) embryos from Kdm1a Vasa control and mutant mothers at e3.5. n = 58 for Kdm1a Vasa M+Z+ embryos from 7 litters. n = 79 for Kdm1a Vasa M-Z+ embryos from 10 litters. ( F ) Litter sizes of Kdm1a Vasa control and mutant mothers. Average litter size for each indicated by red line. Each circle indicates one litter and n=number of litters analyzed. p-values calculated using an unpaired t-test with **** = p

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Derivative Assay, Mutagenesis

    The MZT is impaired in Kdm1a Zp3 mutants. ( A–H ) Differential expression of mRNAs in Kdm1a fl/fl versus Kdm1a Zp3 oocytes ( A , E ), Kdm1a fl/fl M+Z+ versus Kdm1a Zp3 M-Z+ 2C embryos ( B , F ), Kdm1a fl/fl M+Z+ 2C embryos versus Kdm1a fl/fl oocytes ( C , G ), and Kdm1a Zp3 M-Z+ 2C embryos versus Kdm1a fl/fl oocytes ( D , H ) as determined by RNA-seq. Differential expression represented in mean difference plots ( A–D ) and normalized FPKM values on XY scatter plots ( E–H ). Genes/repeats highlighted in red are significant. DOI: http://dx.doi.org/10.7554/eLife.08848.013

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: The MZT is impaired in Kdm1a Zp3 mutants. ( A–H ) Differential expression of mRNAs in Kdm1a fl/fl versus Kdm1a Zp3 oocytes ( A , E ), Kdm1a fl/fl M+Z+ versus Kdm1a Zp3 M-Z+ 2C embryos ( B , F ), Kdm1a fl/fl M+Z+ 2C embryos versus Kdm1a fl/fl oocytes ( C , G ), and Kdm1a Zp3 M-Z+ 2C embryos versus Kdm1a fl/fl oocytes ( D , H ) as determined by RNA-seq. Differential expression represented in mean difference plots ( A–D ) and normalized FPKM values on XY scatter plots ( E–H ). Genes/repeats highlighted in red are significant. DOI: http://dx.doi.org/10.7554/eLife.08848.013

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Expressing, RNA Sequencing Assay

    Principal component analysis of Kdm1a Zp3 2C embryos. ( A ) Principal Component 1 is plotted on x-axis and Principal Component 2 is plotted on y-axis. Variance due to each component for Kdm1a Zp3 M-Z+ 2C embryos (red), Kdm1a fl/fl M+Z+ 2C embryos (green), and Kdm1a fl/fl oocytes (purple) are shown. DOI: http://dx.doi.org/10.7554/eLife.08848.014

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Principal component analysis of Kdm1a Zp3 2C embryos. ( A ) Principal Component 1 is plotted on x-axis and Principal Component 2 is plotted on y-axis. Variance due to each component for Kdm1a Zp3 M-Z+ 2C embryos (red), Kdm1a fl/fl M+Z+ 2C embryos (green), and Kdm1a fl/fl oocytes (purple) are shown. DOI: http://dx.doi.org/10.7554/eLife.08848.014

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques:

    Generation of Kdm1a mutant and control animals. Kdm1a animals were generated by crossing multiple generations of Kdm1a fl/fl animals with either Gdf9-, Zp3 -, or Vasa-Cre transgenic animals. Blue indicates Mus castaneus control animals. Purple indicates Kdm1a mutant females. Green indicates B6/Cast hybrid control progeny. Red indicates Kdm1a maternal effect progeny (MEP). Orange indicates progeny resulting from intercrossing 2 MEP adult animals. All labelled progeny were used in crosses and assays presented in subsequent figures (color-coding matches animals used and graphed in each figure). DOI: http://dx.doi.org/10.7554/eLife.08848.005

    Journal: eLife

    Article Title: Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally

    doi: 10.7554/eLife.08848

    Figure Lengend Snippet: Generation of Kdm1a mutant and control animals. Kdm1a animals were generated by crossing multiple generations of Kdm1a fl/fl animals with either Gdf9-, Zp3 -, or Vasa-Cre transgenic animals. Blue indicates Mus castaneus control animals. Purple indicates Kdm1a mutant females. Green indicates B6/Cast hybrid control progeny. Red indicates Kdm1a maternal effect progeny (MEP). Orange indicates progeny resulting from intercrossing 2 MEP adult animals. All labelled progeny were used in crosses and assays presented in subsequent figures (color-coding matches animals used and graphed in each figure). DOI: http://dx.doi.org/10.7554/eLife.08848.005

    Article Snippet: Immunostaining was performed using rabbit polyclonal anti-KDM1A (1:200, Abcam, Cambridge UK, ab17721) and Alexa fluor conjugated secondary antibodies.

    Techniques: Mutagenesis, Generated, Transgenic Assay

    Interaction with HP1 is required for efficient tethering of KAP1 to DNA. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged M2-KAP1 (this KAP1 construct harbors two amino acid substitutions

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Interaction with HP1 is required for efficient tethering of KAP1 to DNA. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged M2-KAP1 (this KAP1 construct harbors two amino acid substitutions

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FLAG-tag, Stable Transfection, Expressing, Construct

    Interaction with HP1 is required for efficient nuclear retention of KAP1. Immunofluorescence staining was performed with HEK293 cells stably expressing either WT or mutant KAP1 proteins using antibodies against the FLAG tag (green) or KAP1 (red); nuclei

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Interaction with HP1 is required for efficient nuclear retention of KAP1. Immunofluorescence staining was performed with HEK293 cells stably expressing either WT or mutant KAP1 proteins using antibodies against the FLAG tag (green) or KAP1 (red); nuclei

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Immunofluorescence, Staining, Stable Transfection, Expressing, Mutagenesis, FLAG-tag

    Comparison of FLAG-tagged and endogenous KAP1. (A) ChIP-seq binding patterns. Snapshots of ChIP-seq results for endogenous KAP1 in U2OS cells, endogenous KAP1 in HEK293 cells, and FLAG-tagged WT KAP1 in HEK293 cells for a region on chromosome 19. The

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Comparison of FLAG-tagged and endogenous KAP1. (A) ChIP-seq binding patterns. Snapshots of ChIP-seq results for endogenous KAP1 in U2OS cells, endogenous KAP1 in HEK293 cells, and FLAG-tagged WT KAP1 in HEK293 cells for a region on chromosome 19. The

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    The PHD and bromodomain of KAP1 are not required for recruitment to DNA. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged ΔPB-KAP1 (this KAP1 construct contains a deletion of the

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: The PHD and bromodomain of KAP1 are not required for recruitment to DNA. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged ΔPB-KAP1 (this KAP1 construct contains a deletion of the

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FLAG-tag, Stable Transfection, Expressing, Construct

    ChIP-seq analysis of KAP1 lacking both the N- and C-terminal domains. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged Δ(RBBB+PB)-KAP1 (this KAP1 construct contains a deletion of

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: ChIP-seq analysis of KAP1 lacking both the N- and C-terminal domains. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged Δ(RBBB+PB)-KAP1 (this KAP1 construct contains a deletion of

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FLAG-tag, Stable Transfection, Expressing, Construct

    The SETDB1 histone methylation complex is recruited to specific subsets of KAP1 targets. ChIP-qPCR data for H3K9me3 (A), SetDB1 (B), H3K36me3 (C), and H3K4me3 (D) at the chosen binding sites (see ) from HEK293 cells stably expressing FLAG-tagged

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: The SETDB1 histone methylation complex is recruited to specific subsets of KAP1 targets. ChIP-qPCR data for H3K9me3 (A), SetDB1 (B), H3K36me3 (C), and H3K4me3 (D) at the chosen binding sites (see ) from HEK293 cells stably expressing FLAG-tagged

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Stable Transfection, Expressing

    Description and expression of KAP1 mutants. (A) Illustration of endogenous KAP1 protein and the KAP1 mutants used in this study. Well-characterized protein interaction domains of KAP1 are indicated along with their interacting partners. All of the mutant

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Description and expression of KAP1 mutants. (A) Illustration of endogenous KAP1 protein and the KAP1 mutants used in this study. Well-characterized protein interaction domains of KAP1 are indicated along with their interacting partners. All of the mutant

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Expressing, Mutagenesis

    Analysis of KAP1 knockdown cells. (A) Shown is a Western blot analysis of KAP1 levels in control (NT2 GFP) and knockdown (NT2 K2 GFP) cells. (B) ChIP-PCR of a positive KAP1 binding site (ZNF180) and a negative-control region (EVX1) in control cells (NT2

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Analysis of KAP1 knockdown cells. (A) Shown is a Western blot analysis of KAP1 levels in control (NT2 GFP) and knockdown (NT2 K2 GFP) cells. (B) ChIP-PCR of a positive KAP1 binding site (ZNF180) and a negative-control region (EVX1) in control cells (NT2

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Negative Control

    ChIP-seq patterns at genes regulated by KAP1. Shown are ChIP-seq patterns for several genes activated (A and B) or repressed (C and D) by KAP1. The binding of KAP1 to EFEMP1, EPAS1, ZSWIM5, and NRXN2 was also confirmed by ChIP-PCR (data not shown).

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: ChIP-seq patterns at genes regulated by KAP1. Shown are ChIP-seq patterns for several genes activated (A and B) or repressed (C and D) by KAP1. The binding of KAP1 to EFEMP1, EPAS1, ZSWIM5, and NRXN2 was also confirmed by ChIP-PCR (data not shown).

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    KAP1 binds to promoters and nonpromoter regions. (A) Five binding sites identified by ChIP-seq were selected from each of three different types of KAP1 targets, i.e., 3′ ends of ZNF genes, 5′ regions of ZNF genes, and 5′ regions

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: KAP1 binds to promoters and nonpromoter regions. (A) Five binding sites identified by ChIP-seq were selected from each of three different types of KAP1 targets, i.e., 3′ ends of ZNF genes, 5′ regions of ZNF genes, and 5′ regions

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Functional annotation of WT KAP1 versus ΔRBCC-KAP1 binding sites. Gene ontology analyses (right panels) and location analyses (left panels) are shown for all FLAG-tagged WT KAP1 peaks (A), the top 1,000 FLAG-tagged WT KAP1 peaks (B), and all FLAG-tagged

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: Functional annotation of WT KAP1 versus ΔRBCC-KAP1 binding sites. Gene ontology analyses (right panels) and location analyses (left panels) are shown for all FLAG-tagged WT KAP1 peaks (A), the top 1,000 FLAG-tagged WT KAP1 peaks (B), and all FLAG-tagged

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Functional Assay, Binding Assay

    The RBCC domain of KAP1 is required for recruitment to the 3′ ends of ZNF genes. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged ΔRBCC-KAP1 (this KAP1 construct contains

    Journal:

    Article Title: Functional Analysis of KAP1 Genomic Recruitment

    doi: 10.1128/MCB.01331-10

    Figure Lengend Snippet: The RBCC domain of KAP1 is required for recruitment to the 3′ ends of ZNF genes. (A) ChIP-qPCR was performed using an antibody against the FLAG tag and HEK293 cells stably expressing FLAG-tagged ΔRBCC-KAP1 (this KAP1 construct contains

    Article Snippet: The primary antibodies used in this study were FLAG (Sigma F1804), KAP1 (Abcam Ab10483), SetDB1 (Proteintech Group catalog no. 11231-1-AP), H3me3K9 (Abcam Ab8898), H3me3K36 (Cell Signaling Technology catalog no. 9763S), H3me3K4 (Cell Signaling Technology catalog no. 9751), ELK4 (Santa Cruz Biotechnology sc-13030X), and YY1 (Santa Cruz Biotechnology sc-1703X), and nonspecific rabbit IgG was used as a negative control (Alpha Diagnostics catalog nol.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, FLAG-tag, Stable Transfection, Expressing, Construct

    H3K9ac signal and H3K27ac signal at silenced genes and housekeeping genes in thymocytes. ( A–B ) Average H3K9ac and H3K27ac signal ±20 kb around the gene body among gene sets that are silenced in thymocytes ( A ) or housekeeping genes ( B ). Below are example H3K9ac ChIP-seq snapshots of genes within each of the genesets— Csf1r for macrophage, Pax5 for B cell, Cd34 for HSC/progenitor, Hprt for housekeeping, Atp5g1 for mitochondrial, and Rpl15 for ribosomal. Each image depicts an overlay of ChIP-seq tracks between OT-II (blue) and OT-II HDAC3-cKO (orange) mice. Refer to Figure 3—source data 2 for list of genes in these gene sets (macrophage, B cell, HSC/Progenitor, mitochondrial, ribosomal). TSS represents transcription start site; TES represents transcription end site. Scale bar in kb below ChIP-seq tracks identifies scale of snapshot.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: H3K9ac signal and H3K27ac signal at silenced genes and housekeeping genes in thymocytes. ( A–B ) Average H3K9ac and H3K27ac signal ±20 kb around the gene body among gene sets that are silenced in thymocytes ( A ) or housekeeping genes ( B ). Below are example H3K9ac ChIP-seq snapshots of genes within each of the genesets— Csf1r for macrophage, Pax5 for B cell, Cd34 for HSC/progenitor, Hprt for housekeeping, Atp5g1 for mitochondrial, and Rpl15 for ribosomal. Each image depicts an overlay of ChIP-seq tracks between OT-II (blue) and OT-II HDAC3-cKO (orange) mice. Refer to Figure 3—source data 2 for list of genes in these gene sets (macrophage, B cell, HSC/Progenitor, mitochondrial, ribosomal). TSS represents transcription start site; TES represents transcription end site. Scale bar in kb below ChIP-seq tracks identifies scale of snapshot.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Chromatin Immunoprecipitation, Mouse Assay

    Gene expression of select housekeeping genes, CD4-lineage genes, and CD8-lineage genes. RPKM values of indicated genes of Selecting cells from OT-II and OT-II HDAC3-cKO mice. ( A ) Housekeeping genes, ( B ) CD4-lineage genes, and ( C ) CD8-lineage genes. Bar graphs depict mean ± SEM of three mice. P values (***, p

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Gene expression of select housekeeping genes, CD4-lineage genes, and CD8-lineage genes. RPKM values of indicated genes of Selecting cells from OT-II and OT-II HDAC3-cKO mice. ( A ) Housekeeping genes, ( B ) CD4-lineage genes, and ( C ) CD8-lineage genes. Bar graphs depict mean ± SEM of three mice. P values (***, p

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Expressing, Mouse Assay

    HDAC3 ChIP-seq in pro-B cells and human CD4 T cells. ChIP-seq snapshots at Runx3 and Patz1 of publicly available HDAC3 ChIP-seq datasets in pro-B cells (GEO: GSM2096648, genome alignment mm10) and human CD4 T cells (GEO: GSM393952, genome alignment hg18). Alongside the HDAC3 ChIP-seq, DNase-seq (GEO: GSM2195840, genome alignment mm10) and H3K27ac ChIP seq from Immature OT-II and OT-II HDAC3-cKO thymocytes (as shown in Figure 4 ) were provided to visualize where HDAC3 binds in relation to regulatory elements and changes in histone acetylation between WT and HDAC3-cKO mice, respectively. Orange shaded regions demarcate super enhancers (SE) and blue shaded regions highlight where primers were made for qPCR. In pro-B cells, HDAC3 binds 50 kb upstream of Runx3 , at the Runx3 promoter, and approximately 2 kb downstream of the Patz1 promoter. Likewise, HDAC3 binds approximately 57 kb upstream of Runx3 and 2 kb downstream of the Patz1 promoter in human CD4 T cells. These three regions were used to make primers for HDAC3 qChIP performed in Figure 7 .

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: HDAC3 ChIP-seq in pro-B cells and human CD4 T cells. ChIP-seq snapshots at Runx3 and Patz1 of publicly available HDAC3 ChIP-seq datasets in pro-B cells (GEO: GSM2096648, genome alignment mm10) and human CD4 T cells (GEO: GSM393952, genome alignment hg18). Alongside the HDAC3 ChIP-seq, DNase-seq (GEO: GSM2195840, genome alignment mm10) and H3K27ac ChIP seq from Immature OT-II and OT-II HDAC3-cKO thymocytes (as shown in Figure 4 ) were provided to visualize where HDAC3 binds in relation to regulatory elements and changes in histone acetylation between WT and HDAC3-cKO mice, respectively. Orange shaded regions demarcate super enhancers (SE) and blue shaded regions highlight where primers were made for qPCR. In pro-B cells, HDAC3 binds 50 kb upstream of Runx3 , at the Runx3 promoter, and approximately 2 kb downstream of the Patz1 promoter. Likewise, HDAC3 binds approximately 57 kb upstream of Runx3 and 2 kb downstream of the Patz1 promoter in human CD4 T cells. These three regions were used to make primers for HDAC3 qChIP performed in Figure 7 .

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Chromatin Immunoprecipitation, Mouse Assay, Real-time Polymerase Chain Reaction

    H3K9ac signal at CD4-lineage and CD8-lineage genes. ( A–B ) Average H3K9ac ChIP-seq signal at the transcription start site (TSS) between OT-II and OT-II HDAC3-cKO Selecting cells for CD4-lineage gene sets ( A ) and CD8-lineage gene sets ( B ). Box-and-whisker plots depict H3K9ac signal at the TSS at each CD4- or CD8-lineage gene between OT-II and OT-II HDAC3-cKO mice. Snapshots of example ChIP-seq tracks for each gene set are below plots depicting the average signal. P values were calculated using Wilcoxon signed rank test.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: H3K9ac signal at CD4-lineage and CD8-lineage genes. ( A–B ) Average H3K9ac ChIP-seq signal at the transcription start site (TSS) between OT-II and OT-II HDAC3-cKO Selecting cells for CD4-lineage gene sets ( A ) and CD8-lineage gene sets ( B ). Box-and-whisker plots depict H3K9ac signal at the TSS at each CD4- or CD8-lineage gene between OT-II and OT-II HDAC3-cKO mice. Snapshots of example ChIP-seq tracks for each gene set are below plots depicting the average signal. P values were calculated using Wilcoxon signed rank test.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Chromatin Immunoprecipitation, Whisker Assay, Mouse Assay

    Acceleration of CD8-lineage commitment in HDAC3-deficient thymocytes.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Acceleration of CD8-lineage commitment in HDAC3-deficient thymocytes.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques:

    HDAC3 binding at the Il21r promoter in human CD4 T cells and mouse pro-B cells.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: HDAC3 binding at the Il21r promoter in human CD4 T cells and mouse pro-B cells.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Binding Assay

    Heatmaps of CD4-lineage and CD8-lineage genes. Heatmaps depict Log 10 RPKM of genes in CD4-lineage ( A ) and CD8-lineage ( B ) gene sets in Selecting (CD69 + ) thymocytes from OT-II and OT-II HDAC3-cKO. Heatmaps show three biological replicates per mouse group. Scale bar is below each heatmap. Refer to Figure 3—source data 1 for Log 2 fold change between OT-II and OT-II HDAC3-cKO mice.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Heatmaps of CD4-lineage and CD8-lineage genes. Heatmaps depict Log 10 RPKM of genes in CD4-lineage ( A ) and CD8-lineage ( B ) gene sets in Selecting (CD69 + ) thymocytes from OT-II and OT-II HDAC3-cKO. Heatmaps show three biological replicates per mouse group. Scale bar is below each heatmap. Refer to Figure 3—source data 1 for Log 2 fold change between OT-II and OT-II HDAC3-cKO mice.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Mouse Assay

    Acceleration of CD8-lineage commitment in HDAC3-deficient thymocytes. ( A ) Gating scheme outlining two phases of CD4/CD8-lineage commitment—Phase one includes CD69 + CCR7 - cells (Stage 2), CD69 + CCR7 lo cells (Stage 3), and CD69 + CCR7 int cells (Stage 4); Phase two identifies CD69 + CCR7 + cells (Stage 5) and CD69 - CCR7 + cells (Stage 6). Orange shading highlights Phase 1. ( B ) Expression of Runx3 and CD8α during stages of lineage commitment (Stages 1–6), as outlined in ( A ) from OT-I, OT-II, OT-II RB and OT-II RB3 mice. CD69-versus-CCR7 plots were gated from DN-removed, CD45.2 + cells. ( C ) Expression of ThPOK during stages of lineage commitment, gated as in ( B ). Plots in B and C show mean ±SEM of MFI from 5 to 7 mice per group from five independent experiments.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Acceleration of CD8-lineage commitment in HDAC3-deficient thymocytes. ( A ) Gating scheme outlining two phases of CD4/CD8-lineage commitment—Phase one includes CD69 + CCR7 - cells (Stage 2), CD69 + CCR7 lo cells (Stage 3), and CD69 + CCR7 int cells (Stage 4); Phase two identifies CD69 + CCR7 + cells (Stage 5) and CD69 - CCR7 + cells (Stage 6). Orange shading highlights Phase 1. ( B ) Expression of Runx3 and CD8α during stages of lineage commitment (Stages 1–6), as outlined in ( A ) from OT-I, OT-II, OT-II RB and OT-II RB3 mice. CD69-versus-CCR7 plots were gated from DN-removed, CD45.2 + cells. ( C ) Expression of ThPOK during stages of lineage commitment, gated as in ( B ). Plots in B and C show mean ±SEM of MFI from 5 to 7 mice per group from five independent experiments.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Expressing, Mouse Assay

    Very few SP thymocytes are generated in CD2-icre HDAC3-cKO mice.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Very few SP thymocytes are generated in CD2-icre HDAC3-cKO mice.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Generated, Mouse Assay

    IL-21R expression and signaling in CD2-icre HDAC3-cKO mice. ( A ) IL-21R expression in DN (CD4 - CD8 - ), immature SP (ISP; CD4 - CD8 + TCRβ - ), and DP (CD4 + CD8 + ) thymocytes from WT and HDAC3-cKO mice. Bar graph is mean ±SEM of MFI. N = 3 mice/group. ( B ) p-STAT5 expression in ISPs from WT and CD2-icre HDAC3-cKO mice that were stimulated with the indicated cytokines for 10mins. Experimental conditions are the same as performed in Figure 5E . Bar graph shows mean ±SEM of p-STAT5 MFI fold to unstimulated conditions. N = 4–5 mice/group.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: IL-21R expression and signaling in CD2-icre HDAC3-cKO mice. ( A ) IL-21R expression in DN (CD4 - CD8 - ), immature SP (ISP; CD4 - CD8 + TCRβ - ), and DP (CD4 + CD8 + ) thymocytes from WT and HDAC3-cKO mice. Bar graph is mean ±SEM of MFI. N = 3 mice/group. ( B ) p-STAT5 expression in ISPs from WT and CD2-icre HDAC3-cKO mice that were stimulated with the indicated cytokines for 10mins. Experimental conditions are the same as performed in Figure 5E . Bar graph shows mean ±SEM of p-STAT5 MFI fold to unstimulated conditions. N = 4–5 mice/group.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Expressing, Mouse Assay

    Model. HDAC3 is required to restrain CD8-lineage genes for CD4-lineage choice. In WT thymocytes (upper panel), HDAC3 associates with Runx3 and Patz1 in DP thymocytes to restrain CD8-lineage gene expression. After positive selection, HDAC3 stays bound to these regions in CD4SP thymocytes for CD4-lineage commitment, while in CD8SP thymocytes HDAC3 no longer binds to these regions for expression of CD8-lineage genes and CD8-lineage commitment. However, deletion of HDAC3 (lower panel) results in an increase in histone acetylation at CD8-lineage genes ( Runx3 , Patz1 ) and priming DP thymocytes for the CD8-lineage. As a result, Runx3 is pre-maturely expressing during CD4/CD8-lineage choice and cells commit to the CD8-lineage, which is accelerated.

