Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions

Journal: Oncotarget

Article Title: Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer

doi: 10.18632/oncotarget.24091

Figure Lengend Snippet: BC cells were transfected with HSP27 shRNAs by lentivirus infection. shRNA-transfected J82 ( A ), 253J ( B ), and TCCSUP cells ( C ) were isolated by 3 weeks of puromycin selection, and HSP27 expression in each cell line was analyzed by western blot. GAPDH was used as a housekeeping protein control. ct: non-transfected control; sc: scramble shRNA; shRNA: shRNA against HSP27.

Article Snippet: Plasmids containing shRNAs for human HSP27 (TRCN0000008752, TRCN0000008753, TRCN0000011466, TRCN0000342857, and TRCN0000342790, Sigma) or a scramble shRNA (#1864, Addgene, Cambridge, MA) were co-transfected with pVSV-G and a packaging plasmid (SBI, Palo Alto, CA) into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Scientific, Waltham, MA).

Techniques: Transfection, Infection, shRNA, Isolation, Selection, Expressing, Western Blot, Control

Journal: Oncotarget

Article Title: Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer

doi: 10.18632/oncotarget.24091

Figure Lengend Snippet: The same numbers of shRNA-transfected BC cells were seeded onto a 96-well culture plate. The proliferation of BC cells transfected with shRNA as measured by WST-1 assay at 24 and 48 h. ct: non-transfected control, scramble: scramble shRNA, shRNA1-5: shRNA for HSP27. * p < 0.05, ** p < 0.01, Student's t -test. Red lines in the graph indicate mean value of BC proliferation after transfection of control and scramble shRNA plasmid.

Article Snippet: Plasmids containing shRNAs for human HSP27 (TRCN0000008752, TRCN0000008753, TRCN0000011466, TRCN0000342857, and TRCN0000342790, Sigma) or a scramble shRNA (#1864, Addgene, Cambridge, MA) were co-transfected with pVSV-G and a packaging plasmid (SBI, Palo Alto, CA) into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Scientific, Waltham, MA).

Techniques: shRNA, Transfection, WST-1 Assay, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer

doi: 10.18632/oncotarget.24091

Figure Lengend Snippet: shRNA-transfected BC cells were cultured in serum-free culture media for 48 h. Apoptosis and necrosis of these cells were analyzed by staining with Annexin V and propidium iodide (PI). ( A ) Representative staining results from three independent experiments. The percentages of Annexin V- and PI-positive cells are indicated. ( B ) The average percentages of cells showing apoptosis and necrosis from (A) are indicated. ct: non-transfected control, sc: scramble shRNA, shRNA1-5: shRNA for HSP27.

Article Snippet: Plasmids containing shRNAs for human HSP27 (TRCN0000008752, TRCN0000008753, TRCN0000011466, TRCN0000342857, and TRCN0000342790, Sigma) or a scramble shRNA (#1864, Addgene, Cambridge, MA) were co-transfected with pVSV-G and a packaging plasmid (SBI, Palo Alto, CA) into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Scientific, Waltham, MA).

Techniques: shRNA, Transfection, Cell Culture, Staining, Control

Journal: Oncotarget

Article Title: Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer

doi: 10.18632/oncotarget.24091

Figure Lengend Snippet: ShRNA-transfected BC cells were incubated with various concentrations of cisplatin for 48 h. Cell viability was analyzed by WST-1 assay. ct: non-transfected control, scramble: scramble shRNA, shRNA1-5: shRNA for HSP27. * p < 0.05, ** p < 0.01, Student's t -test. Red lines in the graph indicate mean value of BC cell viability after transfection of control and scramble shRNA plasmid.

Article Snippet: Plasmids containing shRNAs for human HSP27 (TRCN0000008752, TRCN0000008753, TRCN0000011466, TRCN0000342857, and TRCN0000342790, Sigma) or a scramble shRNA (#1864, Addgene, Cambridge, MA) were co-transfected with pVSV-G and a packaging plasmid (SBI, Palo Alto, CA) into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Scientific, Waltham, MA).

Techniques: shRNA, Transfection, Incubation, WST-1 Assay, Control, Plasmid Preparation

Journal: Oncotarget

Article Title: Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer

doi: 10.18632/oncotarget.24091

Figure Lengend Snippet: HSP27 shRNA-targeted sequences are underlined and highlighted in the human HSP27 CDR region for each shRNA. TLC1: sigma TLC1 vector based shRNA. TLC2: Sigma TLC2 vector based shRNA.

Article Snippet: Plasmids containing shRNAs for human HSP27 (TRCN0000008752, TRCN0000008753, TRCN0000011466, TRCN0000342857, and TRCN0000342790, Sigma) or a scramble shRNA (#1864, Addgene, Cambridge, MA) were co-transfected with pVSV-G and a packaging plasmid (SBI, Palo Alto, CA) into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Scientific, Waltham, MA).

Techniques: shRNA, Plasmid Preparation