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: Model. HDAC3 is required to restrain CD8-lineage genes for CD4-lineage choice. In WT thymocytes (upper panel), HDAC3 associates with Runx3 and Patz1 in DP thymocytes to restrain CD8-lineage gene expression. After positive selection, HDAC3 stays bound to these regions in CD4SP thymocytes for CD4-lineage commitment, while in CD8SP thymocytes HDAC3 no longer binds to these regions for expression of CD8-lineage genes and CD8-lineage commitment. However, deletion of HDAC3 (lower panel) results in an increase in histone acetylation at CD8-lineage genes ( Runx3 , Patz1 ) and priming DP thymocytes for the CD8-lineage. As a result, Runx3 is pre-maturely expressing during CD4/CD8-lineage choice and cells commit to the CD8-lineage, which is accelerated.

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Expressing, Selection

    DP thymocytes from HDAC3-cKO mice show a CD8-lineage bias at the chromatin level. ( A ) Typical enhancers (TEs) and super enhancers (SEs) in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. ( B ) Average H3K27ac signal (normalized to input) for TE and SE regions from OT-II and OT-II HDAC3-cKO mice. ( C ) Box-and-whisker plot of H3K27ac signal for each super enhancer in OT-II and OT-II HDAC3-cKO mice. ( D ) Median length of super-enhancers between OT-II and OT-II HDAC3-cKO mice. ( E ) Box-and-whisker plots of mRNA expression (reads per kilobase of exon per million mapped reads, RPKM) from super-enhancer-associated genes from OT-II and OT-II HDAC3-cKO mice. ( F ) Venn Diagram of shared and unique SEs between OT-II and OT-II HDAC3-cKO mice. ( G ) H3K27ac signal at super-enhancer regions unique to OT-II mice and the corresponding regions in OT-II HDAC3-cKO mice. ( H ) Box-and-whisker plots of mRNA expression (RPKM) between OT-II and OT-II HDAC3-cKO mice of genes associated with super-enhancers unique to OT-II mice. ( I ) H3K27ac signal at super-enhancer regions unique to OT-II HDAC3-cKO mice and the corresponding regions in OT-II mice. ( J ) Snapshot of H3K27ac ChIP-seq tracks at the Runx3 and Patz1 locus from OT-II (blue) and OT-II HDAC3-cKO (orange) mice. Shaded regions depict TEs and SEs. ( K ) Gene expression (RNA-seq) of Runx3 and Patz1 in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. ( B, G, I ) The x-axis represents a surrounding area that corresponds to 200% of the center of each region. (***, p

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: DP thymocytes from HDAC3-cKO mice show a CD8-lineage bias at the chromatin level. ( A ) Typical enhancers (TEs) and super enhancers (SEs) in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. ( B ) Average H3K27ac signal (normalized to input) for TE and SE regions from OT-II and OT-II HDAC3-cKO mice. ( C ) Box-and-whisker plot of H3K27ac signal for each super enhancer in OT-II and OT-II HDAC3-cKO mice. ( D ) Median length of super-enhancers between OT-II and OT-II HDAC3-cKO mice. ( E ) Box-and-whisker plots of mRNA expression (reads per kilobase of exon per million mapped reads, RPKM) from super-enhancer-associated genes from OT-II and OT-II HDAC3-cKO mice. ( F ) Venn Diagram of shared and unique SEs between OT-II and OT-II HDAC3-cKO mice. ( G ) H3K27ac signal at super-enhancer regions unique to OT-II mice and the corresponding regions in OT-II HDAC3-cKO mice. ( H ) Box-and-whisker plots of mRNA expression (RPKM) between OT-II and OT-II HDAC3-cKO mice of genes associated with super-enhancers unique to OT-II mice. ( I ) H3K27ac signal at super-enhancer regions unique to OT-II HDAC3-cKO mice and the corresponding regions in OT-II mice. ( J ) Snapshot of H3K27ac ChIP-seq tracks at the Runx3 and Patz1 locus from OT-II (blue) and OT-II HDAC3-cKO (orange) mice. Shaded regions depict TEs and SEs. ( K ) Gene expression (RNA-seq) of Runx3 and Patz1 in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. ( B, G, I ) The x-axis represents a surrounding area that corresponds to 200% of the center of each region. (***, p

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Mouse Assay, Whisker Assay, Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay

    MHC class II restricted thymocytes are redirected to the CD8-lineage when HDAC3 is absent. ( A ) CD4/CD8 profile of mature thymic SP (Vβ5 + Vα2 + H2K + CD24 lo ) and splenic T cells (Vβ5 + Vα2 + ) from OT-II, OT-II RORγt-KO Bcl-xl tg (RB) and OT-II RB3 mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–5/group) ( B ) Runx3 and ThPOK expression in mature thymic SP cells from OT-II, OT-II RB, and OT-II RB3 mice (n = 4/group from four independent experiments). ( C ) CD4/CD8 profile of mature thymic SP from OT-II RORγt-KO and OT-II RORγt-KO HDAC3-cKO mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–4/group). ( D ) Granzyme b and perforin expression in SP thymocytes from OT-II, OT-I, OT-II RB and OT-II RB3 mice. FACS plots were gated on CD4SP cells for OT-II and OT-II RB mice and CD8SP cells for OT-I and OT-II RB3 mice. Bar graphs depict mean ± SEM (n = 3/group from three independent experiments) of the fold change in MFI between unstimulated and TCR/CD2 stimulated conditions. ( E ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from straight BMCs, where bone marrow from OT-II or OT-II RB3 mice were transplanted into B6.SJL or β2m-KO recipients. Mature SP thymocytes were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice (n = 3–5/group from three independent experiments) were analyzed 8–10 weeks after transfer. ( F ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from mixed BMCs from OT-II (CD45.1 - )/B6.SJL (CD45.1 + ) and OT-II RB3 (CD45.1 - )/B6.SJL (CD45.1 + ) mice. Mature SP thymocytes from CD45.1 + cells were gated as H2K + CD24 lo ; mature SP thymocytes from CD45.1 - cells were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice were analyzed 8–10 weeks after transfer. (n = 5/group from three independent experiments).

    Journal: eLife

    Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4+CD8+ thymocytes for CD4-lineage commitment

    doi: 10.7554/eLife.43821

    Figure Lengend Snippet: MHC class II restricted thymocytes are redirected to the CD8-lineage when HDAC3 is absent. ( A ) CD4/CD8 profile of mature thymic SP (Vβ5 + Vα2 + H2K + CD24 lo ) and splenic T cells (Vβ5 + Vα2 + ) from OT-II, OT-II RORγt-KO Bcl-xl tg (RB) and OT-II RB3 mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–5/group) ( B ) Runx3 and ThPOK expression in mature thymic SP cells from OT-II, OT-II RB, and OT-II RB3 mice (n = 4/group from four independent experiments). ( C ) CD4/CD8 profile of mature thymic SP from OT-II RORγt-KO and OT-II RORγt-KO HDAC3-cKO mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–4/group). ( D ) Granzyme b and perforin expression in SP thymocytes from OT-II, OT-I, OT-II RB and OT-II RB3 mice. FACS plots were gated on CD4SP cells for OT-II and OT-II RB mice and CD8SP cells for OT-I and OT-II RB3 mice. Bar graphs depict mean ± SEM (n = 3/group from three independent experiments) of the fold change in MFI between unstimulated and TCR/CD2 stimulated conditions. ( E ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from straight BMCs, where bone marrow from OT-II or OT-II RB3 mice were transplanted into B6.SJL or β2m-KO recipients. Mature SP thymocytes were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice (n = 3–5/group from three independent experiments) were analyzed 8–10 weeks after transfer. ( F ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from mixed BMCs from OT-II (CD45.1 - )/B6.SJL (CD45.1 + ) and OT-II RB3 (CD45.1 - )/B6.SJL (CD45.1 + ) mice. Mature SP thymocytes from CD45.1 + cells were gated as H2K + CD24 lo ; mature SP thymocytes from CD45.1 - cells were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice were analyzed 8–10 weeks after transfer. (n = 5/group from three independent experiments).

    Article Snippet: Unless stated in other Materials and methods section, the following clones for flow cytometry were used: CD4 (clone RM4-5), CD8α (clone 53–6.7), TCR Vβ5.1/5.2 (clone MR9-4), TCR Vα2 (clone B20.1), Runx3 (clone R3-5G4), ThPOK (clone T43-94), Granzyme B (clone NGZB), CD24 (clone M1/69), H-2Kb (clone AF6-88.5), CCR7 (clone 4B12), CD132 (common γ chain) (clone TUGm2), IL-4Rα (clone I015F8), IL-7Rα (clone A7R34), IL-15Rα (clone DNT15Ra), CD122 (IL-2Rβ) (clone 5H4), IL-21R (clone 4A9), ROR gamma (t) (clone B2D), Bcl-xl (clone 7B2.5), CD45.2 (clone 104), HDAC3 (ab7030).

    Techniques: Mouse Assay, Expressing, FACS

    HDAC3 is neither induced nor required for brown adipogenesis, but required for cell-autonomous Ucp1 expression (a) Gene expression spanning differentiation of cultured wild type primary brown adipocytes (n= 5 replicates per time point). (b) Depletion of HDAC3 in Day 8 cultured mature brown adipocytes following addition of 2μm 4-hydroxytamixofen (4-OHT) during Days 0–2 of differentiation to Rosa26 -CreER-positive (HDAC3 KO) and Rosa26 -CreER-negative (Control) cells derived from littermates (n= 3, 3). (c) Adipocyte-specific gene expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (d) Assessment of lipid accumulation (evaluated by Oil Red-O staining) in cultured HDAC3 KO versus control primary brown adipocytes. (e) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (f) UCP1 protein expression in cultured primary brown adipocytes following depletion of HDAC3 versus control. (n= 3, 3). (g) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control and treated with vehicle (ethanol) or isoproterenol (1μm) for 3 h (n= 4 per group). VCL, vinculin. * P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 is neither induced nor required for brown adipogenesis, but required for cell-autonomous Ucp1 expression (a) Gene expression spanning differentiation of cultured wild type primary brown adipocytes (n= 5 replicates per time point). (b) Depletion of HDAC3 in Day 8 cultured mature brown adipocytes following addition of 2μm 4-hydroxytamixofen (4-OHT) during Days 0–2 of differentiation to Rosa26 -CreER-positive (HDAC3 KO) and Rosa26 -CreER-negative (Control) cells derived from littermates (n= 3, 3). (c) Adipocyte-specific gene expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (d) Assessment of lipid accumulation (evaluated by Oil Red-O staining) in cultured HDAC3 KO versus control primary brown adipocytes. (e) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (f) UCP1 protein expression in cultured primary brown adipocytes following depletion of HDAC3 versus control. (n= 3, 3). (g) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control and treated with vehicle (ethanol) or isoproterenol (1μm) for 3 h (n= 4 per group). VCL, vinculin. * P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Expressing, Cell Culture, Gene Knockout, Negative Control, Derivative Assay, Staining

    HDAC3 functions as an ERRα coactivator in BAT (a) Average H3K27ac ChIP-seq profiles of Adipoq- Cre HDAC3 KO mice versus control littermates (n= 3, 3; pooled biological replicates/library) at enhancers bound by HDAC3 within 100 kb of transcription start sites of HDAC3 KO-regulated genes by GRO-seq (fold-change > 1.5 or

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 functions as an ERRα coactivator in BAT (a) Average H3K27ac ChIP-seq profiles of Adipoq- Cre HDAC3 KO mice versus control littermates (n= 3, 3; pooled biological replicates/library) at enhancers bound by HDAC3 within 100 kb of transcription start sites of HDAC3 KO-regulated genes by GRO-seq (fold-change > 1.5 or

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Chromatin Immunoprecipitation, Gene Knockout, Mouse Assay

    Ablation of HDAC3 in adipose tissue depots (a) Immunoblot analysis of interscapular BAT, inguinal WAT, and epididymal WAT of Adipoq -Cre HDAC3 KO and control littermates, or Ucp1 -Cre HDAC3 KO and control littermates maintained at 22°C ( n = 2, all groups) demonstrating tissue-specific conditional knockout of HDAC3. (b) Interscapular BAT mass, (c) Relative BAT mitochondrial number, and (d) Total body mass from Adipoq -Cre HDAC3 KO and Ucp1 -Cre HDAC3 KO versus control littermates maintained at 22°C (n= 13 Adipoq -Cre, n= 9 control; n= 9 Ucp1 -Cre, n= 10 control). (e) Representative hematoxylin and eosin (H E) staining of inguinal white adipose from 10–12 week old Adipoq -Cre HDAC3 KO, Ucp1 -Cre HDAC3 KO, Ucp1 KO, or control mice housed at 22°C. Scale bars, 100μm. Data are represented as mean ± s.e.m.

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: Ablation of HDAC3 in adipose tissue depots (a) Immunoblot analysis of interscapular BAT, inguinal WAT, and epididymal WAT of Adipoq -Cre HDAC3 KO and control littermates, or Ucp1 -Cre HDAC3 KO and control littermates maintained at 22°C ( n = 2, all groups) demonstrating tissue-specific conditional knockout of HDAC3. (b) Interscapular BAT mass, (c) Relative BAT mitochondrial number, and (d) Total body mass from Adipoq -Cre HDAC3 KO and Ucp1 -Cre HDAC3 KO versus control littermates maintained at 22°C (n= 13 Adipoq -Cre, n= 9 control; n= 9 Ucp1 -Cre, n= 10 control). (e) Representative hematoxylin and eosin (H E) staining of inguinal white adipose from 10–12 week old Adipoq -Cre HDAC3 KO, Ucp1 -Cre HDAC3 KO, Ucp1 KO, or control mice housed at 22°C. Scale bars, 100μm. Data are represented as mean ± s.e.m.

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Gene Knockout, Knock-Out, Staining, Mouse Assay

    BAT HDAC3 is required for cold-mediated induction of Ucp1 expression and HDAC3 expression is not altered by acute cold a–b BAT Ucp1 mRNA levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C in (a) Adipoq -Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (b) Ucp1 -Cre HDAC3 KO versus control (n= 5, 5, per temperature). c–d iWAT Ucp1 mRNA levels following 3 h exposure to 4°C, versus control littermates maintained at 22°C in (c) Adipoq -Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (d) Ucp1 -Cre HDAC3 KO versus control (n= 5, 5, per temperature). (e) BAT HDAC3 mRNA expression levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C (n= 5, 5, per temperature). (f) BAT HDAC3 protein levels following 3 h acute cold exposure at 4°C (from 22°C) versus control littermates maintained at 22°C. VCL, vinculin. NS, not significant, * P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: BAT HDAC3 is required for cold-mediated induction of Ucp1 expression and HDAC3 expression is not altered by acute cold a–b BAT Ucp1 mRNA levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C in (a) Adipoq -Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (b) Ucp1 -Cre HDAC3 KO versus control (n= 5, 5, per temperature). c–d iWAT Ucp1 mRNA levels following 3 h exposure to 4°C, versus control littermates maintained at 22°C in (c) Adipoq -Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (d) Ucp1 -Cre HDAC3 KO versus control (n= 5, 5, per temperature). (e) BAT HDAC3 mRNA expression levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C (n= 5, 5, per temperature). (f) BAT HDAC3 protein levels following 3 h acute cold exposure at 4°C (from 22°C) versus control littermates maintained at 22°C. VCL, vinculin. NS, not significant, * P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Expressing, Gene Knockout

    Role of HDAC3 on PGC-1α acetylation and function (a) Co-immunoprecipitation of HDAC3 and PGC-1α with ERRα from 293FT cells. (b) Luciferase reporter assay of transcription driven by the major Ucp1 enhancer (−6 kb) following transfection of ERRα, PGC-1α , GCN5, and/or HDAC3 (n= 3 replicates per condition). (c–d) Primary brown pre-adipocytes from Rosa26 -CreER-positive HDAC3 F/F and HDAC3 F/F control littermates transduced with MSCV retroviruses: Control, PGC-1α WT, or non-acetylatable PGC-1α R13 mutant, and treated with 2μm 4-OHT during days 0–2 of differentiation to deplete HDAC3, and studied at Day 8 of differentiation. (c) Immunoblot analysis of exogenous PGC-1α expression in primary brown adipocytes (n= 2 replicates pooled per lane). (d) RT-qPCR analysis of Ucp1 and Fasn expression in control and HDAC3 KO primary brown adipocytes following transduction with MSCV-Control (n= 4 control, 3 HDAC3 KO), MSCV-PGC-1α WT (n= 4 control, 4 HDAC3 KO), or MSCV-PGC-1α R13 (non-acetylatable mutant) (n= 3 Control, 4 HDAC3 KO). *P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: Role of HDAC3 on PGC-1α acetylation and function (a) Co-immunoprecipitation of HDAC3 and PGC-1α with ERRα from 293FT cells. (b) Luciferase reporter assay of transcription driven by the major Ucp1 enhancer (−6 kb) following transfection of ERRα, PGC-1α , GCN5, and/or HDAC3 (n= 3 replicates per condition). (c–d) Primary brown pre-adipocytes from Rosa26 -CreER-positive HDAC3 F/F and HDAC3 F/F control littermates transduced with MSCV retroviruses: Control, PGC-1α WT, or non-acetylatable PGC-1α R13 mutant, and treated with 2μm 4-OHT during days 0–2 of differentiation to deplete HDAC3, and studied at Day 8 of differentiation. (c) Immunoblot analysis of exogenous PGC-1α expression in primary brown adipocytes (n= 2 replicates pooled per lane). (d) RT-qPCR analysis of Ucp1 and Fasn expression in control and HDAC3 KO primary brown adipocytes following transduction with MSCV-Control (n= 4 control, 3 HDAC3 KO), MSCV-PGC-1α WT (n= 4 control, 4 HDAC3 KO), or MSCV-PGC-1α R13 (non-acetylatable mutant) (n= 3 Control, 4 HDAC3 KO). *P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Pyrolysis Gas Chromatography, Immunoprecipitation, Luciferase, Reporter Assay, Transfection, Transduction, Mutagenesis, Expressing, Quantitative RT-PCR, Gene Knockout

    HDAC3 is required for expression of UCP1 and OXPHOS genes in BAT (a) Scatterplot of RNA-seq data showing HDAC3 regulated BAT genes from Adipoq -Cre HDAC3 KO versus control littermates ( n = 4, 4) adapted to thermoneutrality (fold-change > 1.5 up (red) or down (blue) and FDR

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 is required for expression of UCP1 and OXPHOS genes in BAT (a) Scatterplot of RNA-seq data showing HDAC3 regulated BAT genes from Adipoq -Cre HDAC3 KO versus control littermates ( n = 4, 4) adapted to thermoneutrality (fold-change > 1.5 up (red) or down (blue) and FDR

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Expressing, RNA Sequencing Assay, Gene Knockout

    HDAC3 and ERRα activate Ppargc1 β enhancers and transcription (a) Genome browser tracks of the Ppargc1β locus highlighting GRO-seq and ChIP-seq data from HDAC3 KO and control BAT (y-axis scales, normalized reads, reads per million) demonstrating co-binding of HDAC3, ERRα, and NCoR at functional enhancers. (b) BAT PGC-1 β mRNA levels in Adipoq -Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7). (c–d) RT-qPCR of eRNAs found at HDAC3 and ERRαenhancers in Adipoq -Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7) and Ucp1 -Cre HDAC3 KO BAT versus control littermates (29°C: n= 5, 6; 22°C, n= 5, 7). (e) RT-qPCR analysis of Ucp1 mRNA expression in mature brown adipocytes following combinatorial siRNA knockdown of Pgc-1 α, Pgc-1 β and/or ERRα versus scramble siRNA (n= 5 replicates per condition). Statistical analysis performed amongst groups transfected with siRNAs. (f) Quantification of Ucp1 and Pgc-1 a nascent gene body transcription (GRO-seq) at 22°C and 29°C in Adipoq -Cre HDAC3 KO BAT versus control littermates (n= 10, 10, pooled biological replicates per library). * P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 and ERRα activate Ppargc1 β enhancers and transcription (a) Genome browser tracks of the Ppargc1β locus highlighting GRO-seq and ChIP-seq data from HDAC3 KO and control BAT (y-axis scales, normalized reads, reads per million) demonstrating co-binding of HDAC3, ERRα, and NCoR at functional enhancers. (b) BAT PGC-1 β mRNA levels in Adipoq -Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7). (c–d) RT-qPCR of eRNAs found at HDAC3 and ERRαenhancers in Adipoq -Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7) and Ucp1 -Cre HDAC3 KO BAT versus control littermates (29°C: n= 5, 6; 22°C, n= 5, 7). (e) RT-qPCR analysis of Ucp1 mRNA expression in mature brown adipocytes following combinatorial siRNA knockdown of Pgc-1 α, Pgc-1 β and/or ERRα versus scramble siRNA (n= 5 replicates per condition). Statistical analysis performed amongst groups transfected with siRNAs. (f) Quantification of Ucp1 and Pgc-1 a nascent gene body transcription (GRO-seq) at 22°C and 29°C in Adipoq -Cre HDAC3 KO BAT versus control littermates (n= 10, 10, pooled biological replicates per library). * P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Chromatin Immunoprecipitation, Gene Knockout, Binding Assay, Functional Assay, Pyrolysis Gas Chromatography, Quantitative RT-PCR, Expressing, Transfection

    HDAC3 coactivation of ERRα is mediated by PGC-1α deacetylation (a) Luciferase reporter assay of transcription driven by an identified Ucp1 enhancer (−6 kb), demonstrating effects of ERRα, HDAC3, wild type PGC-1α, and/or a PGC-1α LXXLL mutant (L1/2/3A) unable to interact with ERRα (n= 3 replicates/condition). (b) Immunoblot analysis of PGC-1α lysine acetylation following immunoprecipitation from co-transfected 293FT cells. (c) Immunoblot analysis of an in vitro deacetylation reaction of purified acetylated-PGC-1α by recombinant human HDAC3, with or without trichostatin A (TSA). (d) Genome browser tracks of the Ppargc1α locus highlighting GRO-seq and RNA-seq data, and eRNAs at HDAC3, ERRα, and NCoR co-bound distal enhancers (boxed). (e) Luciferase reporter assay as in (a) for identified Ppargc1α distal enhancer (−38 kb), (n= 3 replicates/condition). (f) BAT Pgc-1α mRNA from Adipoq- Cre HDAC3 KO mice versus control littermates ( n = 9, 6) and Ucp1- Cre HDAC3 KO mice versus control littermates ( n = 5, 6). (g) Immunoblot of PGC-1α in BAT nuclear extract of Adipoq -Cre HDAC3 KO versus control littermates (n= 5, 5 pooled replicates per lane). (h) BAT HDAC3 deacetylates PGC-1 α to co-activate an ERR-driven transcriptional loop of Ppargc1α/β Ucp1, and OXPHOS genes. (i) Flow chart depicting unbiased hierarchical gene clustering of GRO-seq gene transcription by temperature and genotype. Boxplot displays gene cluster requiring HDAC3 coactivator function, where line denotes median, top/bottom of boxes represents first/third quartiles, and whiskers denote 1.5x the interquartile range from first/third quartiles. (j) Heatmap of thermogenic and OXPHOS genes identified in (i). *** P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 coactivation of ERRα is mediated by PGC-1α deacetylation (a) Luciferase reporter assay of transcription driven by an identified Ucp1 enhancer (−6 kb), demonstrating effects of ERRα, HDAC3, wild type PGC-1α, and/or a PGC-1α LXXLL mutant (L1/2/3A) unable to interact with ERRα (n= 3 replicates/condition). (b) Immunoblot analysis of PGC-1α lysine acetylation following immunoprecipitation from co-transfected 293FT cells. (c) Immunoblot analysis of an in vitro deacetylation reaction of purified acetylated-PGC-1α by recombinant human HDAC3, with or without trichostatin A (TSA). (d) Genome browser tracks of the Ppargc1α locus highlighting GRO-seq and RNA-seq data, and eRNAs at HDAC3, ERRα, and NCoR co-bound distal enhancers (boxed). (e) Luciferase reporter assay as in (a) for identified Ppargc1α distal enhancer (−38 kb), (n= 3 replicates/condition). (f) BAT Pgc-1α mRNA from Adipoq- Cre HDAC3 KO mice versus control littermates ( n = 9, 6) and Ucp1- Cre HDAC3 KO mice versus control littermates ( n = 5, 6). (g) Immunoblot of PGC-1α in BAT nuclear extract of Adipoq -Cre HDAC3 KO versus control littermates (n= 5, 5 pooled replicates per lane). (h) BAT HDAC3 deacetylates PGC-1 α to co-activate an ERR-driven transcriptional loop of Ppargc1α/β Ucp1, and OXPHOS genes. (i) Flow chart depicting unbiased hierarchical gene clustering of GRO-seq gene transcription by temperature and genotype. Boxplot displays gene cluster requiring HDAC3 coactivator function, where line denotes median, top/bottom of boxes represents first/third quartiles, and whiskers denote 1.5x the interquartile range from first/third quartiles. (j) Heatmap of thermogenic and OXPHOS genes identified in (i). *** P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Pyrolysis Gas Chromatography, Luciferase, Reporter Assay, Mutagenesis, Immunoprecipitation, Transfection, In Vitro, Purification, Recombinant, RNA Sequencing Assay, Gene Knockout, Mouse Assay, Flow Cytometry

    Transcriptional Role of HDAC3 and ERRα in BAT (a) Heat map demonstrating correlation of RNA-seq and GRO-seq data. Differentially expressed genes in RNA-seq or GRO-seq data were sorted by log 2 FC in RNA-seq. (b) De novo motif enrichment at repressed eRNAs in Adipoq -Cre, HDAC3 KO mice versus control littermates (n= 10, 10; pooled biological replicates/library) maintained at 22°C and ranked by P -value. (c) Endogenous HDAC3 co-immunoprecipitation of ERRα in differentiated mature brown adipocytes. (d–e) RT-qPCRs of BAT Ucp1 eRNA expression and (f) Ucp1 mRNA at 22°C and 29°C in Adipoq -Cre and Ucp1 -Cre HDAC3 KO mice versus control littermates, 29°C (n= 9 Adipoq -Cre, 6 control; n= 5 Ucp1 -Cre, 6 control) and 22°C (n= 9 Adipoq -Cre, 7 control; n= 5 Ucp1 -Cre, 7 control). (g) ChIP-qPCR of ERRα at Ucp1 enhancers in Adipoq -Cre HDAC3 KO versus control littermates (n= 3, 3) adapted to 29°C. (h) RT-qPCR of ERRα and Ucp1 mRNA expression and (i) Ucp1 eRNA expression in ERRα KO BAT versus control littermates (n= 8, 7). (j) RT-qPCR analysis of ERRα and Ucp1 mRNA expression in mature brown adipocytes following siRNA mediated knockdown of ERRα versus scramble 72 h post-transfection (n= 3, 3). *P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: Transcriptional Role of HDAC3 and ERRα in BAT (a) Heat map demonstrating correlation of RNA-seq and GRO-seq data. Differentially expressed genes in RNA-seq or GRO-seq data were sorted by log 2 FC in RNA-seq. (b) De novo motif enrichment at repressed eRNAs in Adipoq -Cre, HDAC3 KO mice versus control littermates (n= 10, 10; pooled biological replicates/library) maintained at 22°C and ranked by P -value. (c) Endogenous HDAC3 co-immunoprecipitation of ERRα in differentiated mature brown adipocytes. (d–e) RT-qPCRs of BAT Ucp1 eRNA expression and (f) Ucp1 mRNA at 22°C and 29°C in Adipoq -Cre and Ucp1 -Cre HDAC3 KO mice versus control littermates, 29°C (n= 9 Adipoq -Cre, 6 control; n= 5 Ucp1 -Cre, 6 control) and 22°C (n= 9 Adipoq -Cre, 7 control; n= 5 Ucp1 -Cre, 7 control). (g) ChIP-qPCR of ERRα at Ucp1 enhancers in Adipoq -Cre HDAC3 KO versus control littermates (n= 3, 3) adapted to 29°C. (h) RT-qPCR of ERRα and Ucp1 mRNA expression and (i) Ucp1 eRNA expression in ERRα KO BAT versus control littermates (n= 8, 7). (j) RT-qPCR analysis of ERRα and Ucp1 mRNA expression in mature brown adipocytes following siRNA mediated knockdown of ERRα versus scramble 72 h post-transfection (n= 3, 3). *P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: RNA Sequencing Assay, Flow Cytometry, Gene Knockout, Mouse Assay, Immunoprecipitation, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection

    Metabolic studies of Adiponectin -Cre and Ucp1 -Cre HDAC3 KO mouse models a–b NMR analysis of body composition, (a) Adipoq- Cre mice versus control littermates (n= 8, 11) , (b) Ucp1- Cre mice versus control littermates (n= 7, 9). c–n , CLAMS metabolic cage analysis. c–d Oxygen consumption (VO 2 ), (c) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (d) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). e–f ANCOVA VO 2 analysis (linear regression analysis of total body mass and oxygen consumption) (e) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5) , (f) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). g–h Respiratory Exchange Ratio (RER), (g) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (h) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). i–j Heat measurements (kcal/hr), (i) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5). (j) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). k–l Food Intake, (k) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (l) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). m–n Physical activity, (m) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (n) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). P -values shown in italics. CLAMS data is graphed as rolling averages. NS, not significant, * P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: Metabolic studies of Adiponectin -Cre and Ucp1 -Cre HDAC3 KO mouse models a–b NMR analysis of body composition, (a) Adipoq- Cre mice versus control littermates (n= 8, 11) , (b) Ucp1- Cre mice versus control littermates (n= 7, 9). c–n , CLAMS metabolic cage analysis. c–d Oxygen consumption (VO 2 ), (c) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (d) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). e–f ANCOVA VO 2 analysis (linear regression analysis of total body mass and oxygen consumption) (e) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5) , (f) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). g–h Respiratory Exchange Ratio (RER), (g) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (h) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). i–j Heat measurements (kcal/hr), (i) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5). (j) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). k–l Food Intake, (k) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (l) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). m–n Physical activity, (m) Adipoq -Cre HDAC3 KO vs. control littermates (n= 6, 5), (n) Ucp1 -Cre HDAC3 KO vs. control littermates (n= 6, 6). P -values shown in italics. CLAMS data is graphed as rolling averages. NS, not significant, * P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Gene Knockout, Nuclear Magnetic Resonance, Mouse Assay, Activity Assay

    HDAC3 controls BAT thermogenesis (a, b) Effect of acute cold exposure from standard housing at 22°C to 4°C on Adipoq- Cre HDAC3 knockout (KO) mice versus control littermates (n= 15, 8), Ucp1- Cre HDAC3 KO mice versus control littermates (n= 15, 7), and Ucp1 − / − mice ( n = 15): a, core body temperature; b , survival. (c) Oxygen consumption rates of anesthetized Adipoq- Cre HDAC3 KO mice versus control littermates (n= 12, 5), Ucp1- Cre HDAC3 KO mice versus control littermates (n= 6, 5), and Ucp1 − / − mice ( n = 5) following injection of 1 mg kg −1 norepinephrine. (d) Mitochondrial respiration of purified BAT homogenates from Adipoq- Cre HDAC3 KO mice versus control littermates (n= 5, 6), and Ucp1- Cre HDAC3 KO mice versus control littermates (n= 6, 5), following brief acclimation to thermoneutrality. Mitochondria were provided palmitoylcarnitine and pyruvate substrates. UCP1-dependent respiration was assessed upon addition of guanosine diphosphate (GDP), and coupled respiration rates of complex I, II, and IV were determined in the presence of adenosine diphosphate (ADP). (e) Representative hematoxylin and eosin staining of interscapular brown adipose from 10–12 week old mice at 22°C. Scale bars, 100μm. * P

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 controls BAT thermogenesis (a, b) Effect of acute cold exposure from standard housing at 22°C to 4°C on Adipoq- Cre HDAC3 knockout (KO) mice versus control littermates (n= 15, 8), Ucp1- Cre HDAC3 KO mice versus control littermates (n= 15, 7), and Ucp1 − / − mice ( n = 15): a, core body temperature; b , survival. (c) Oxygen consumption rates of anesthetized Adipoq- Cre HDAC3 KO mice versus control littermates (n= 12, 5), Ucp1- Cre HDAC3 KO mice versus control littermates (n= 6, 5), and Ucp1 − / − mice ( n = 5) following injection of 1 mg kg −1 norepinephrine. (d) Mitochondrial respiration of purified BAT homogenates from Adipoq- Cre HDAC3 KO mice versus control littermates (n= 5, 6), and Ucp1- Cre HDAC3 KO mice versus control littermates (n= 6, 5), following brief acclimation to thermoneutrality. Mitochondria were provided palmitoylcarnitine and pyruvate substrates. UCP1-dependent respiration was assessed upon addition of guanosine diphosphate (GDP), and coupled respiration rates of complex I, II, and IV were determined in the presence of adenosine diphosphate (ADP). (e) Representative hematoxylin and eosin staining of interscapular brown adipose from 10–12 week old mice at 22°C. Scale bars, 100μm. * P

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Knock-Out, Gene Knockout, Mouse Assay, Injection, Purification, Staining

    Effect of high fat diet on Adiponectin -Cre and Ucp1 -Cre HDAC3 KO mouse models 12-week old weight-matched HDAC3 KO and control littermates were fed high-fat diet (HFD) for 12 weeks ( a ) Weekly body weights, (n= 8 Adipoq -Cre, n= 10 control), ( b ) Body composition analysis by NMR, (n= 8 Adipoq -Cre, n= 10 control), ( c ) Weekly body weights, (n= 7 Ucp1 -Cre, n= 7 control), ( d ) Body composition analysis by NMR, (n= 7 Ucp1 -Cre, n= 7 control). (e) RT-qPCR of BAT HDAC3 mRNA expression following 12 weeks HFD versus regular chow fed controls (n= 7, 5, respectively). Data are represented as mean ± s.e.m.

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: Effect of high fat diet on Adiponectin -Cre and Ucp1 -Cre HDAC3 KO mouse models 12-week old weight-matched HDAC3 KO and control littermates were fed high-fat diet (HFD) for 12 weeks ( a ) Weekly body weights, (n= 8 Adipoq -Cre, n= 10 control), ( b ) Body composition analysis by NMR, (n= 8 Adipoq -Cre, n= 10 control), ( c ) Weekly body weights, (n= 7 Ucp1 -Cre, n= 7 control), ( d ) Body composition analysis by NMR, (n= 7 Ucp1 -Cre, n= 7 control). (e) RT-qPCR of BAT HDAC3 mRNA expression following 12 weeks HFD versus regular chow fed controls (n= 7, 5, respectively). Data are represented as mean ± s.e.m.

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Gene Knockout, Nuclear Magnetic Resonance, Quantitative RT-PCR, Expressing

    HDAC3 is required for expression of mitochondrial OXPHOS and TCA cycle genes (a) Bioinformatic extension of identified Gene Ontology categories ( Fig 2c .) to all oxidative phosphorylation and TCA cycle genes as retrieved by HUGO gene nomenclature database. Asterisk designates gene expression change in RNA-seq dataset with an FDR

    Journal: Nature

    Article Title: Histone Deacetylase 3 Prepares Brown Adipose Tissue For Acute Thermogenic Challenge

    doi: 10.1038/nature22819

    Figure Lengend Snippet: HDAC3 is required for expression of mitochondrial OXPHOS and TCA cycle genes (a) Bioinformatic extension of identified Gene Ontology categories ( Fig 2c .) to all oxidative phosphorylation and TCA cycle genes as retrieved by HUGO gene nomenclature database. Asterisk designates gene expression change in RNA-seq dataset with an FDR

    Article Snippet: The following antibodies were used for ChIP: HDAC3 (ABCAM, 7030, Lot GR121157-6), H3K27ac (ABCAM, 4729, Lot GR251958-1), ERRα (ABCAM, 16363, Lot GR263385-1), NCoR (previously described , raised in rabbit against a.a.1944–2453, affinity purified), H3K4me1 (ABCAM 8895), and H3 (ABCAM 1791).

    Techniques: Expressing, RNA Sequencing Assay

    Mitochondrial membrane potential and cellular respiration in Foxg1-transfected HN9.10e cells. ( A ) Exemplificative pictures of untagged FL-Foxg1-expressing and mt-Foxg1-expressing (272–481) HN9.10e cells, loaded with TMRM. (Scale bar, 5 µm.)

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Mitochondrial membrane potential and cellular respiration in Foxg1-transfected HN9.10e cells. ( A ) Exemplificative pictures of untagged FL-Foxg1-expressing and mt-Foxg1-expressing (272–481) HN9.10e cells, loaded with TMRM. (Scale bar, 5 µm.)

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Transfection, Expressing

    Subcellular localization of transiently transfected Foxg1-GFP and GFP-Foxg1 in different cell lines. The pictures ( A ) indicate that only Foxg1-GFP localizes both in the nucleus and at mitochondria. It is worth noting that the GFP N-terminal fusion can

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Subcellular localization of transiently transfected Foxg1-GFP and GFP-Foxg1 in different cell lines. The pictures ( A ) indicate that only Foxg1-GFP localizes both in the nucleus and at mitochondria. It is worth noting that the GFP N-terminal fusion can

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Transfection

    Western blot assays on tagged and untagged Foxg1 overexpressing cells. ( A ) Western blot assay on untransfected NIH 3T3 cells (control) and GFP-Foxg1- and Foxg1-GFP-overexpressing cells (whole lysate) processed 24 h after transfection; the filters were

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Western blot assays on tagged and untagged Foxg1 overexpressing cells. ( A ) Western blot assay on untransfected NIH 3T3 cells (control) and GFP-Foxg1- and Foxg1-GFP-overexpressing cells (whole lysate) processed 24 h after transfection; the filters were

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Western Blot, Transfection

    Foxg1-GFP and GFP-Foxg1 display different subcellular localizations. ( A ) Schematic representation of Foxg1-GFP and representative images of Foxg1-GFP-expressing cells loaded with TMRM. ( B ) Schematic representation of GFP-Foxg1 and representative images

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Foxg1-GFP and GFP-Foxg1 display different subcellular localizations. ( A ) Schematic representation of Foxg1-GFP and representative images of Foxg1-GFP-expressing cells loaded with TMRM. ( B ) Schematic representation of GFP-Foxg1 and representative images

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Expressing

    Submitochondrial localization of Foxg1-GFP in transfected living cells. ( A ) Representative time-lapse of a Foxg1-GFP overexpressing living NIH 3T3 cell permeabilized with digitonin and treated with trypan blue. (Scale bar, 5 µm.) These experiments

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Submitochondrial localization of Foxg1-GFP in transfected living cells. ( A ) Representative time-lapse of a Foxg1-GFP overexpressing living NIH 3T3 cell permeabilized with digitonin and treated with trypan blue. (Scale bar, 5 µm.) These experiments

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Transfection

    Looking for Foxg1 mitochondrial localization domain. ( A ) Schemes representing the constructs generated to unmask the critical aa for Foxg1 mitochondrial localization. The length of the shortest ones is shown on the bottom left; their GFP tag is omitted

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Looking for Foxg1 mitochondrial localization domain. ( A ) Schemes representing the constructs generated to unmask the critical aa for Foxg1 mitochondrial localization. The length of the shortest ones is shown on the bottom left; their GFP tag is omitted

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Construct, Generated

    Mitochondrial localization of Foxg1. Immunocytochemical istribution of Foxg1 and Sod2 in representative confocal images of HN9.10e cells and primary glia. ( A ) HN9.10e cells expressing endogenous Foxg1. ( B ) HN9.10e cells transfected with untagged Foxg1.

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Mitochondrial localization of Foxg1. Immunocytochemical istribution of Foxg1 and Sod2 in representative confocal images of HN9.10e cells and primary glia. ( A ) HN9.10e cells expressing endogenous Foxg1. ( B ) HN9.10e cells transfected with untagged Foxg1.

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Expressing, Transfection

    Mitochondrial import assay in isolated rat liver mitochondria and Foxg1 subcellular localization in mouse brain. ( A ) Import assay of in vitro synthesized full-length and aa 272–481 fragment of mouse Foxg1. Incubation of Foxg1 with isolated rat

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Mitochondrial import assay in isolated rat liver mitochondria and Foxg1 subcellular localization in mouse brain. ( A ) Import assay of in vitro synthesized full-length and aa 272–481 fragment of mouse Foxg1. Incubation of Foxg1 with isolated rat

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Isolation, In Vitro, Synthesized, Incubation

    Cellular and mitochondrial morphology of HN9.10e cells overexpressing full-length and truncated Foxg1. ( A ) Exemplificative pictures of HN9.10e cells loaded with TMRM and expressing mt-Foxg1 (272–481) and cyt-Foxg1 (315–481) fused to GFP

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Cellular and mitochondrial morphology of HN9.10e cells overexpressing full-length and truncated Foxg1. ( A ) Exemplificative pictures of HN9.10e cells loaded with TMRM and expressing mt-Foxg1 (272–481) and cyt-Foxg1 (315–481) fused to GFP

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Expressing

    Subcellular localization of Foxg1 in HN9 cells. Western blot analysis of HN9.10e cells subcellular fractions: total lysate (Tot), nuclear (Nuc), cytoplasmic (Cyt), mitochondrial (Mit). Similar to the cortex, three immunoreactive bands of 58, 45, and 24

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Subcellular localization of Foxg1 in HN9 cells. Western blot analysis of HN9.10e cells subcellular fractions: total lysate (Tot), nuclear (Nuc), cytoplasmic (Cyt), mitochondrial (Mit). Similar to the cortex, three immunoreactive bands of 58, 45, and 24

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Western Blot

    Imaging and WB assays on tagged and untagged Foxg1 overexpressing cells. ( A ) Schematic representation of CFP-Foxg1-YFP double-fusion protein and representative image of HN9.10e cells transiently transfected with the YFP-Foxg1-CFP construct. (Scale bar,

    Journal:

    Article Title: Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

    doi: 10.1073/pnas.1515190112

    Figure Lengend Snippet: Imaging and WB assays on tagged and untagged Foxg1 overexpressing cells. ( A ) Schematic representation of CFP-Foxg1-YFP double-fusion protein and representative image of HN9.10e cells transiently transfected with the YFP-Foxg1-CFP construct. (Scale bar,

    Article Snippet: Membranes were incubated 1 h in TBS-Tween 20 containing 5% (wt/vol) nonfat dry milk and then exposed to a 1:1,000 dilution of rabbit polyclonal antiserum anti-Foxg1 (Abcam, catalog no. ab18259) at 4 °C overnight.

    Techniques: Imaging, Western Blot, Transfection, Construct

    PFKFB4 functions as a protein kinase by phosphorylating SRC-3 at the S857 residue a , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, fructose-6-phosphate (F6P), ATP and increasing concentration of recombinant PFKFB4 enzyme followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of phosphorylated Ser/Thr-SRC-3 protein. b , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, PFKFB4 enzyme and varying concentrations of F6P and ATP followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of p-SRC-3 protein. c , Coomassie blue stain showing the levels of GST-fused SRC-3 fragments used in in vitro kinase reactions performed in Fig. 2b . d , Proteomics analysis of in vitro kinase assay using the GST-SRC-3-CID fragment in the presence of PFKFB4 enzyme and ATP followed by mass spectrometric analyses. Mass spectrum shows the green phosphorylation peak. e , Proteomics analysis of an in vitro kinase assay using a S857A-mutated GST-SRC-3-CID protein in the presence of PFKFB4 enzyme and ATP, followed by mass spectrometric analyses. Mass spectrum failed to detect phosphorylation peaks in the S857A mutated SRC-3-CID protein. f , Expression of PFKFB1, PFKFB2, PFKFB3 and PFKFB4 in MDA-MB-231 cells expressing shRNAs targeting PFKFB4 (sh#09 and sh#20). mRNA levels were normalized to internal housekeeping gene actin. [Mean ± s.d., n =3, biological replicates, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4 functions as a protein kinase by phosphorylating SRC-3 at the S857 residue a , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, fructose-6-phosphate (F6P), ATP and increasing concentration of recombinant PFKFB4 enzyme followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of phosphorylated Ser/Thr-SRC-3 protein. b , In vitro PFKFB4 kinase assay in presence of purified SRC-3 protein, PFKFB4 enzyme and varying concentrations of F6P and ATP followed by SDS-PAGE. Immunoblotting with p-Ser/Thr antibody shows the level of p-SRC-3 protein. c , Coomassie blue stain showing the levels of GST-fused SRC-3 fragments used in in vitro kinase reactions performed in Fig. 2b . d , Proteomics analysis of in vitro kinase assay using the GST-SRC-3-CID fragment in the presence of PFKFB4 enzyme and ATP followed by mass spectrometric analyses. Mass spectrum shows the green phosphorylation peak. e , Proteomics analysis of an in vitro kinase assay using a S857A-mutated GST-SRC-3-CID protein in the presence of PFKFB4 enzyme and ATP, followed by mass spectrometric analyses. Mass spectrum failed to detect phosphorylation peaks in the S857A mutated SRC-3-CID protein. f , Expression of PFKFB1, PFKFB2, PFKFB3 and PFKFB4 in MDA-MB-231 cells expressing shRNAs targeting PFKFB4 (sh#09 and sh#20). mRNA levels were normalized to internal housekeeping gene actin. [Mean ± s.d., n =3, biological replicates, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: In Vitro, Kinase Assay, Purification, Concentration Assay, Recombinant, SDS Page, Staining, Expressing, Multiple Displacement Amplification

    SRC-3 phosphorylation by PFKFB4 enhances gene expression of metabolic enzymes a , Relative levels of metabolites altered by sh-PFKFB4 or sh-SRC-3 compared to control shNT in MDA-MB231 cells. [Mean ± s.d., n =3 biologically independent samples; two-way ANOVA with Tukey’s Multiple comparisons test] b , Relative proliferation of MDA-MB-231 and MCF-7 cells four days after treatment with siRNA-GFP (control) or SRC-3 under conditions indicated. [Mean ± s.d., n =5 biologically independent replicates; one-way ANOVA with Tukey’s Multiple comparisons test]. c , Expression of metabolic enzymes TKT, XDH , and AMPD1 in MDA-MB-231 cells after treatment with siRNAs-GFP (control), PFKFB4, or SRC-3. [Mean ± s.d., n =3 biologically independent samples; one-way ANOVA with Tukey’s Multiple comparisons test]. d , Immunoprecipitation of ATF4 from MDA-MB-231 cells grown in 5mM or 25mM glucose expressing shPFKFB4, shSRC-3, control- NTshRNA or expression of SRC-3-S857A in shSRC-3 cells. Levels of pSRC-3-S857 associated with ATF4 were detected by immunoblotting. IgG light chain-HRP was used to probe ATF4 in immunoblotting. e–g , Chromatin immunoprecipitation (ChIP) of ATF4, total SRC-3, and pSRC-3-S857 followed by qPCR from MDA-MB-231 cells treated with 5 mM or 25 mM glucose compared to an IgG isotype control. e , TKT . f , AMPD1 . [Mean ± s.d., n =3 biologically independent samples used for ChIP; one-way ANOVA with Tukey's multiple comparisons test compared to 5 mM glucose groups]. For exact P -values please refer to source data.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: SRC-3 phosphorylation by PFKFB4 enhances gene expression of metabolic enzymes a , Relative levels of metabolites altered by sh-PFKFB4 or sh-SRC-3 compared to control shNT in MDA-MB231 cells. [Mean ± s.d., n =3 biologically independent samples; two-way ANOVA with Tukey’s Multiple comparisons test] b , Relative proliferation of MDA-MB-231 and MCF-7 cells four days after treatment with siRNA-GFP (control) or SRC-3 under conditions indicated. [Mean ± s.d., n =5 biologically independent replicates; one-way ANOVA with Tukey’s Multiple comparisons test]. c , Expression of metabolic enzymes TKT, XDH , and AMPD1 in MDA-MB-231 cells after treatment with siRNAs-GFP (control), PFKFB4, or SRC-3. [Mean ± s.d., n =3 biologically independent samples; one-way ANOVA with Tukey’s Multiple comparisons test]. d , Immunoprecipitation of ATF4 from MDA-MB-231 cells grown in 5mM or 25mM glucose expressing shPFKFB4, shSRC-3, control- NTshRNA or expression of SRC-3-S857A in shSRC-3 cells. Levels of pSRC-3-S857 associated with ATF4 were detected by immunoblotting. IgG light chain-HRP was used to probe ATF4 in immunoblotting. e–g , Chromatin immunoprecipitation (ChIP) of ATF4, total SRC-3, and pSRC-3-S857 followed by qPCR from MDA-MB-231 cells treated with 5 mM or 25 mM glucose compared to an IgG isotype control. e , TKT . f , AMPD1 . [Mean ± s.d., n =3 biologically independent samples used for ChIP; one-way ANOVA with Tukey's multiple comparisons test compared to 5 mM glucose groups]. For exact P -values please refer to source data.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Expressing, Multiple Displacement Amplification, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Kinome-wide screen identified potential kinases regulating SRC-3 intrinsic transcriptional activity a , HeLa cells expressing varying concentrations of pBIND or pBIND-SRC-3 construct were used to measure SRC-3 activity. [Mean ± s.d., n =4 biologically independent samples, * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: Kinome-wide screen identified potential kinases regulating SRC-3 intrinsic transcriptional activity a , HeLa cells expressing varying concentrations of pBIND or pBIND-SRC-3 construct were used to measure SRC-3 activity. [Mean ± s.d., n =4 biologically independent samples, * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Activity Assay, Expressing, Construct

    PFKFB4, the top hit from the kinase screen, enhances the transcriptional activity of SRC-3 a , Effect of PFKFB4 knockdown on SRC-3 transcriptional activity in various breast cancer cell lines. [Mean ± s.d., n =3 or n =4 (siGFP + pBIND-SRC-3), biologically independent cells; * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4, the top hit from the kinase screen, enhances the transcriptional activity of SRC-3 a , Effect of PFKFB4 knockdown on SRC-3 transcriptional activity in various breast cancer cell lines. [Mean ± s.d., n =3 or n =4 (siGFP + pBIND-SRC-3), biologically independent cells; * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Activity Assay

    PFKFB4-SRC-3 stabilizes ATF4 transcription factor to promote purine synthesis a , Chromatin localization peaks of SRC-3 and ATF4 on TKT, XDH and AMPD1 genes in mouse liver. b , ATF4 binding peaks are conserved on three SRC-3 target purine biosynthetic genes in both mouse and human genomes. c , Chromatin immunoprecipitation (ChIP) of ATF4, total SRC-3, and pSRC-3-S857 from MDA-MB-231 cells treated with 5 mM or 25 mM glucose compared to an IgG isotype control. qPCR was performed to determine amount of promoter enrichment. d , ChIP-qPCR was performed from MDA-MB-231 cells cultured in 25 mM glucose expressing shSRC-3, shPFKFB4, or control-shNT [Mean ± s.d., n =3 biological cell samples; one-way ANOVA with Tukey's multiple comparisons test compared to 5 mM glucose groups (3e, f, g); and compared to shNT group (3h)]. e , ChIP of ATF4, total SRC-3 (BD Biosciences-Ab), and pSRC-3-S857 from MDA-MB-231 cells on AMPD1 promoter treated with non-targeting siRNA (NTsiRNA) or siRNA against ATF4 and cultured in presence of 25 mM glucose compared to an IgG isotype control. qPCR was performed to determine amount of promoter enrichment. [Mean ± s.d., n =3 biological cell samples; one-way ANOVA with Tukey’s Multiple comparisons]. f , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), adenosine monophosphate dehydrogenase 1 (AMPD1) and SRC-3 in MDA-MB-231 cells expressing siRNA targeting control (NT), or siRNA-ATF4. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Sidak’s Multiple comparisons test]. For exact P -values please refer to source data.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4-SRC-3 stabilizes ATF4 transcription factor to promote purine synthesis a , Chromatin localization peaks of SRC-3 and ATF4 on TKT, XDH and AMPD1 genes in mouse liver. b , ATF4 binding peaks are conserved on three SRC-3 target purine biosynthetic genes in both mouse and human genomes. c , Chromatin immunoprecipitation (ChIP) of ATF4, total SRC-3, and pSRC-3-S857 from MDA-MB-231 cells treated with 5 mM or 25 mM glucose compared to an IgG isotype control. qPCR was performed to determine amount of promoter enrichment. d , ChIP-qPCR was performed from MDA-MB-231 cells cultured in 25 mM glucose expressing shSRC-3, shPFKFB4, or control-shNT [Mean ± s.d., n =3 biological cell samples; one-way ANOVA with Tukey's multiple comparisons test compared to 5 mM glucose groups (3e, f, g); and compared to shNT group (3h)]. e , ChIP of ATF4, total SRC-3 (BD Biosciences-Ab), and pSRC-3-S857 from MDA-MB-231 cells on AMPD1 promoter treated with non-targeting siRNA (NTsiRNA) or siRNA against ATF4 and cultured in presence of 25 mM glucose compared to an IgG isotype control. qPCR was performed to determine amount of promoter enrichment. [Mean ± s.d., n =3 biological cell samples; one-way ANOVA with Tukey’s Multiple comparisons]. f , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), adenosine monophosphate dehydrogenase 1 (AMPD1) and SRC-3 in MDA-MB-231 cells expressing siRNA targeting control (NT), or siRNA-ATF4. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Sidak’s Multiple comparisons test]. For exact P -values please refer to source data.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Cell Culture, Expressing

    PFKFB4 phosphorylates SRC-3 by functioning as a protein kinase a , Upper panel-Recombinant GST-fused PFKFB4 incubated with full-length SRC-3 (SRC-3 FL) in presence of [ 32 P]ATP in an in vitro kinase assay. Lower panels- SRC-3 and PFKFB4 protein levels were analyzed by immunoblotting. b , In vitro kinase assay of PFKFB4 in the presence of SRC-3 fragments expressing different domains or full length SRC-3-FL. c , HEK293T cells expressing Flag-tagged-SRC-3 and PFKFB4 cultured in different concentrations of glucose and immunoprecipitated by Flag or p-Ser/Thr antibodies followed by immunoblotting. d , MDA-MB-231 cells stably expressing shRNAs targeting PFKFB4 (sh-PFK#09 and sh-PFK#20) or control NT-shRNA grown in presence of 5mM, 25mM glucose or glucose withdrawn from cells grown in 25mM of glucose and replaced with 5mM (WD) for 6 hours. Protein levels of pSRC-3-S857, PFKFB4 and β-actin were detected by immunoblotting. e , HEK293T cells expressing pBIND, pBIND-SRC-3 or pBIND-SRC-3-S857A were transduced with Adv. GFP or PFKFB4, and cultured in 5mM or 25mM glucose followed by luciferase assay. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean, n =6 biologically independent experiments; Two-way ANOVA with Tukey’s Multiple comparisons test]. Data shown in (a–e) are representative of 3 biologically independent experiments with similar results. For exact P -values please refer to source data.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4 phosphorylates SRC-3 by functioning as a protein kinase a , Upper panel-Recombinant GST-fused PFKFB4 incubated with full-length SRC-3 (SRC-3 FL) in presence of [ 32 P]ATP in an in vitro kinase assay. Lower panels- SRC-3 and PFKFB4 protein levels were analyzed by immunoblotting. b , In vitro kinase assay of PFKFB4 in the presence of SRC-3 fragments expressing different domains or full length SRC-3-FL. c , HEK293T cells expressing Flag-tagged-SRC-3 and PFKFB4 cultured in different concentrations of glucose and immunoprecipitated by Flag or p-Ser/Thr antibodies followed by immunoblotting. d , MDA-MB-231 cells stably expressing shRNAs targeting PFKFB4 (sh-PFK#09 and sh-PFK#20) or control NT-shRNA grown in presence of 5mM, 25mM glucose or glucose withdrawn from cells grown in 25mM of glucose and replaced with 5mM (WD) for 6 hours. Protein levels of pSRC-3-S857, PFKFB4 and β-actin were detected by immunoblotting. e , HEK293T cells expressing pBIND, pBIND-SRC-3 or pBIND-SRC-3-S857A were transduced with Adv. GFP or PFKFB4, and cultured in 5mM or 25mM glucose followed by luciferase assay. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean, n =6 biologically independent experiments; Two-way ANOVA with Tukey’s Multiple comparisons test]. Data shown in (a–e) are representative of 3 biologically independent experiments with similar results. For exact P -values please refer to source data.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Recombinant, Incubation, In Vitro, Kinase Assay, Expressing, Cell Culture, Immunoprecipitation, Multiple Displacement Amplification, Stable Transfection, shRNA, Transduction, Luciferase

    PFKFB4-SRC-3 axis promotes breast tumor growth and metastasis a , Primary tumors resected out after six weeks. b , Ki-67 staining of primary tumors from animals injected with MDA-MB-231 cells stably expressing NT-shRNA, shSRC-3 or shPFKFB4. Data shown representative of five fields per slide from n =5 animals per group with similar findings. c , Quantification of Ki67 positive cells in the tumor. [Mean ± s.d., n =5 animals per group, average of five fields counted from each slide, one-way ANOVA with Dunnett's multiple comparisons test, **** P =0.0001] d , Primary tumor growth in animals injected with MDA-MB-231 cells stably expressing shRNA targeting SRC-3 (shSRC-3), PFKFB4 (shPFKFB4), or expression of wildtype SRC-3 (WT-SRC-3) or S857A mutant in the shSRC-3 knockdown cells. [Mean ± s.d., n =5 animals per group, two-way ANOVA with Tukey’s multiple comparisons test, * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4-SRC-3 axis promotes breast tumor growth and metastasis a , Primary tumors resected out after six weeks. b , Ki-67 staining of primary tumors from animals injected with MDA-MB-231 cells stably expressing NT-shRNA, shSRC-3 or shPFKFB4. Data shown representative of five fields per slide from n =5 animals per group with similar findings. c , Quantification of Ki67 positive cells in the tumor. [Mean ± s.d., n =5 animals per group, average of five fields counted from each slide, one-way ANOVA with Dunnett's multiple comparisons test, **** P =0.0001] d , Primary tumor growth in animals injected with MDA-MB-231 cells stably expressing shRNA targeting SRC-3 (shSRC-3), PFKFB4 (shPFKFB4), or expression of wildtype SRC-3 (WT-SRC-3) or S857A mutant in the shSRC-3 knockdown cells. [Mean ± s.d., n =5 animals per group, two-way ANOVA with Tukey’s multiple comparisons test, * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Staining, Injection, Multiple Displacement Amplification, Stable Transfection, Expressing, shRNA, Mutagenesis

    Activation of the PFKFB4-SRC-3 axis drives breast tumor primary growth and metastasis a–g , MDA-MB-231 cells stably expressing shSRC-3, shPFKFB4, or wildtype SRC-3 (WT-SRC-3) or the SRC-3 S857A mutant in the shSRC-3 cells were injected into nude female mice. a , Schematics of the in vivo orthotopic xenograft experiment. Tumor cells were injected in the mammary fat pad ( n =5 mice) and after six weeks primary tumors were resected out and animals were monitored by bioluminescence. b , Tumor volume. One-way ANOVA with Tukey’s Multiple comparisons test. n =5 [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean.] c , Bioluminescence imaging of animals four weeks post-surgery. Representative images of three animals are shown from n =5 mice for SRC-3-WT, S857A, and shPFKFB4; and n =4 mice for shSRC-3. Residual or recurrence tumors at primary sites were masked with black paper to visualize lung lesions. d , Histology images showing lung sections stained with hematoxylin and eosin (H E). Arrows indicate micro metastasis lesions. Scale bar 100µm. Data shown are representative of four fields per slide from n =5 animals per group. e , Immunohistochemical (IHC) images from primary tumors demonstrating pSRC-3-S857 expression (red) co-stained with DAPI (blue). Scale bar: 100 µm. Magnified image in the box shows the tumor boundary as indicated by the dotted line. Scale bar: 200 µm. f , Quantification of nuclear-stained pSRC-3-S857 in each groups. Average of four fields per slide from n =5 mice per group. [Mean (center) ± s.d. (errors), One-way ANOVA with Dunnett's multiple comparisons test.]. For exact P -values please refer to source data.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: Activation of the PFKFB4-SRC-3 axis drives breast tumor primary growth and metastasis a–g , MDA-MB-231 cells stably expressing shSRC-3, shPFKFB4, or wildtype SRC-3 (WT-SRC-3) or the SRC-3 S857A mutant in the shSRC-3 cells were injected into nude female mice. a , Schematics of the in vivo orthotopic xenograft experiment. Tumor cells were injected in the mammary fat pad ( n =5 mice) and after six weeks primary tumors were resected out and animals were monitored by bioluminescence. b , Tumor volume. One-way ANOVA with Tukey’s Multiple comparisons test. n =5 [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points, + indicates mean.] c , Bioluminescence imaging of animals four weeks post-surgery. Representative images of three animals are shown from n =5 mice for SRC-3-WT, S857A, and shPFKFB4; and n =4 mice for shSRC-3. Residual or recurrence tumors at primary sites were masked with black paper to visualize lung lesions. d , Histology images showing lung sections stained with hematoxylin and eosin (H E). Arrows indicate micro metastasis lesions. Scale bar 100µm. Data shown are representative of four fields per slide from n =5 animals per group. e , Immunohistochemical (IHC) images from primary tumors demonstrating pSRC-3-S857 expression (red) co-stained with DAPI (blue). Scale bar: 100 µm. Magnified image in the box shows the tumor boundary as indicated by the dotted line. Scale bar: 200 µm. f , Quantification of nuclear-stained pSRC-3-S857 in each groups. Average of four fields per slide from n =5 mice per group. [Mean (center) ± s.d. (errors), One-way ANOVA with Dunnett's multiple comparisons test.]. For exact P -values please refer to source data.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Activation Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Mutagenesis, Injection, Mouse Assay, In Vivo, Imaging, Staining, Immunohistochemistry

    Growth defect due to loss of SRC-3 or PFKFB4 is rescued by exogenous purines a , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), adenosine monophosphate dehydrogenase 1 (AMPD1) and SRC-3 in MDA-MB-231 cells expressing shRNA targeting control-shNT, shSRC-3-21 or re-expression of shRNA –resistant wildtype SRC-3 protein in SRC-3 depleted cells (shSRC-3-21+WT-SRC-3). [Mean ± s.d., n =4 biological cell samples, two-way ANOVA with Tukey’s Multiple comparisons test]. b , Relative proliferation of MDA-MB-231 expressing shRNA targeting SRC-3 (shSRC-3-1 and shSRC-3-2) or NT after treatment with siRNAs targeting luciferase (siLuc) as control or PFKFB4 under conditions indicated. [Mean ± s.d., n =6 samples from biologically independent experiments, two-way ANOVA with Tukey’s Multiple comparisons test, **** P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: Growth defect due to loss of SRC-3 or PFKFB4 is rescued by exogenous purines a , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), adenosine monophosphate dehydrogenase 1 (AMPD1) and SRC-3 in MDA-MB-231 cells expressing shRNA targeting control-shNT, shSRC-3-21 or re-expression of shRNA –resistant wildtype SRC-3 protein in SRC-3 depleted cells (shSRC-3-21+WT-SRC-3). [Mean ± s.d., n =4 biological cell samples, two-way ANOVA with Tukey’s Multiple comparisons test]. b , Relative proliferation of MDA-MB-231 expressing shRNA targeting SRC-3 (shSRC-3-1 and shSRC-3-2) or NT after treatment with siRNAs targeting luciferase (siLuc) as control or PFKFB4 under conditions indicated. [Mean ± s.d., n =6 samples from biologically independent experiments, two-way ANOVA with Tukey’s Multiple comparisons test, **** P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Expressing, Multiple Displacement Amplification, shRNA, Luciferase

    The PFKFB4-SRC-3 axis drives transcriptional programming in breast cancer patients a, b , Expression of p-SRC-3, SRC-3 and PFKFB4 in ER(+) breast tumor specimens and matched adjoining normal tissues as detected by immunoblotting n =14, ER-positive breast cancer patients. c , Semi-quantitative levels of each band shown in a and b , were analyzed by densitometry using UVP Vision Works LS software, and the relative values normalized to actin were used to calculate the fold change (tumor/normal) and plotted to obtain the correlation between PFKFB4 and pSRC-3-Ser857 expression [ n =14 normal and tumor tissues, R =0.63, p=0.02 Spearman's rank correlation coefficient]. d , Log fold change in protein expression of the PFKFB4-SRC-3 signature compared to the control knockdown (NTshRNA) as determined using a parametric t-test as implemented in the python (spicy) statistical system. Significance was assessed for P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: The PFKFB4-SRC-3 axis drives transcriptional programming in breast cancer patients a, b , Expression of p-SRC-3, SRC-3 and PFKFB4 in ER(+) breast tumor specimens and matched adjoining normal tissues as detected by immunoblotting n =14, ER-positive breast cancer patients. c , Semi-quantitative levels of each band shown in a and b , were analyzed by densitometry using UVP Vision Works LS software, and the relative values normalized to actin were used to calculate the fold change (tumor/normal) and plotted to obtain the correlation between PFKFB4 and pSRC-3-Ser857 expression [ n =14 normal and tumor tissues, R =0.63, p=0.02 Spearman's rank correlation coefficient]. d , Log fold change in protein expression of the PFKFB4-SRC-3 signature compared to the control knockdown (NTshRNA) as determined using a parametric t-test as implemented in the python (spicy) statistical system. Significance was assessed for P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Expressing, Software

    Increased glucose and purines are required for SRC-3 dependent growth a , Real time measurement of MCF10A cell proliferation transduced with adenovirus Adv. GFP or Adv. SRC-3 in presence of 93 different metabolites. n =3 independent plates run for each sample. Mean (center) ± s.d. (error bars), b , Relative growth of MCF10A cells transduced with Adv. GFP or Adv. SRC-3 in presence of D-glucose, c , adenosine and d , inosine. Mean ± s.d., n =6 biological cell samples, Unpaired t-test two tailed. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points]. e–f , Relative levels of intermediary metabolites in MDA-MB-231 cells after treatment with shRNAs targeting PFKFB4 or SRC-3 compared to control shRNA (NT). e , Glycolytic and pentose phosphate pathway (PPP) metabolites. f , Nucleotides. [Mean ± s.d., n =3 biological independent samples, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: Increased glucose and purines are required for SRC-3 dependent growth a , Real time measurement of MCF10A cell proliferation transduced with adenovirus Adv. GFP or Adv. SRC-3 in presence of 93 different metabolites. n =3 independent plates run for each sample. Mean (center) ± s.d. (error bars), b , Relative growth of MCF10A cells transduced with Adv. GFP or Adv. SRC-3 in presence of D-glucose, c , adenosine and d , inosine. Mean ± s.d., n =6 biological cell samples, Unpaired t-test two tailed. [Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points]. e–f , Relative levels of intermediary metabolites in MDA-MB-231 cells after treatment with shRNAs targeting PFKFB4 or SRC-3 compared to control shRNA (NT). e , Glycolytic and pentose phosphate pathway (PPP) metabolites. f , Nucleotides. [Mean ± s.d., n =3 biological independent samples, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Transduction, Two Tailed Test, Multiple Displacement Amplification, shRNA

    PFKFB4 is an essential activator of transcriptional coregulator SRC-3 a , Schematics showing the RNAi kinome library screening with SRC-3 transcriptional activity assay using GAL4 DNA binding site-luciferase reporter (pG5-luc) along with GAL4-DNA binding domain (DBD)-full-length SRC-3 fusion (pBIND-SRC-3) or control pBIND as readout. b , Log 2 fold change in SRC-3 activity with three siRNAs/kinase represented as Set A, Set B and Set C in the 3D plot ( n =3, biologically independent samples targeted by siRNAs). Suppression of kinases increasing SRC-3 activity with 2/3 siRNAs (red) or 3/3 siRNAs (purple), and reducing SRC-3 activity with 3/3 siRNAs (green) or 2/3 (blue). c , Kinases scoring reproducibly in modulating SRC-3 activity. d , SRC-3 activity in MCF-7 cells transduced with adv. GFP or PFKFB4 and co-transfected with pBIND ( n =3) or pBIND-SRC-3 ( n =4). [Mean ± s.d., one-way ANOVA with Tukey’s Multiple comparisons test]. g , Protein expression of SRC-3, PFKFB4 and actin in MCF-7 cells overexpressing PFKFB4 or control-GFP. For exact P -values please refer to source data, and n represents biologically independent samples.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: PFKFB4 is an essential activator of transcriptional coregulator SRC-3 a , Schematics showing the RNAi kinome library screening with SRC-3 transcriptional activity assay using GAL4 DNA binding site-luciferase reporter (pG5-luc) along with GAL4-DNA binding domain (DBD)-full-length SRC-3 fusion (pBIND-SRC-3) or control pBIND as readout. b , Log 2 fold change in SRC-3 activity with three siRNAs/kinase represented as Set A, Set B and Set C in the 3D plot ( n =3, biologically independent samples targeted by siRNAs). Suppression of kinases increasing SRC-3 activity with 2/3 siRNAs (red) or 3/3 siRNAs (purple), and reducing SRC-3 activity with 3/3 siRNAs (green) or 2/3 (blue). c , Kinases scoring reproducibly in modulating SRC-3 activity. d , SRC-3 activity in MCF-7 cells transduced with adv. GFP or PFKFB4 and co-transfected with pBIND ( n =3) or pBIND-SRC-3 ( n =4). [Mean ± s.d., one-way ANOVA with Tukey’s Multiple comparisons test]. g , Protein expression of SRC-3, PFKFB4 and actin in MCF-7 cells overexpressing PFKFB4 or control-GFP. For exact P -values please refer to source data, and n represents biologically independent samples.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Library Screening, Activity Assay, Binding Assay, Luciferase, Transduction, Transfection, Expressing

    SRC-3 drives the purine synthesis program under conditions of active glycolysis a , MDA-MB231 cells stably expressing NTshRNA, shPFKFB4 and shSRC-3 were fed with [6-C 13 glucose]. Ribulose/Xylulose-5P (m+1) labeling from [6-C 13 glucose] were shown. [Mean ± s.d., n =3 biological cell samples, One-way ANOVA with Tukey’s Multiple comparisons test, *** P =0.00013; **** P =0.000078]. b , Genes involved in oxidative and non-oxidative PPP. [Mean ± s.d., n =3 biological cell samples, Two-way ANOVA with Sidak's multiple comparisons test, * P =0.0431]. c , Genes involved in nucleotide synthesis. [Mean ± s.d., n =3 biological cell samples, Two-way ANOVA with Sidak's multiple comparisons test]. d , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), and adenosine monophosphate dehydrogenase 1 (AMPD1) in MDA-MB-231 cells transduced with adenovirus expressing GFP (control) and PFKFB4 cultured in presence of 5mM, 15mM or 25mM glucose. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Dunnett's multiple comparisons test]. e–f , MDA-MB231 cells stably expressing NTshRNA, shPFKFB4 and shSRC-3 were fed with [6-C 13 glucose]. e , Seduheptulose-7P (m+1). and f , Erythrose-4P labeling from [6-C 13 glucose] were shown. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Dunnett's multiple comparisons test (e) or with Tukey’s multiple comparison test (f), Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points]. For exact P -values please refer to source data.

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: SRC-3 drives the purine synthesis program under conditions of active glycolysis a , MDA-MB231 cells stably expressing NTshRNA, shPFKFB4 and shSRC-3 were fed with [6-C 13 glucose]. Ribulose/Xylulose-5P (m+1) labeling from [6-C 13 glucose] were shown. [Mean ± s.d., n =3 biological cell samples, One-way ANOVA with Tukey’s Multiple comparisons test, *** P =0.00013; **** P =0.000078]. b , Genes involved in oxidative and non-oxidative PPP. [Mean ± s.d., n =3 biological cell samples, Two-way ANOVA with Sidak's multiple comparisons test, * P =0.0431]. c , Genes involved in nucleotide synthesis. [Mean ± s.d., n =3 biological cell samples, Two-way ANOVA with Sidak's multiple comparisons test]. d , Expression of metabolic enzymes transketolase (TKT), xanthine dehydrogenase (XDH), and adenosine monophosphate dehydrogenase 1 (AMPD1) in MDA-MB-231 cells transduced with adenovirus expressing GFP (control) and PFKFB4 cultured in presence of 5mM, 15mM or 25mM glucose. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Dunnett's multiple comparisons test]. e–f , MDA-MB231 cells stably expressing NTshRNA, shPFKFB4 and shSRC-3 were fed with [6-C 13 glucose]. e , Seduheptulose-7P (m+1). and f , Erythrose-4P labeling from [6-C 13 glucose] were shown. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Dunnett's multiple comparisons test (e) or with Tukey’s multiple comparison test (f), Boxes represent 25 th to 75 th percentile, line in the middle represents median, whiskers showing min to max all points]. For exact P -values please refer to source data.

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Multiple Displacement Amplification, Stable Transfection, Expressing, Labeling, Transduction, Cell Culture

    Ser-857 phosphorylation enhances SRC-3 transcriptional activity a , Relative luciferase activity (RLU) showing the activity of SRC-3 WT , SRC-3 S857A and SRC-3 S857E in MDA-MB-231 cells transduced with lentivirus expressing NTshRNA or shPFKFB4 cultured in presence of 5mM or 25mM glucose. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Journal: Nature

    Article Title: Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer

    doi: 10.1038/s41586-018-0018-1

    Figure Lengend Snippet: Ser-857 phosphorylation enhances SRC-3 transcriptional activity a , Relative luciferase activity (RLU) showing the activity of SRC-3 WT , SRC-3 S857A and SRC-3 S857E in MDA-MB-231 cells transduced with lentivirus expressing NTshRNA or shPFKFB4 cultured in presence of 5mM or 25mM glucose. [Mean ± s.d., n =3 biological cell samples, two-way ANOVA with Tukey’s Multiple comparisons test, * P

    Article Snippet: The following antibodies were used for ChIP: SRC-3 (Cell Signaling or BD Biosciences), ATF4 (Santa Cruz C-20, and Cat# 11815 Cell Signaling), pSRC-3-S857 (Cell Signaling), and rabbit IgG.

    Techniques: Activity Assay, Luciferase, Multiple Displacement Amplification, Transduction, Expressing, Cell Culture

    Identification of genes significantly dysregulated with deletion of Asxl1 alone and in concert with deletion of Tet2 and their functional impact. (A) Volcano plot of differentially expressed transcripts from RNA-Seq data of 1-yr-old control versus littermate Asxl1 KO ( Mx1-cre Asxl1 fl/fl ) LSK and MP (lineage − Sca-1 − c-Kit + ) cells (experiment included cells from two individual mice per genotype). (B) Venn diagrams of genes significantly up- and down-regulated with Asxl1 loss in LSK and MP (lineage − , Sca-1 − , cKit + ) cells from 1-yr-old Mx1-cre Asxl1 fl/fl mice and littermate Cre − controls as identified in A. (C) qRT-PCR analysis of HoxA and Hox -associated transcription factor genes in LSK cells of 1-yr-old Cre − Asxl1 fl/fl control versus littermate Vav-cre Asxl1 fl/fl . (D) qRT-PCR analysis of p16 INK4a in LT-HSCs (lineage − , Sca-1 + , c-Kit + , CD150 + , CD48 − ) and MPP cells (lineage − , Sca-1 + , c-Kit + , CD150 − , CD48 + ) from 6-wk- and 6-mo-old control (C) versus littermate Vav-cre Asxl1 fl/fl (KO) mice. (E) Cell cycle analysis of MPPs from 72-wk-old Vav-cre Asxl1 fl/fl or littermate Cre − Asxl1 fl/fl control mice with in vivo BrdU administration. Representative FACS plot analysis showing gating on MPP cells followed by BrdU versus DAPI stain is shown on the left (parent gate is LSK cells). Relative quantification of the percentage of MPP cells in S, G2/M, and G0/1 phase is shown on the right ( n = 5 mice per group). (F) Assessment of the proportion of HSPCs undergoing apoptosis was performed by Annexin V/DAPI stain of LSK cells from 72-wk-old Vav-cre Asxl1 fl/fl mice or Cre − Asxl1 fl/fl littermate controls. Representative FACS plot analysis showing gating on LSK cells followed by Annexin V versus DAPI stain is shown on the left (parent gate is lineage − cells). Relative quantification of the percentage of Annexin V + /DAPI − and Annexin V + /DAPI + LSK cells is shown on the right ( n = 5 mice per group). (G) Comparison of significant differentially expressed genes in LSK cells from 6-wk-old Mx1-cre Asxl1 fl/fl , Mx1-cre Tet2 fl/fl , or Mx1-cre Asxl1 fl/fl Tet2 fl/fl relative to controls (or Mx1-cre Asxl1 WT Tet2 WT). 99 genes are uniquely down-regulated in Asxl1 / Tet2 DKO mice relative to all other genotypes (left), whereas 49 genes are significantly up-regulated (right). (H) GSEA of overlapping and statistically significant gene sets enriched in the LSK cells of mice with deletion of Asxl1 alone or with combined Asxl1 and Tet2 deletion. (I) Gene sets uniquely enriched in mice with concomitant deletion of Asxl1 and Tet2 relative to all other genotypes as determined by GSEA. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Identification of genes significantly dysregulated with deletion of Asxl1 alone and in concert with deletion of Tet2 and their functional impact. (A) Volcano plot of differentially expressed transcripts from RNA-Seq data of 1-yr-old control versus littermate Asxl1 KO ( Mx1-cre Asxl1 fl/fl ) LSK and MP (lineage − Sca-1 − c-Kit + ) cells (experiment included cells from two individual mice per genotype). (B) Venn diagrams of genes significantly up- and down-regulated with Asxl1 loss in LSK and MP (lineage − , Sca-1 − , cKit + ) cells from 1-yr-old Mx1-cre Asxl1 fl/fl mice and littermate Cre − controls as identified in A. (C) qRT-PCR analysis of HoxA and Hox -associated transcription factor genes in LSK cells of 1-yr-old Cre − Asxl1 fl/fl control versus littermate Vav-cre Asxl1 fl/fl . (D) qRT-PCR analysis of p16 INK4a in LT-HSCs (lineage − , Sca-1 + , c-Kit + , CD150 + , CD48 − ) and MPP cells (lineage − , Sca-1 + , c-Kit + , CD150 − , CD48 + ) from 6-wk- and 6-mo-old control (C) versus littermate Vav-cre Asxl1 fl/fl (KO) mice. (E) Cell cycle analysis of MPPs from 72-wk-old Vav-cre Asxl1 fl/fl or littermate Cre − Asxl1 fl/fl control mice with in vivo BrdU administration. Representative FACS plot analysis showing gating on MPP cells followed by BrdU versus DAPI stain is shown on the left (parent gate is LSK cells). Relative quantification of the percentage of MPP cells in S, G2/M, and G0/1 phase is shown on the right ( n = 5 mice per group). (F) Assessment of the proportion of HSPCs undergoing apoptosis was performed by Annexin V/DAPI stain of LSK cells from 72-wk-old Vav-cre Asxl1 fl/fl mice or Cre − Asxl1 fl/fl littermate controls. Representative FACS plot analysis showing gating on LSK cells followed by Annexin V versus DAPI stain is shown on the left (parent gate is lineage − cells). Relative quantification of the percentage of Annexin V + /DAPI − and Annexin V + /DAPI + LSK cells is shown on the right ( n = 5 mice per group). (G) Comparison of significant differentially expressed genes in LSK cells from 6-wk-old Mx1-cre Asxl1 fl/fl , Mx1-cre Tet2 fl/fl , or Mx1-cre Asxl1 fl/fl Tet2 fl/fl relative to controls (or Mx1-cre Asxl1 WT Tet2 WT). 99 genes are uniquely down-regulated in Asxl1 / Tet2 DKO mice relative to all other genotypes (left), whereas 49 genes are significantly up-regulated (right). (H) GSEA of overlapping and statistically significant gene sets enriched in the LSK cells of mice with deletion of Asxl1 alone or with combined Asxl1 and Tet2 deletion. (I) Gene sets uniquely enriched in mice with concomitant deletion of Asxl1 and Tet2 relative to all other genotypes as determined by GSEA. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Functional Assay, RNA Sequencing Assay, Gene Knockout, Mouse Assay, Quantitative RT-PCR, Cell Cycle Assay, In Vivo, FACS, Staining

    Effect of Asxl1 loss in vivo on H3K27me3 and identification of Asxl1-regulated genes ChIP-Seq. (A) Western blot analysis of H3K27me3 and total histone H3 in splenocytes of 6-wk-old Vav-cre Asxl1 fl/fl mice relative to littermate control. (B) Levels of core PRC2 members Ezh2, Suz12, and Eed in splenocytes of same mice as shown in A. (C) Characterization of Asxl1-binding sites identified by anti-Asxl1 ChIP-Seq analysis in mouse WT BMDMs. (D) Heat map representation of Asxl1 ChIP-Seq signal centered around TSSs (±2 kb) of CpG (left) and non-CpG (right) promoters. (E) Mean Asxl1 ChIP-Seq signal density of CpG and non-CpG promoters centered around the TSS ± 10 kb. (F) Motif enrichment analysis of Asxl1-binding sites identified significant enrichment of Ets transcription factor binding sites (P = 1 ×10 −59 , percent target = 40.1%, and percent background = 21.4%).

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Effect of Asxl1 loss in vivo on H3K27me3 and identification of Asxl1-regulated genes ChIP-Seq. (A) Western blot analysis of H3K27me3 and total histone H3 in splenocytes of 6-wk-old Vav-cre Asxl1 fl/fl mice relative to littermate control. (B) Levels of core PRC2 members Ezh2, Suz12, and Eed in splenocytes of same mice as shown in A. (C) Characterization of Asxl1-binding sites identified by anti-Asxl1 ChIP-Seq analysis in mouse WT BMDMs. (D) Heat map representation of Asxl1 ChIP-Seq signal centered around TSSs (±2 kb) of CpG (left) and non-CpG (right) promoters. (E) Mean Asxl1 ChIP-Seq signal density of CpG and non-CpG promoters centered around the TSS ± 10 kb. (F) Motif enrichment analysis of Asxl1-binding sites identified significant enrichment of Ets transcription factor binding sites (P = 1 ×10 −59 , percent target = 40.1%, and percent background = 21.4%).

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: In Vivo, Chromatin Immunoprecipitation, Western Blot, Mouse Assay, Binding Assay

    Conditional deletion of Asxl1 results in age-dependent leukopenia and anemia. (A) qRT-PCR showing relative expression level of Asxl1 in purified progenitor and mature mouse hematopoietic stem and progenitor subsets. (B) Verification of Mx1-cre – and Vav-cre –mediated deletion of Asxl1 at the level of protein expression in Western blot of splenocytes. (C) Enumeration of nucleated cells in bilateral femurs and tibiae or whole spleens of control ( Asxl1 fl/fl ) and Asxl1 hematopoietic-specific KO mice ( Vav-cre Asxl1 fl/fl ) at 6 as well as 24 wk of age ( n = 6–10 mice per genotype at each time point examined). (D and E) Enumeration of peripheral WBCs (D) and Hb (E) with postnatal deletion of Asxl1 (performed using Mx1-cre Asxl1 fl/fl mice or Cre − Asxl1 fl/fl controls). Counts in aged Asxl1 KO mice are compared with age-matched controls as well as younger KO and control mice ( n = 6–12 mice per genotype at each time point examined). (F and G) Flow cytometric enumeration of B220 + , CD11b + /Gr1 + , CD3 + , and CD11b + /Gr1 − cells in the peripheral blood of > 6-mo-old Mx1-cre Asxl1 fl/fl (KO) and Asxl1 fl/fl (C) mice ( n = 5 mice per genotype were used for FACS analysis of peripheral blood). The right panel reveals peripheral blood FACS analysis. Antibody stainings are as indicated, and cells were gated on live cells in the parent gate. (A and C–F) Error bars represent ±SD (A and F); mean ± SEM is shown (C–E); *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Conditional deletion of Asxl1 results in age-dependent leukopenia and anemia. (A) qRT-PCR showing relative expression level of Asxl1 in purified progenitor and mature mouse hematopoietic stem and progenitor subsets. (B) Verification of Mx1-cre – and Vav-cre –mediated deletion of Asxl1 at the level of protein expression in Western blot of splenocytes. (C) Enumeration of nucleated cells in bilateral femurs and tibiae or whole spleens of control ( Asxl1 fl/fl ) and Asxl1 hematopoietic-specific KO mice ( Vav-cre Asxl1 fl/fl ) at 6 as well as 24 wk of age ( n = 6–10 mice per genotype at each time point examined). (D and E) Enumeration of peripheral WBCs (D) and Hb (E) with postnatal deletion of Asxl1 (performed using Mx1-cre Asxl1 fl/fl mice or Cre − Asxl1 fl/fl controls). Counts in aged Asxl1 KO mice are compared with age-matched controls as well as younger KO and control mice ( n = 6–12 mice per genotype at each time point examined). (F and G) Flow cytometric enumeration of B220 + , CD11b + /Gr1 + , CD3 + , and CD11b + /Gr1 − cells in the peripheral blood of > 6-mo-old Mx1-cre Asxl1 fl/fl (KO) and Asxl1 fl/fl (C) mice ( n = 5 mice per genotype were used for FACS analysis of peripheral blood). The right panel reveals peripheral blood FACS analysis. Antibody stainings are as indicated, and cells were gated on live cells in the parent gate. (A and C–F) Error bars represent ±SD (A and F); mean ± SEM is shown (C–E); *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Quantitative RT-PCR, Expressing, Purification, Western Blot, Gene Knockout, Mouse Assay, Flow Cytometry, FACS

    Concomitant deletion of Asxl1 and Tet2 results in myelodysplasia in mice. (A) Kaplan-Meier survival curve of primary Cre − Asxl1 fl/fl ( n = 5), Mx1-cre Asxl1 fl/fl ( n = 12), Mx1-cre Tet2 fl/fl ( n = 6), Mx1-cre Asxl1 fl/fl Tet2 fl/fl ( n = 10 mice per genotype). Mice were treated with polyI:polyC at 4 wk after birth and then followed for 50 wk. (B) Peripheral WBC count and differential of recipient mice transplanted with BM from 6-wk-old Mx1-cre Asxl1 WT Tet2 WT (control; C), Mx1-cre Asxl1 fl/fl (Asxl1 KO), Mx1-cre Tet2 fl/fl (Tet2 KO), and Mx1-cre Asxl1 fl/fl Tet2 fl/fl ( Asxl1 / Tet2 DKO) mice 66 wk after transplantation (68 wk after polyI:polyC administration to recipient mice; n = 10 mice per genotype). Differential was determined by flow cytometric analysis of peripheral blood. (C and D) Hematocrit (C) and total number (D) of nucleated BM cells of same mice as shown in B. Horizontal lines indicate the mean. (E) Representative flow cytometric assessment of relative frequencies of MP and LSK cells in 72-wk-old mice. Parent population was live, lineage − cells. (F) Total numbers of LSK and MP cells (lineage − Sca-1 − c-Kit + ) in mice from each genotype at 72 wk of age. This was determined by flow cytometric quantification of living LSK and MP cells from c-KIT–enriched BM cells harvested from spine plus bilateral femurs, tibiae, and humeri of each mouse from each genotype at 72 wk of age ( n = 3 mice per group). (G) Wright-Giemsa stain of BM representative erythroid precursor from cytospins of 72-wk-old control, Asxl1 KO, Tet2 KO, or Asx1/Tet2 DKO mice. Arrows indicate multinuclearity and nuclear fragmentation in erythroid precursors. (H) Representation histological sections of liver from 72-wk-old control, Asxl1 KO, Tet2 KO, or Asx1/Tet2 DKO mice Bars: (G) 5 µm; (H) 50 µm. For A and B, n = 10 mice per group; for C–H, n = 3 mice per group. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Concomitant deletion of Asxl1 and Tet2 results in myelodysplasia in mice. (A) Kaplan-Meier survival curve of primary Cre − Asxl1 fl/fl ( n = 5), Mx1-cre Asxl1 fl/fl ( n = 12), Mx1-cre Tet2 fl/fl ( n = 6), Mx1-cre Asxl1 fl/fl Tet2 fl/fl ( n = 10 mice per genotype). Mice were treated with polyI:polyC at 4 wk after birth and then followed for 50 wk. (B) Peripheral WBC count and differential of recipient mice transplanted with BM from 6-wk-old Mx1-cre Asxl1 WT Tet2 WT (control; C), Mx1-cre Asxl1 fl/fl (Asxl1 KO), Mx1-cre Tet2 fl/fl (Tet2 KO), and Mx1-cre Asxl1 fl/fl Tet2 fl/fl ( Asxl1 / Tet2 DKO) mice 66 wk after transplantation (68 wk after polyI:polyC administration to recipient mice; n = 10 mice per genotype). Differential was determined by flow cytometric analysis of peripheral blood. (C and D) Hematocrit (C) and total number (D) of nucleated BM cells of same mice as shown in B. Horizontal lines indicate the mean. (E) Representative flow cytometric assessment of relative frequencies of MP and LSK cells in 72-wk-old mice. Parent population was live, lineage − cells. (F) Total numbers of LSK and MP cells (lineage − Sca-1 − c-Kit + ) in mice from each genotype at 72 wk of age. This was determined by flow cytometric quantification of living LSK and MP cells from c-KIT–enriched BM cells harvested from spine plus bilateral femurs, tibiae, and humeri of each mouse from each genotype at 72 wk of age ( n = 3 mice per group). (G) Wright-Giemsa stain of BM representative erythroid precursor from cytospins of 72-wk-old control, Asxl1 KO, Tet2 KO, or Asx1/Tet2 DKO mice. Arrows indicate multinuclearity and nuclear fragmentation in erythroid precursors. (H) Representation histological sections of liver from 72-wk-old control, Asxl1 KO, Tet2 KO, or Asx1/Tet2 DKO mice Bars: (G) 5 µm; (H) 50 µm. For A and B, n = 10 mice per group; for C–H, n = 3 mice per group. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Mouse Assay, Gene Knockout, Transplantation Assay, Flow Cytometry, Giemsa Stain

    Asxl1 −/− mice have increased stem/progenitor cells but impaired self-renewal. (A) Flow cytometric enumeration of BM LSK cells, LT-HSCs (LSK CD150 + CD48 − ), and MPP cells (LSK CD150 − CD48 + ) in WT ( Asxl1 fl/fl ) and KO ( Vav-cre Asxl1 fl/fl ) mice at 6 wk of age ( n = 4–6 mice per genotype as indicated). Data are expressed as total number of live cells per femur. (B) Representative FACS analysis of BM stem cell populations of Asxl1 −/− ( Vav-cre Asxl1 fl/fl ) and WT ( Asxl1 fl/fl ) at 6 wk. Antibody stains are as indicated and parent gate is live, lineage − cells. (C) Schematic depiction of the competitive transplantation assay. Asxl1 fl/fl and Vav-cre Asxl1 fl/fl are positive for CD45.2, whereas WT competitor cells are positive for CD45.1. Recipient mice are also CD45.1. Representative FACS plots of the percentage of CD45.1 versus CD45.2 total chimerism in the peripheral blood of recipient animals at 16 wk after competitive transplantation is shown. (D) Percentage of CD45.1 versus CD45.2 total chimerism in the peripheral blood of recipient animals at 4 and 16 wk in primary competitive transplant and serial secondary competitive transplants are shown ( n = 5 recipient mice for each genotype; C, control; KO, Asxl1 KO). The experiment was performed in biological duplicate. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Asxl1 −/− mice have increased stem/progenitor cells but impaired self-renewal. (A) Flow cytometric enumeration of BM LSK cells, LT-HSCs (LSK CD150 + CD48 − ), and MPP cells (LSK CD150 − CD48 + ) in WT ( Asxl1 fl/fl ) and KO ( Vav-cre Asxl1 fl/fl ) mice at 6 wk of age ( n = 4–6 mice per genotype as indicated). Data are expressed as total number of live cells per femur. (B) Representative FACS analysis of BM stem cell populations of Asxl1 −/− ( Vav-cre Asxl1 fl/fl ) and WT ( Asxl1 fl/fl ) at 6 wk. Antibody stains are as indicated and parent gate is live, lineage − cells. (C) Schematic depiction of the competitive transplantation assay. Asxl1 fl/fl and Vav-cre Asxl1 fl/fl are positive for CD45.2, whereas WT competitor cells are positive for CD45.1. Recipient mice are also CD45.1. Representative FACS plots of the percentage of CD45.1 versus CD45.2 total chimerism in the peripheral blood of recipient animals at 16 wk after competitive transplantation is shown. (D) Percentage of CD45.1 versus CD45.2 total chimerism in the peripheral blood of recipient animals at 4 and 16 wk in primary competitive transplant and serial secondary competitive transplants are shown ( n = 5 recipient mice for each genotype; C, control; KO, Asxl1 KO). The experiment was performed in biological duplicate. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Mouse Assay, Flow Cytometry, Gene Knockout, FACS, Transplantation Assay

    Deletion of Asxl1 results in myeloid and erythroid dysplasia and impaired progenitor differentiation consistent with myelodysplasia. (A) Relative frequency of CD71 + /Ter119 − erythroid precursors in BM and spleen of 6.5-mo-old Mx1-cre Asxl1 fl/fl (KO) and Cre − Asxl1 fl/fl control mice (expressed as percentage of live cells; n = 3–5 mice per genotype in each tissue type examined by FACS analysis). (B) Histological (H E) analysis of Mx1-cre Asxl1 fl/fl and Cre − Asxl1 fl/fl control BM from 6-mo-old littermate mice. (C) BM cytospins (Wright-Giemsa) from the same mice (arrows indicate erythroid precursors with prominent irregular nuclear contours). (D) Representative morphology of peripheral blood myeloid cells (top) and nucleated RBCs (bottom) in KO mice (Wright-Giemsa stain). (E) Number of colonies formed 7 d after plating of 1,000 CMP, GMP, or MEP cells into methylcellulose from 6-wk-old Vav-cre Asxl1 fl/fl and littermate control (Cre − Asxl1 1fl/fl control) mice. The experiment was performed in biological duplicate. (F) Photograph of methylcellulose colony plate 7 d after plating of MEP cells from 6-wk-old KO and control mice. (G) Histological analysis by H E staining of liver from 72-wk-old Mx1-cre Asxl1 fl/fl mice and littermates. (H) Number of colonies formed 7 d after plating of 200,000 nucleated cells harvested from the liver of 72-wk-old Mx1-cre Asxl1 fl/fl or littermate control mice in methylcellulose containing rmIL-3, rm-SCF, rh-IL6, rh-EPO (liver cells from n = 5 mice per genotype plated in methylcellulose). (I) Photomicrograph of colonies grown from cells taken from the liver and plated in methylcellulose is shown on right. Bars: (B) 50 µm; (C) 10 µm; (D) 5 µm; (G) 100 µm; (I) 200 µm. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Deletion of Asxl1 results in myeloid and erythroid dysplasia and impaired progenitor differentiation consistent with myelodysplasia. (A) Relative frequency of CD71 + /Ter119 − erythroid precursors in BM and spleen of 6.5-mo-old Mx1-cre Asxl1 fl/fl (KO) and Cre − Asxl1 fl/fl control mice (expressed as percentage of live cells; n = 3–5 mice per genotype in each tissue type examined by FACS analysis). (B) Histological (H E) analysis of Mx1-cre Asxl1 fl/fl and Cre − Asxl1 fl/fl control BM from 6-mo-old littermate mice. (C) BM cytospins (Wright-Giemsa) from the same mice (arrows indicate erythroid precursors with prominent irregular nuclear contours). (D) Representative morphology of peripheral blood myeloid cells (top) and nucleated RBCs (bottom) in KO mice (Wright-Giemsa stain). (E) Number of colonies formed 7 d after plating of 1,000 CMP, GMP, or MEP cells into methylcellulose from 6-wk-old Vav-cre Asxl1 fl/fl and littermate control (Cre − Asxl1 1fl/fl control) mice. The experiment was performed in biological duplicate. (F) Photograph of methylcellulose colony plate 7 d after plating of MEP cells from 6-wk-old KO and control mice. (G) Histological analysis by H E staining of liver from 72-wk-old Mx1-cre Asxl1 fl/fl mice and littermates. (H) Number of colonies formed 7 d after plating of 200,000 nucleated cells harvested from the liver of 72-wk-old Mx1-cre Asxl1 fl/fl or littermate control mice in methylcellulose containing rmIL-3, rm-SCF, rh-IL6, rh-EPO (liver cells from n = 5 mice per genotype plated in methylcellulose). (I) Photomicrograph of colonies grown from cells taken from the liver and plated in methylcellulose is shown on right. Bars: (B) 50 µm; (C) 10 µm; (D) 5 µm; (G) 100 µm; (I) 200 µm. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Gene Knockout, Mouse Assay, FACS, Giemsa Stain, Staining

    Serial noncompetitive transplantation of Asxl1 -null cells results in lethal myelodysplastic disorder. (A) Kaplan-Meier survival curve of recipient mice transplanted with 70-wk-old Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM after secondary and tertiary transplantation. Also shown is the survival of mice transplanted with purified LSK cells in tertiary transplantation (tertiary transplant of Asxl1 fl/fl control LSK cells is not shown; no recipient mice from this group died by 40 wk [ n = 5]). Cre − Asxl1 fl/fl littermate controls were similarly transplanted in parallel in each experiment. Four to six recipient mice were transplanted in each experiment. (B) Hematocrit over time of secondary recipient mice transplanted with Asxl1 -null or littermate control whole BM in a noncompetitive manner. The dashed line represents the lower limit of normal hematocrit for C57BL/6 mice ( n = 4–6 mice per genotype at each time point). (C) Body weight of secondarily transplanted mice at 50 wk after transplantation ( n = 4 mice per genotype). (D) BM histopathology of secondary recipient mice transplanted with Asxl1 -null or littermate control whole BM at 50 wk. (E) Relative frequency of LSK cells, MPP cells (LSK + , CD150 − , CD48 + ), and LT-HSCs (LSK + CD150 + CD48 − ) in BM and spleen at 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as frequency of live cells ( n = 4 mice per genotype examined for FACS experiments). (F) Relative frequency of MP (lineage − c-Kit + Sca-1 − ), CMP (lineage − , c-Kit + , Sca-1 − , FcγR − , CD34 + ), GMP (lineage − c-Kit + Sca-1 − , FcγR + CD34 + ), and MEP (lineage − c-Kit + Sca-1 − FcγR − CD34 − ) cells at 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as a frequency of live cells. (G) Photographs of spleens from secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after lethal irradiation. (H) Weight of the same spleens as shown in G ( n = 4 mice per genotype). (I) Histopathology of spleens from secondary recipient mice transplanted with Asxl1 -null or WT littermate control whole BM 50 wk after noncompetitive secondary transplantation revealing loss of normal splenic architecture. (J) Photographs of representative femur (top) and tibia (bottom) from secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after noncompetitive secondary transplantation. Bars: (D and I) 50 µm; (J) 2 mm. (K) Relative quantification of CD71 + /Ter119 − and CD71/Ter119 double-positive cells from BM and spleen of secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as a percentage of live cells ( n = 4 mice per genotype examined by FACS analysis). (L) Representative FACS plots of data shown in K from splenocytes. Staining is as shown, and live cells were gated in parent gate. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Serial noncompetitive transplantation of Asxl1 -null cells results in lethal myelodysplastic disorder. (A) Kaplan-Meier survival curve of recipient mice transplanted with 70-wk-old Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM after secondary and tertiary transplantation. Also shown is the survival of mice transplanted with purified LSK cells in tertiary transplantation (tertiary transplant of Asxl1 fl/fl control LSK cells is not shown; no recipient mice from this group died by 40 wk [ n = 5]). Cre − Asxl1 fl/fl littermate controls were similarly transplanted in parallel in each experiment. Four to six recipient mice were transplanted in each experiment. (B) Hematocrit over time of secondary recipient mice transplanted with Asxl1 -null or littermate control whole BM in a noncompetitive manner. The dashed line represents the lower limit of normal hematocrit for C57BL/6 mice ( n = 4–6 mice per genotype at each time point). (C) Body weight of secondarily transplanted mice at 50 wk after transplantation ( n = 4 mice per genotype). (D) BM histopathology of secondary recipient mice transplanted with Asxl1 -null or littermate control whole BM at 50 wk. (E) Relative frequency of LSK cells, MPP cells (LSK + , CD150 − , CD48 + ), and LT-HSCs (LSK + CD150 + CD48 − ) in BM and spleen at 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as frequency of live cells ( n = 4 mice per genotype examined for FACS experiments). (F) Relative frequency of MP (lineage − c-Kit + Sca-1 − ), CMP (lineage − , c-Kit + , Sca-1 − , FcγR − , CD34 + ), GMP (lineage − c-Kit + Sca-1 − , FcγR + CD34 + ), and MEP (lineage − c-Kit + Sca-1 − FcγR − CD34 − ) cells at 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as a frequency of live cells. (G) Photographs of spleens from secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after lethal irradiation. (H) Weight of the same spleens as shown in G ( n = 4 mice per genotype). (I) Histopathology of spleens from secondary recipient mice transplanted with Asxl1 -null or WT littermate control whole BM 50 wk after noncompetitive secondary transplantation revealing loss of normal splenic architecture. (J) Photographs of representative femur (top) and tibia (bottom) from secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after noncompetitive secondary transplantation. Bars: (D and I) 50 µm; (J) 2 mm. (K) Relative quantification of CD71 + /Ter119 − and CD71/Ter119 double-positive cells from BM and spleen of secondary recipient mice transplanted with Vav-cre Asxl1 fl/fl or Cre − Asxl1 fl/fl littermate control whole BM 50 wk after noncompetitive secondary transplantation. Frequencies are expressed as a percentage of live cells ( n = 4 mice per genotype examined by FACS analysis). (L) Representative FACS plots of data shown in K from splenocytes. Staining is as shown, and live cells were gated in parent gate. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Transplantation Assay, Mouse Assay, Purification, Histopathology, FACS, Irradiation, Staining

    Generation of a conditional Asxl1 allele and characterization of mice with constitutive Asxl1 loss. (A) Schematic depiction of the targeted Asxl1 allele. Exons 5–10 are targeted and flanked by LoxP sites upon Frt-mediated deletion of the Neo cassette. (B) Verification of correct homologous recombination of Asxl1-targeted allele using Southern blots on targeted ES cells. (C) Enumeration of offspring derived from mating EIIa-cre Asxl1 +/Δ parents. (D and E) Gross pathology (D) and tissue sections (E) of Asxl1 Δ/Δ mice at 14.5 and 18.5 d postcoitus (dpc). (F) Analysis of skeletal preparations from germline Asxl1 -null mice surviving to E20.5 including hypoplastic mandibles (asterisk), lack of hyoid bone (arrowhead), and lower lumbar/sacral posterior homeotic transformations (arrow). (G) Gross phenotype of EIIa-cre Asxl1 +/Δ and littermate control mice on bilateral microphthalmia. Bars: (D) 2 mm; (E and F [top]) 1 mm; (F, bottom) 2.5 mm; (G) 0.5 cm. (H) Immunophenotyping of fetal liver at 14.5 dpc on relative frequency of LSK cells, MPP cells (LSK, CD48 + , CD150 − cells), and LT-HSCs (LSK, CD48 − , CD150 + cells) between mice with germline loss of 0, 1, or 2 copies of Asxl1 . FACS analysis was performed with three to five independent fetal liver samples per genotype. (I) FACS analysis of fetal liver at 14.5 dpc reveals relative frequency of CD71 + single-positive, CD71/Ter119 double-positive, or Ter119 single-positive cells with constitutive loss of Asxl1 . Antibody stainings are as indicated, and cells were gated on live cells in the parent gate. Error bars represent ±SD.

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Generation of a conditional Asxl1 allele and characterization of mice with constitutive Asxl1 loss. (A) Schematic depiction of the targeted Asxl1 allele. Exons 5–10 are targeted and flanked by LoxP sites upon Frt-mediated deletion of the Neo cassette. (B) Verification of correct homologous recombination of Asxl1-targeted allele using Southern blots on targeted ES cells. (C) Enumeration of offspring derived from mating EIIa-cre Asxl1 +/Δ parents. (D and E) Gross pathology (D) and tissue sections (E) of Asxl1 Δ/Δ mice at 14.5 and 18.5 d postcoitus (dpc). (F) Analysis of skeletal preparations from germline Asxl1 -null mice surviving to E20.5 including hypoplastic mandibles (asterisk), lack of hyoid bone (arrowhead), and lower lumbar/sacral posterior homeotic transformations (arrow). (G) Gross phenotype of EIIa-cre Asxl1 +/Δ and littermate control mice on bilateral microphthalmia. Bars: (D) 2 mm; (E and F [top]) 1 mm; (F, bottom) 2.5 mm; (G) 0.5 cm. (H) Immunophenotyping of fetal liver at 14.5 dpc on relative frequency of LSK cells, MPP cells (LSK, CD48 + , CD150 − cells), and LT-HSCs (LSK, CD48 − , CD150 + cells) between mice with germline loss of 0, 1, or 2 copies of Asxl1 . FACS analysis was performed with three to five independent fetal liver samples per genotype. (I) FACS analysis of fetal liver at 14.5 dpc reveals relative frequency of CD71 + single-positive, CD71/Ter119 double-positive, or Ter119 single-positive cells with constitutive loss of Asxl1 . Antibody stainings are as indicated, and cells were gated on live cells in the parent gate. Error bars represent ±SD.

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Mouse Assay, Homologous Recombination, Derivative Assay, FACS

    Combined loss of Asxl1 and Tet2 rescues the impaired self-renewal of Asxl1 -deficient HSCs. (A) Enumeration of colonies and serial replating capacity of 20,000 whole BM cells from 6-wk-old littermate mice with hematopoietic-specific deletion of Asxl1 ( Vav-cre Asxl1 fl/fl ), Tet2 (Vav-cre Tet2 fl/fl ), or both ( Vav-cre Asxl1 fl/fl Tet2 fl/fl ). (B) Schematic depiction of the competitive transplantation experiment. Control, Vav-cre Asxl1 fl/fl , Vav-cre Tet2 fl/fl , and Vav-cre Asxl1 fl/fl Tet2 fl/fl cells are positive for CD45.2, whereas WT competitor cells are positive for CD45.1. On the right, monthly assessment of donor chimerism in the peripheral blood of recipient animals is shown up to 16 wk after transplant ( n = 5 recipient mice were used for each genotype and experiment was performed in biological duplicate). 16-wk chimerism was significantly higher in Tet2 −/− transplanted mice compared with all other genotypes. (C) Representative FACS analysis of peripheral blood of mice transplanted with each genotype at 16 wk. Staining schemes are as indicated and parental gate was live cells. (D) Proportion of CD45.2 + peripheral blood cells of each lineage at 16 wk in mice transplanted with each genotype ( n = 5 mice analyzed for each genotype) as determined by FACS analysis. Each competitive transplantation experiment was performed in biological duplicate with five recipient mice per genotype in each experiment. Error bars represent ±SD; *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

    doi: 10.1084/jem.20131141

    Figure Lengend Snippet: Combined loss of Asxl1 and Tet2 rescues the impaired self-renewal of Asxl1 -deficient HSCs. (A) Enumeration of colonies and serial replating capacity of 20,000 whole BM cells from 6-wk-old littermate mice with hematopoietic-specific deletion of Asxl1 ( Vav-cre Asxl1 fl/fl ), Tet2 (Vav-cre Tet2 fl/fl ), or both ( Vav-cre Asxl1 fl/fl Tet2 fl/fl ). (B) Schematic depiction of the competitive transplantation experiment. Control, Vav-cre Asxl1 fl/fl , Vav-cre Tet2 fl/fl , and Vav-cre Asxl1 fl/fl Tet2 fl/fl cells are positive for CD45.2, whereas WT competitor cells are positive for CD45.1. On the right, monthly assessment of donor chimerism in the peripheral blood of recipient animals is shown up to 16 wk after transplant ( n = 5 recipient mice were used for each genotype and experiment was performed in biological duplicate). 16-wk chimerism was significantly higher in Tet2 −/− transplanted mice compared with all other genotypes. (C) Representative FACS analysis of peripheral blood of mice transplanted with each genotype at 16 wk. Staining schemes are as indicated and parental gate was live cells. (D) Proportion of CD45.2 + peripheral blood cells of each lineage at 16 wk in mice transplanted with each genotype ( n = 5 mice analyzed for each genotype) as determined by FACS analysis. Each competitive transplantation experiment was performed in biological duplicate with five recipient mice per genotype in each experiment. Error bars represent ±SD; *, P

    Article Snippet: The antibody used for Asxl1 ChIP-Seq experiments was obtained from Santa Cruz Biotechnology, Inc. ChIP was performed as described previously ( ).

    Techniques: Mouse Assay, Transplantation Assay, FACS, Staining

    R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation, Gene Knockout

    Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Chromatin Immunoprecipitation, Gene Knockout, Expressing

    H3K9me2 and H3 levels over mouse β-actin gene in G9a/GLP double KO mouse ES cells and Ago2 KO MEFs a . Top and bottom panels: H3K9me2 and H3 ChIP performed on mouse β-actin gene in wild type and G9a/GLP double KO ES cells. H3K9me2 occupancy depends on presence of G9a/GLP HKMTs. Right panels: H3K9me2 and H3 ChIP performed on mouse major satellites in wild type and G9a/GLP KO cells. b . ChIP analyses using H3K9me2 (top panel) and H3 (bottom panel) antibodies performed on mouse β-actin gene in wild type and Ago2 KO cells. ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: H3K9me2 and H3 levels over mouse β-actin gene in G9a/GLP double KO mouse ES cells and Ago2 KO MEFs a . Top and bottom panels: H3K9me2 and H3 ChIP performed on mouse β-actin gene in wild type and G9a/GLP double KO ES cells. H3K9me2 occupancy depends on presence of G9a/GLP HKMTs. Right panels: H3K9me2 and H3 ChIP performed on mouse major satellites in wild type and G9a/GLP KO cells. b . ChIP analyses using H3K9me2 (top panel) and H3 (bottom panel) antibodies performed on mouse β-actin gene in wild type and Ago2 KO cells. ChIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Gene Knockout, Chromatin Immunoprecipitation

    Cellular localisation of R-loops, dsRNA and H3K9me2 a. Immunofluorescence imaging of dsRNA (J2 antibody) and R-loops (S9.6 antibody), using paraformaldehyde (PFA) and methanol (MeOH) as fixing reagents. Fixation with methanol allowed visualisation of R-loops and dsRNA in HeLa cell nuclei. Enlarged boxes (1 and 2) shown in right panels. b. Whole cell images showing immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Enlarged versions (1 and 2) are shown in Fig. 2h .

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Cellular localisation of R-loops, dsRNA and H3K9me2 a. Immunofluorescence imaging of dsRNA (J2 antibody) and R-loops (S9.6 antibody), using paraformaldehyde (PFA) and methanol (MeOH) as fixing reagents. Fixation with methanol allowed visualisation of R-loops and dsRNA in HeLa cell nuclei. Enlarged boxes (1 and 2) shown in right panels. b. Whole cell images showing immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Enlarged versions (1 and 2) are shown in Fig. 2h .

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Immunofluorescence, Imaging

    R-loops and H3K9me2 repressive mark are not specifically enriched over the CoTC terminators of human Cyclin B1 and Akirin 1 genes a . DIP on endogenous Cyclin B1 and Akirin 1 genes. No detection of R-loops was observed over their CoTC terminators. Human β-actin gene was used as a positive control. For Cyclin B1 and Akirin 1 genes, 3′ end 1 and 3′ end 2 amplicons amplify two different regions within the CoTC terminator of each gene. 3′ end 1 and 3′ end 2 amplicons for β-actin gene amplify the 5′ pause and pause amplicons, respectively. b . Ratio of H3K9me2 signal over the 3′ ends versus intron 3 signal in Cyclin B1, Akirin 1 and β-actin human genes. DIP and ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: R-loops and H3K9me2 repressive mark are not specifically enriched over the CoTC terminators of human Cyclin B1 and Akirin 1 genes a . DIP on endogenous Cyclin B1 and Akirin 1 genes. No detection of R-loops was observed over their CoTC terminators. Human β-actin gene was used as a positive control. For Cyclin B1 and Akirin 1 genes, 3′ end 1 and 3′ end 2 amplicons amplify two different regions within the CoTC terminator of each gene. 3′ end 1 and 3′ end 2 amplicons for β-actin gene amplify the 5′ pause and pause amplicons, respectively. b . Ratio of H3K9me2 signal over the 3′ ends versus intron 3 signal in Cyclin B1, Akirin 1 and β-actin human genes. DIP and ChIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Positive Control, Chromatin Immunoprecipitation

    Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Over Expression, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunofluorescence

    Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Positive Control, Quantitative RT-PCR, Chromatin Immunoprecipitation

    H3K9me2 and H3 levels over human β-actin gene a . Left panel: H3K9me2 ChIP on β-actin gene. Right panel: H3K9me2 ChIP analysis on human centromere 9 (positive control). b . Left panel: H3 ChIP on β-actin gene. Right panel: H3 ChIP analysis on human centromere 9. c . Left panel: H3K9me2 ChIP +/− BIX treatment. Right panel: H3 ChIP +/− BIX treatment. ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: H3K9me2 and H3 levels over human β-actin gene a . Left panel: H3K9me2 ChIP on β-actin gene. Right panel: H3K9me2 ChIP analysis on human centromere 9 (positive control). b . Left panel: H3 ChIP on β-actin gene. Right panel: H3 ChIP analysis on human centromere 9. c . Left panel: H3K9me2 ChIP +/− BIX treatment. Right panel: H3 ChIP +/− BIX treatment. ChIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Chromatin Immunoprecipitation, Positive Control

    RNAi-dependent H3K9me2 repressive mark formed over human β-actin terminator in HeLa cells a . RT-qPCR of β-actin antisense transcription. RT with region-specific forward primers. b . Sense and antisense transcripts levels by RT-qPCR from J2 immuno-selected dsRNA. Samples either untreated (grey bars), treated with V1 RNase (black bars) or S1 nuclease (white bars). All RT-qPCR values are average +/− SD from three to four biological repeats. c-e, g . ChIP analysis using Dicer, Ago1, G9a and HP1γ antibodies respectively. f . Ratio of H3K9me2 ChIP versus H3 on β-actin gene and centromere 9 (right panel). ChIP values +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: RNAi-dependent H3K9me2 repressive mark formed over human β-actin terminator in HeLa cells a . RT-qPCR of β-actin antisense transcription. RT with region-specific forward primers. b . Sense and antisense transcripts levels by RT-qPCR from J2 immuno-selected dsRNA. Samples either untreated (grey bars), treated with V1 RNase (black bars) or S1 nuclease (white bars). All RT-qPCR values are average +/− SD from three to four biological repeats. c-e, g . ChIP analysis using Dicer, Ago1, G9a and HP1γ antibodies respectively. f . Ratio of H3K9me2 ChIP versus H3 on β-actin gene and centromere 9 (right panel). ChIP values +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation

    H3K9me2, H3K9me3 and H3 levels over the endogenous mouse β-actin gene a . H3K9me2 and H3K9me3 ChIP on mouse β-actin gene in MEF cells. Right panel: H3K9me2 and H3K9me3 ChIP on mouse major satellites (positive control). b . Total H3 ChIP on mouse β-actin gene. Major satellites were used as a positive control. ChIP values are +/− SD from three biological repeats.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: H3K9me2, H3K9me3 and H3 levels over the endogenous mouse β-actin gene a . H3K9me2 and H3K9me3 ChIP on mouse β-actin gene in MEF cells. Right panel: H3K9me2 and H3K9me3 ChIP on mouse major satellites (positive control). b . Total H3 ChIP on mouse β-actin gene. Major satellites were used as a positive control. ChIP values are +/− SD from three biological repeats.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques: Chromatin Immunoprecipitation, Positive Control

    Model for how R-loops and RNAi-dependent H3K9me2 chromatin mediate pause-dependent transcriptional termination in mammalian genes Mammalian genes possessing pause elements downstream of their PAS form R-loops in termination regions. This facilitates generation of an antisense transcript that hybridises with the sense transcript to form dsRNA. This triggers recruitment of the RNAi factors, Dicer, Ago1 and Ago2. G9a/GLP HKMTs and HP1γ are then recruited forming and maintaining H3K9me2 repressive marks. R-loops and H3K9me2 facilitate Pol II pausing prior to termination. DNA is shown as grey lines and RNA as a red line. Points of contact between the DNA strand and nascent RNA indicates R-loop formation, whereas points of contact between sense and antisense RNA indicate dsRNA formation. Pol II is shown as a blue icon with arrow indicating transcription direction. Nucleosomes are shown in green except over H3K9me2 region where they are coloured red.

    Journal: Nature

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    doi: 10.1038/nature13787

    Figure Lengend Snippet: Model for how R-loops and RNAi-dependent H3K9me2 chromatin mediate pause-dependent transcriptional termination in mammalian genes Mammalian genes possessing pause elements downstream of their PAS form R-loops in termination regions. This facilitates generation of an antisense transcript that hybridises with the sense transcript to form dsRNA. This triggers recruitment of the RNAi factors, Dicer, Ago1 and Ago2. G9a/GLP HKMTs and HP1γ are then recruited forming and maintaining H3K9me2 repressive marks. R-loops and H3K9me2 facilitate Pol II pausing prior to termination. DNA is shown as grey lines and RNA as a red line. Points of contact between the DNA strand and nascent RNA indicates R-loop formation, whereas points of contact between sense and antisense RNA indicate dsRNA formation. Pol II is shown as a blue icon with arrow indicating transcription direction. Nucleosomes are shown in green except over H3K9me2 region where they are coloured red.

    Article Snippet: The following antibodies were used for ChIP: anti-H3K9me2 (Abcam), anti-H3K9me3 (Abcam), anti-H3 (Abcam), anti-Dicer (13D6) (Abcam), anti-KMT1C/G9a (Abcam), anti-Ago1 (Millipore), anti-Ago2 (Abcam) and anti-Pol II (H-224) (Santa Cruz Biotechnology).

    Techniques:

    Schematic illustrating three models for ZNF217 and CtBP2 interaction

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Schematic illustrating three models for ZNF217 and CtBP2 interaction

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    Localization of ZNF217-binding sites in the ENCODE regions

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Localization of ZNF217-binding sites in the ENCODE regions

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques: Binding Assay

    Identification of ZNF217 target genes using promoter arrays

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Identification of ZNF217 target genes using promoter arrays

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 can function as a transcriptional repressor

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 can function as a transcriptional repressor

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 can function as a transcriptional repressor

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 can function as a transcriptional repressor

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 can function as a transcriptional repressor

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 can function as a transcriptional repressor

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    De novo motif analysis using ZNF217 ChIP-chip data

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: De novo motif analysis using ZNF217 ChIP-chip data

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques: Chromatin Immunoprecipitation

    Identification of ZNF217 target promoters in Ntera2 cells

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Identification of ZNF217 target promoters in Ntera2 cells

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    Cell type-specificity of ZNF217 binding

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Cell type-specificity of ZNF217 binding

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques: Binding Assay

    Characterization of ZNF217 targets expressed at different levels

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Characterization of ZNF217 targets expressed at different levels

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 and CtBP co-localize at promoters

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 and CtBP co-localize at promoters

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 is down-regulated by retinoic acid in Ntera2cells

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 is down-regulated by retinoic acid in Ntera2cells

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    ZNF217 can function as a transcriptional repressor

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: ZNF217 can function as a transcriptional repressor

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    Identification of ZNF217 target genes using promoter arrays

    Journal:

    Article Title: Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays

    doi: 10.1074/jbc.M611752200

    Figure Lengend Snippet: Identification of ZNF217 target genes using promoter arrays

    Article Snippet: For ZNF217 knockdown ChIP assays, ZNF217 siRNA (SMARTpool; Dharmacon, cat# M-004987-00) or si-GLO RISC-Free (Dharmacon, cat# D-001600-01) as a non-specific control, was transiently transfected into Ntera2 cells (100nM) plated on 100mm dishes.

    Techniques:

    The Brf2 Molecular Pin (A) The Brf2 TBP anchor domain but not the molecular pin is essential for Brf2-TBP interaction in absence of the DNA, as shown by a pull-down assay. (B) The Brf2 TBP anchor domain and the molecular pin are essential for the formation of a Brf2-TBP/DNA complex, as shown in an EMSA. (C) Close-up view of the Brf2 molecular pin at the interface between the Brf2 C-cyclin repeat, TBP, and the DNA. See also Figures S3 and S7 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: The Brf2 Molecular Pin (A) The Brf2 TBP anchor domain but not the molecular pin is essential for Brf2-TBP interaction in absence of the DNA, as shown by a pull-down assay. (B) The Brf2 TBP anchor domain and the molecular pin are essential for the formation of a Brf2-TBP/DNA complex, as shown in an EMSA. (C) Close-up view of the Brf2 molecular pin at the interface between the Brf2 C-cyclin repeat, TBP, and the DNA. See also Figures S3 and S7 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Pull Down Assay

    Brf2 Redox Regulation (A) Close-up view of C361 at the ternary interface between the Brf2 C-cyclin repeat, TBP, and the upstream edge of the TATA box. Yellow dots represent the van der Waals radius of the sulfur atom. (B) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with the alkylating agent iodoacetamide. The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (C) Representative EMSA of Brf2-TBP/DNA complexes upon removal of reducing agent (DTT) and incubation over time. The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (D) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with H 2 O 2 . The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (E) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with gradients of oxidized/reduced glutathione (GSSG:GSH). The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. See also Figures S3 , S4 , and S7 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Brf2 Redox Regulation (A) Close-up view of C361 at the ternary interface between the Brf2 C-cyclin repeat, TBP, and the upstream edge of the TATA box. Yellow dots represent the van der Waals radius of the sulfur atom. (B) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with the alkylating agent iodoacetamide. The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (C) Representative EMSA of Brf2-TBP/DNA complexes upon removal of reducing agent (DTT) and incubation over time. The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (D) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with H 2 O 2 . The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. (E) Representative EMSA of Brf2-TBP/DNA complexes upon pre-incubation of Brf2 proteins with gradients of oxidized/reduced glutathione (GSSG:GSH). The IC band was used for loading normalization. ∗ Indicates addition of the reducing agent after the oxidative treatment during complex assembly. See also Figures S3 , S4 , and S7 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Incubation

    Architecture of the Human Pol III PIC Model of a Pol III PIC ( Vannini and Cramer, 2012 ) generated using the Brf2-TBP/DNA complex reveals that the path of the downstream DNA points toward the Pol III-specific subunits C39 and C62, and resembles the path observed in yeast and human Pol II PIC ( He et al., 2013 , Mühlbacher et al., 2014 ). See also Figure S7 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Architecture of the Human Pol III PIC Model of a Pol III PIC ( Vannini and Cramer, 2012 ) generated using the Brf2-TBP/DNA complex reveals that the path of the downstream DNA points toward the Pol III-specific subunits C39 and C62, and resembles the path observed in yeast and human Pol II PIC ( He et al., 2013 , Mühlbacher et al., 2014 ). See also Figure S7 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Generated

    Mass Spectrometry Analysis of Brf2 Redox Modifications, Related to Figure 5 Biologically relevant oxidation states of C361 were confirmed by MS/MS as either unmodified, trapped with glutathione (-SS-Glu) or dimedone (Dmd). The precursor ions, errors and ion scores are indicated below the annotated fragmentation mass spectra. For clarity, only prominent fragment ions are marked.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Mass Spectrometry Analysis of Brf2 Redox Modifications, Related to Figure 5 Biologically relevant oxidation states of C361 were confirmed by MS/MS as either unmodified, trapped with glutathione (-SS-Glu) or dimedone (Dmd). The precursor ions, errors and ion scores are indicated below the annotated fragmentation mass spectra. For clarity, only prominent fragment ions are marked.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Mass Spectrometry

    Brf2-Dependent Reduction of SeCys p-tRNA and Enhanced Apoptosis in A549 Cells, Related to Figure 7 (A) Two individual Brf2 siRNAs cause a severe reduction of SeCys p-tRNA in A549 cells challenged with t-BHP, an effect that is fully rescued by concomitant overexpression of a siRNA resistant form of Brf2. (B) Two individual Brf2 siRNAs elicit a strong sensitization towards t-BHP in A549 cells, an effect that is fully rescued by concomitant overexpression of a siRNA resistant form of Brf2.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Brf2-Dependent Reduction of SeCys p-tRNA and Enhanced Apoptosis in A549 Cells, Related to Figure 7 (A) Two individual Brf2 siRNAs cause a severe reduction of SeCys p-tRNA in A549 cells challenged with t-BHP, an effect that is fully rescued by concomitant overexpression of a siRNA resistant form of Brf2. (B) Two individual Brf2 siRNAs elicit a strong sensitization towards t-BHP in A549 cells, an effect that is fully rescued by concomitant overexpression of a siRNA resistant form of Brf2.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Over Expression

    Selenoproteins Levels and Resistance to Oxidative Stress Are Regulated in a Brf2-Dependent Manner (A) Manipulation of Brf2 protein levels affects selenoproteins expression levels during oxidative stress in MRC5 and A549 cells. In the upper insets, a western blot analysis of Nrf2 confirms induction of oxidative stress with 50 μM and 100 μM t-BHP in MRC5 and A549 cells, respectively. A western blot analysis of Brf2 immunoprecipitation from 10 7 MRC5 or A549 cells is shown in the lower insets (IP). (B) Overexpression of Brf2 in MRC5 cells challenged with t-BHP results in decreased apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. (C) Effects of overexpression of Brf2 wild-type and mutants on acquired resistance to apoptosis in MRC5 cells as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. (D) Lowering Brf2 protein levels by siRNA in A549 cells challenged with t-BHP results in an increased cellular commitment to apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. Inset: a western blot analysis of Brf2 immunoprecipitation from 10 7 A549 cells shows siRNA-induced Brf2 protein level reduction. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. See also Figures S5 and S7 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Selenoproteins Levels and Resistance to Oxidative Stress Are Regulated in a Brf2-Dependent Manner (A) Manipulation of Brf2 protein levels affects selenoproteins expression levels during oxidative stress in MRC5 and A549 cells. In the upper insets, a western blot analysis of Nrf2 confirms induction of oxidative stress with 50 μM and 100 μM t-BHP in MRC5 and A549 cells, respectively. A western blot analysis of Brf2 immunoprecipitation from 10 7 MRC5 or A549 cells is shown in the lower insets (IP). (B) Overexpression of Brf2 in MRC5 cells challenged with t-BHP results in decreased apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. (C) Effects of overexpression of Brf2 wild-type and mutants on acquired resistance to apoptosis in MRC5 cells as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. (D) Lowering Brf2 protein levels by siRNA in A549 cells challenged with t-BHP results in an increased cellular commitment to apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. Inset: a western blot analysis of Brf2 immunoprecipitation from 10 7 A549 cells shows siRNA-induced Brf2 protein level reduction. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis. See also Figures S5 and S7 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Immunoprecipitation, Over Expression, FACS, Staining

    Brf2 is a Redox Sensor in Living Cells, Related to Figures 6 and 7 (A) SeCys m-tRNA levels are reduced during oxidative stress in a Brf2-dependent manner, as monitored via four-leaf clover PCR (Honda et al., 2015). Samples labeled empty vector and Brf2 represent transient over-expressions. (B) Overexpression of Brf2 in MCF10A cells challenged with t-BHP results in decreased apoptosis as measured by FACs analysis via Annexin V-FITC/PI staining. (C) Overexpression of Brf2 affects selenoproteins expression levels during oxidative stress in MCF10A cells. (D) Effects of overexpression of Brf2 and Brf2 mutants on acquired resistance to apoptosis in MCF10A cells as measured by FACs analysis via Annexin V-FITC/PI staining.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Brf2 is a Redox Sensor in Living Cells, Related to Figures 6 and 7 (A) SeCys m-tRNA levels are reduced during oxidative stress in a Brf2-dependent manner, as monitored via four-leaf clover PCR (Honda et al., 2015). Samples labeled empty vector and Brf2 represent transient over-expressions. (B) Overexpression of Brf2 in MCF10A cells challenged with t-BHP results in decreased apoptosis as measured by FACs analysis via Annexin V-FITC/PI staining. (C) Overexpression of Brf2 affects selenoproteins expression levels during oxidative stress in MCF10A cells. (D) Effects of overexpression of Brf2 and Brf2 mutants on acquired resistance to apoptosis in MCF10A cells as measured by FACs analysis via Annexin V-FITC/PI staining.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Polymerase Chain Reaction, Labeling, Plasmid Preparation, Over Expression, FACS, Staining, Expressing

    Brf2/DNA Sequence-Specific Interactions (A) Close-up view of the TATA box (yellow), downstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively. (B) Substitutions at positions +3 and +4 of the wild-type (circled in red) U6-2 promoter decrease binding of a R110A mutant, in particular when a T nucleobase is present at position +3 on the non-template strand (in cyan). R110A versus wild-type (WT) is the ratio between the percentage of binding of the mutant versus wild-type Brf2 proteins. (C) Close-up view of the TATA box (yellow), upstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively. (D) Substitutions at positions −3 and −4 of the wild-type (circled in red) U6-2 promoter reveal more efficient complex formation with a pyrimidine nucleobase and a C nucleobase at positions −3 and −4 of the template strand, respectively. (B and D) The intensity of the complex formed with TBP, U6-2 non mutated sequence and wild-type Brf2 (lane 1) was used as a reference for relative quantification. ∗ Indicates samples that were quantified relative to a distinct wild-type sequence reference not shown on the figure. Representative gels of three independent experiments. The data shown are the mean values and SE of three independent experiments. In the insets, 10 μl of a typical binding reaction (25 μl total) with Brf2 wild-type or Brf2 mutants were loaded on a SDS-PAGE gel and stained with Coomassie-blue, confirming that equal amounts of protein of comparable quality were used for EMSA assays. See also Figures S2 and S7 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Brf2/DNA Sequence-Specific Interactions (A) Close-up view of the TATA box (yellow), downstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively. (B) Substitutions at positions +3 and +4 of the wild-type (circled in red) U6-2 promoter decrease binding of a R110A mutant, in particular when a T nucleobase is present at position +3 on the non-template strand (in cyan). R110A versus wild-type (WT) is the ratio between the percentage of binding of the mutant versus wild-type Brf2 proteins. (C) Close-up view of the TATA box (yellow), upstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively. (D) Substitutions at positions −3 and −4 of the wild-type (circled in red) U6-2 promoter reveal more efficient complex formation with a pyrimidine nucleobase and a C nucleobase at positions −3 and −4 of the template strand, respectively. (B and D) The intensity of the complex formed with TBP, U6-2 non mutated sequence and wild-type Brf2 (lane 1) was used as a reference for relative quantification. ∗ Indicates samples that were quantified relative to a distinct wild-type sequence reference not shown on the figure. Representative gels of three independent experiments. The data shown are the mean values and SE of three independent experiments. In the insets, 10 μl of a typical binding reaction (25 μl total) with Brf2 wild-type or Brf2 mutants were loaded on a SDS-PAGE gel and stained with Coomassie-blue, confirming that equal amounts of protein of comparable quality were used for EMSA assays. See also Figures S2 and S7 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Sequencing, Binding Assay, Mutagenesis, SDS Page, Staining

    Sequence Alignments of Brf2 Promoters and Protein, Related to Figures 1 and 3 (A) Brf2-dependent promoters (40 nucleobase long centered around putative TATA boxes) were aligned using MEME (Bailey et al., 2009) by searching for a 16 nucleobase long consensus motif. (B) Sequence and domain conservation between Brf2, Brf1 and TFIIB. Color-coding is as in Fig. 1. Brf2-Brf1 alignments are based on sequence conservation, while Brf2-TFIIB is based on a structural alignment. (C) Two views of the specific interaction of Brf2 R110 and A108 with a TG (U6-2 in pale green) and TC (RPPH1 in light grey) dinucleotide step. In presence of the TG sequence, the T on the nontemplate strand (in red) is left unstacked at its downstream edge. In presence of a TC sequence, no local distortions of the DNA are observed. The two structures were superimposed by structurally aligning the Brf2 N-terminal cyclin repeats.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Sequence Alignments of Brf2 Promoters and Protein, Related to Figures 1 and 3 (A) Brf2-dependent promoters (40 nucleobase long centered around putative TATA boxes) were aligned using MEME (Bailey et al., 2009) by searching for a 16 nucleobase long consensus motif. (B) Sequence and domain conservation between Brf2, Brf1 and TFIIB. Color-coding is as in Fig. 1. Brf2-Brf1 alignments are based on sequence conservation, while Brf2-TFIIB is based on a structural alignment. (C) Two views of the specific interaction of Brf2 R110 and A108 with a TG (U6-2 in pale green) and TC (RPPH1 in light grey) dinucleotide step. In presence of the TG sequence, the T on the nontemplate strand (in red) is left unstacked at its downstream edge. In presence of a TC sequence, no local distortions of the DNA are observed. The two structures were superimposed by structurally aligning the Brf2 N-terminal cyclin repeats.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Sequencing

    Mechanism of the Redox-Dependent Brf2 Blockade during Oxidative Stress and Carcinogenesis, Related to Figures 1 , 2 , 3 , 4 , 5 , 6 , and 7 During normal growth conditions (A) Brf2- and Nrf2-dependent transcripts are synthesized at basal levels. Upon moderate oxidative stress (B), Nrf2 is activated and Nrf2-dependent transcripts upregulated. Concomitantly, Brf2-dependent transcription, including SeCys tRNAs, is rapidly downregulated via redox-dependent modifications of Brf2. The pre-existing pool of SeCys tRNA is sufficient to sustain synthesis of selenoproteins. Upon prolonged oxidative stress (C), SeCys tRNA levels become limiting while, simultaneously, selenoprotein’s mRNAs continue to be highly upregulated by Nrf2. In this scenario, compromised synthesis of selenoproteins drives the cells into apoptosis. In cancer cells (D), the Nrf2 pathway is constitutively activated and contributes to the observed resistance of cancerous cells to higher than normal concentrations of reactive oxygen species. Under these circumstances, Brf2 overexpression is required to overcome the innate redoxdependent blockade, ensuring elevated synthesis of SeCys tRNAs and, ultimately, enabling cancer cells to evade apoptosis under prolonged oxidative stress.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Mechanism of the Redox-Dependent Brf2 Blockade during Oxidative Stress and Carcinogenesis, Related to Figures 1 , 2 , 3 , 4 , 5 , 6 , and 7 During normal growth conditions (A) Brf2- and Nrf2-dependent transcripts are synthesized at basal levels. Upon moderate oxidative stress (B), Nrf2 is activated and Nrf2-dependent transcripts upregulated. Concomitantly, Brf2-dependent transcription, including SeCys tRNAs, is rapidly downregulated via redox-dependent modifications of Brf2. The pre-existing pool of SeCys tRNA is sufficient to sustain synthesis of selenoproteins. Upon prolonged oxidative stress (C), SeCys tRNA levels become limiting while, simultaneously, selenoprotein’s mRNAs continue to be highly upregulated by Nrf2. In this scenario, compromised synthesis of selenoproteins drives the cells into apoptosis. In cancer cells (D), the Nrf2 pathway is constitutively activated and contributes to the observed resistance of cancerous cells to higher than normal concentrations of reactive oxygen species. Under these circumstances, Brf2 overexpression is required to overcome the innate redoxdependent blockade, ensuring elevated synthesis of SeCys tRNAs and, ultimately, enabling cancer cells to evade apoptosis under prolonged oxidative stress.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Synthesized, Over Expression

    Brf2-Dependent Transcription Is Redox Regulated in Living Cells (A) qRT-PCR analysis shows that Brf2-dependent-transcripts (SeCys p-tRNA, RPPH1, RNA7SK, and U6 snRNA) are globally downregulated during oxidative stress, while a Brf1-dependent transcript (Leu p-tRNA) remains unchanged. (B) SeCys p-tRNA levels are strongly reduced in cells challenged with t-BHP relative to the unchallenged cells (as highlighted by gray and black lines, respectively) in a dose- and time-dependent manner, as measured by qRT-PCR. (C) SeCys p-tRNA levels rapidly recover upon removal of the exogenous oxidative stress inducer, as measured by qRT-PCR. Wash indicates replacement of media containing t-BHP with fresh media. (D) Effects of overexpression of Brf2 and Brf2 mutants (inset) on SeCys p-tRNA levels during oxidative stress, as measured by qRT-PCR. The numbers indicated on the histograms represent the percentage of reduction of selenocysteine tRNA levels, while if numbers are indicated above the histograms they represent the percentage of increase. Cumulative data of at least three experiments, mean + SEM. Unpaired t test: ∗ p

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Brf2-Dependent Transcription Is Redox Regulated in Living Cells (A) qRT-PCR analysis shows that Brf2-dependent-transcripts (SeCys p-tRNA, RPPH1, RNA7SK, and U6 snRNA) are globally downregulated during oxidative stress, while a Brf1-dependent transcript (Leu p-tRNA) remains unchanged. (B) SeCys p-tRNA levels are strongly reduced in cells challenged with t-BHP relative to the unchallenged cells (as highlighted by gray and black lines, respectively) in a dose- and time-dependent manner, as measured by qRT-PCR. (C) SeCys p-tRNA levels rapidly recover upon removal of the exogenous oxidative stress inducer, as measured by qRT-PCR. Wash indicates replacement of media containing t-BHP with fresh media. (D) Effects of overexpression of Brf2 and Brf2 mutants (inset) on SeCys p-tRNA levels during oxidative stress, as measured by qRT-PCR. The numbers indicated on the histograms represent the percentage of reduction of selenocysteine tRNA levels, while if numbers are indicated above the histograms they represent the percentage of increase. Cumulative data of at least three experiments, mean + SEM. Unpaired t test: ∗ p

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Quantitative RT-PCR, Over Expression

    General Conservation of the Architecture of TFIIB and TFIIB-like Factors, Related to Figure 1 (A) Schematic of the architecture of the Brf2-TBP/DNA complexes and sequences of the DNA scaffold used for crystallization. (B) Superimposition of Brf2-TBP/DNA and TFIIB-TBP/DNA (PDB: 1C9B ). The structures were superimposed using TBP as a template for structural alignment. (C) Superimposition of the Brf2-TBP/U6-2 (blue), Brf2-TBP/TRNAU1 (grey) and Brf2-TBP/RPPH1 (green) structures. The structures were superimposed using TBP as a template for structural alignment. (D) Final electron density contoured at 1.2 s surrounding a tract of double-stranded DNA. (E) Brf2 sequence conservation and domain organization of Homo sapiens (H.s.) Mus musculus (M.m.), and Danio rerio (D.r.).

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: General Conservation of the Architecture of TFIIB and TFIIB-like Factors, Related to Figure 1 (A) Schematic of the architecture of the Brf2-TBP/DNA complexes and sequences of the DNA scaffold used for crystallization. (B) Superimposition of Brf2-TBP/DNA and TFIIB-TBP/DNA (PDB: 1C9B ). The structures were superimposed using TBP as a template for structural alignment. (C) Superimposition of the Brf2-TBP/U6-2 (blue), Brf2-TBP/TRNAU1 (grey) and Brf2-TBP/RPPH1 (green) structures. The structures were superimposed using TBP as a template for structural alignment. (D) Final electron density contoured at 1.2 s surrounding a tract of double-stranded DNA. (E) Brf2 sequence conservation and domain organization of Homo sapiens (H.s.) Mus musculus (M.m.), and Danio rerio (D.r.).

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Crystallization Assay, Sequencing

    Structure of the Brf2-TBP/DNA Ternary Complex (A) Overview of the Brf2-TBP/U6 promoter structure. DNA template and non-template strands are in blue and cyan respectively. Dashed lines represent disordered regions or regions not present in the crystallization construct. (B) Schematic of Brf2 domain organization. See also Figures S1 , S2 , and S7 and Tables S1 and S2 .

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Structure of the Brf2-TBP/DNA Ternary Complex (A) Overview of the Brf2-TBP/U6 promoter structure. DNA template and non-template strands are in blue and cyan respectively. Dashed lines represent disordered regions or regions not present in the crystallization construct. (B) Schematic of Brf2 domain organization. See also Figures S1 , S2 , and S7 and Tables S1 and S2 .

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Crystallization Assay, Construct

    Modular Functions of the Brf2 CTD, Related to Figures 4 and 5 (A) EMSA with serial Brf2 C-terminal deletion mutants showing that the region comprised between residues 289-311 of Brf2 is involved in direct binding to the upstream transcription factor SNAPc. (B) A conserved surface of TBP is utilized by different TBP associated factors. The TBP surface buried upon interaction with the associated factor is colored in pink for human Brf2 (orange), in cyan for yeast Brf1 (PDB id: 1NGM , turquoise), in green for yeast TFIIA (PDB id: 1NH2 , green) and in blue for yeast TAF1 (PDB id: 4B0A , blue). (C) Structural conservation between Brf2 C361 part of the molecular pin, and C59 part of a short helical motif of the p-50 subunit of the NF-kB transcription factor (PDB id: 1NFK ). (D) Brf2 oxidative-mimic mutation C361D does not hinder Brf2-TBP complex formation in absence of the DNA, as shown by pull-down assay. “IN” indicates the input and “empty resin” the eluted untagged TBP binding non-specifically to the resin. (E) EMSA shows that formation a functional Brf2-TBP/DNA complex is severely impaired in Brf2 oxidative-mimic mutant C361D. “IN” indicates the input and “empty resin” the eluted untagged TBP binding non-specifically to the resin. (F) Fluorescence polarization saturation binding assay shows virtually no reduction in affinity of the Brf2 C361A mutant and an approximately 50-fold reduction in affinity of Brf2 C361D mutant for TBP/DNA complexes.

    Journal: Cell

    Article Title: Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2

    doi: 10.1016/j.cell.2015.11.005

    Figure Lengend Snippet: Modular Functions of the Brf2 CTD, Related to Figures 4 and 5 (A) EMSA with serial Brf2 C-terminal deletion mutants showing that the region comprised between residues 289-311 of Brf2 is involved in direct binding to the upstream transcription factor SNAPc. (B) A conserved surface of TBP is utilized by different TBP associated factors. The TBP surface buried upon interaction with the associated factor is colored in pink for human Brf2 (orange), in cyan for yeast Brf1 (PDB id: 1NGM , turquoise), in green for yeast TFIIA (PDB id: 1NH2 , green) and in blue for yeast TAF1 (PDB id: 4B0A , blue). (C) Structural conservation between Brf2 C361 part of the molecular pin, and C59 part of a short helical motif of the p-50 subunit of the NF-kB transcription factor (PDB id: 1NFK ). (D) Brf2 oxidative-mimic mutation C361D does not hinder Brf2-TBP complex formation in absence of the DNA, as shown by pull-down assay. “IN” indicates the input and “empty resin” the eluted untagged TBP binding non-specifically to the resin. (E) EMSA shows that formation a functional Brf2-TBP/DNA complex is severely impaired in Brf2 oxidative-mimic mutant C361D. “IN” indicates the input and “empty resin” the eluted untagged TBP binding non-specifically to the resin. (F) Fluorescence polarization saturation binding assay shows virtually no reduction in affinity of the Brf2 C361A mutant and an approximately 50-fold reduction in affinity of Brf2 C361D mutant for TBP/DNA complexes.

    Article Snippet: Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody (ab17011, Abcam) covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.

    Techniques: Binding Assay, Mutagenesis, Pull Down Assay, Functional Assay, Fluorescence, Saturation Assay

    Microarray analysis of Nurr1-overexpressing MN9D cells. ( A ) Western blot revealing high levels of Nurr1 protein upon treatment of MN9D- Nurr1 Tet On 13N cells with doxycyclin (Dox). ( B ) The relative percentage increase or decrease of a selection of transcripts

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Microarray analysis of Nurr1-overexpressing MN9D cells. ( A ) Western blot revealing high levels of Nurr1 protein upon treatment of MN9D- Nurr1 Tet On 13N cells with doxycyclin (Dox). ( B ) The relative percentage increase or decrease of a selection of transcripts

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Microarray, Western Blot, Selection

    Expression of Ptpru , Klhl1 and Dlk1 in E14.5 Pitx3 -/- embryos. ( A-D ′) Sagittal sections of Pitx3 +/+ (A-D) and Pitx3 -/- (A′-D′) mouse embryos demonstrating expression of Nurr1 (A), Ptpru (B), Klhl1 (C) and Dlk1 (D). Expression of

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Expression of Ptpru , Klhl1 and Dlk1 in E14.5 Pitx3 -/- embryos. ( A-D ′) Sagittal sections of Pitx3 +/+ (A-D) and Pitx3 -/- (A′-D′) mouse embryos demonstrating expression of Nurr1 (A), Ptpru (B), Klhl1 (C) and Dlk1 (D). Expression of

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Expressing

    Embryonic expression of potential Nurr1-regulated genes. To the left is shown a sagittal section of an E14.5 mouse embryo. The mdDA area is indicated in red and the boxed area corresponds to that shown in A-I. ( A ) Embryonic expression of Nurr1. ( B-I )

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Embryonic expression of potential Nurr1-regulated genes. To the left is shown a sagittal section of an E14.5 mouse embryo. The mdDA area is indicated in red and the boxed area corresponds to that shown in A-I. ( A ) Embryonic expression of Nurr1. ( B-I )

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Expressing

    Adult expression of potential Nurr1-regulated genes. To the left is shown a coronal section of the adult mouse brain. The mdDA area is indicated in red and the boxed area corresponds to that shown in A-F. ( A , B ) Expression of Nurr1 (A) and Th (B) in the

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Adult expression of potential Nurr1-regulated genes. To the left is shown a coronal section of the adult mouse brain. The mdDA area is indicated in red and the boxed area corresponds to that shown in A-F. ( A , B ) Expression of Nurr1 (A) and Th (B) in the

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Expressing

    Identification of Nurr1-regulated genes by Nurr1 loss-of-function analysis

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Identification of Nurr1-regulated genes by Nurr1 loss-of-function analysis

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques:

    Nurr1 gain-of-function analysis in MN9D cells

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Nurr1 gain-of-function analysis in MN9D cells

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques:

    ChIP-on-chip analysis identifies Nurr1 target sites in the mouse genome . ( A ) Western blot showing immunoprecipitation of endogenous Nurr1 after ChIP with Nurr1 antibodies. ( B ) DNA was sheared to an average size of 500 bp by sonication and subjected to

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: ChIP-on-chip analysis identifies Nurr1 target sites in the mouse genome . ( A ) Western blot showing immunoprecipitation of endogenous Nurr1 after ChIP with Nurr1 antibodies. ( B ) DNA was sheared to an average size of 500 bp by sonication and subjected to

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Chromatin Immunoprecipitation, Western Blot, Immunoprecipitation, Sonication

    Microarray analysis of Nurr1 -/- mouse embryos. ( A ) Coronal sections of E14.5 Nurr1 +/+ (top row) and Nurr1 -/- (bottom row) embryos from rostral (left) to caudal (right) positions in the midbrain. Note that Nurr1 -truncated transcripts are still detected

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Microarray analysis of Nurr1 -/- mouse embryos. ( A ) Coronal sections of E14.5 Nurr1 +/+ (top row) and Nurr1 -/- (bottom row) embryos from rostral (left) to caudal (right) positions in the midbrain. Note that Nurr1 -truncated transcripts are still detected

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Microarray

    Model of gene activation by Nurr1 and Pitx3 in mdDA neurons. Nurr1 and Pitx3 regulate genes involved in DA metabolism, terminal differentiation, axon outgrowth, neuronal patterning and survival. Gray arrows indicate concerted regulation by Nurr1 and Pitx3.

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Model of gene activation by Nurr1 and Pitx3 in mdDA neurons. Nurr1 and Pitx3 regulate genes involved in DA metabolism, terminal differentiation, axon outgrowth, neuronal patterning and survival. Gray arrows indicate concerted regulation by Nurr1 and Pitx3.

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Activation Assay

    In vivo ChIP-on-chip analysis reveals binding of Nurr1 to promoters of Nurr1-regulated genes

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: In vivo ChIP-on-chip analysis reveals binding of Nurr1 to promoters of Nurr1-regulated genes

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: In Vivo, Chromatin Immunoprecipitation, Binding Assay

    Expression of potential Nurr1-regulated genes in E14.5 Nurr1 -/- embryos . ( A-H ′) Sagittal sections (as illustrated to the left) of Nurr1 +/+ (A-H) and Nurr1 -/- (A′-H′) mouse embryos demonstrating expression of Nurr1 (A), Dlk1 (B),

    Journal:

    Article Title: Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons

    doi: 10.1242/dev.037556

    Figure Lengend Snippet: Expression of potential Nurr1-regulated genes in E14.5 Nurr1 -/- embryos . ( A-H ′) Sagittal sections (as illustrated to the left) of Nurr1 +/+ (A-H) and Nurr1 -/- (A′-H′) mouse embryos demonstrating expression of Nurr1 (A), Dlk1 (B),

    Article Snippet: Briefly, five ventral midbrains from C57BL6-Jico E14.5 embryos were used for each ChIP using ChIP-grade Nurr1 antibodies (Santa Cruz E20).

    Techniques: Expressing