Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Circular maps show fold enrichment profiles of EcTopoI (ChIP-Seq, light orange; Topo-Seq, cyan and dark orange for two separate DNA strands), RNAP (ChIP-Seq, green), and DNA gyrase (Topo-Seq, dark-red). Additionally, GC-content (%, purple) and mean expression levels (FPKM, RNA-Seq, gray) for annotated TUs (inner blue segments) are also shown. Blue asterisks indicate positions of rRNA operons on the innermost orange ring representing E. coli DY330 genome. The numbers on the outside of the orange ring indicate genome coordinates in megabase pairs (Mbs). Three gaps around ~0.3, ~0.8, and ~1.2 Mb correspond to deletions in the E. coli DY330 genome relative to the E. coli W3110 reference genome. Insets provide a zoom-in view of representative regions with high EcTopoI signals. Coordinates in kb are indicated on top of each inset. For ChIP-Seq, fold enrichment is given relative to the input sample in all figures. The maps were constructed with the Circos tool , and the insets were prepared using IGV .

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Construct

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Representative regions of the E. coli chromosome with EcTopoI ChIP-Seq peaks matching the EcTopoI Topo-Seq TCSs ( dps , potF ) and a region lacking EcTopoI-binding and activity ( nuoN ). EcTopoI cleavage activity is shown strand-specifically. A control, non-induced culture, is shown. Positions of regions used for ChIP-qPCR and affinity measurements are indicated by gray rectangles. b Affinity of purified EcTopoI to three amplified genomic regions from the panel a measured by EMSA. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected. c Metagene plot of EcTopoI ChIP-Seq enrichment (untreated condition, black curve) and EcTopoI Topo-Seq cleavage signal (blue and red curves for coding and template strands, respectively). Confidence bands around the mean metagene signal are represented by ±SEM. Analysis was performed for the HETU set. Regions used for further quantification of enrichment in panel d are shown by colored areas on the plot. d Mean EcTopoI cleavage signal in different regions relative to HETUs. Means were compared by a two-sided Welch t -test. P values <4e-3 are indicated by asterisks and Bonferroni correction for multiple testing was applied. Bars represent mean values ±SEM, n = 200 TUs. e Metagene plot of EcTopoI Topo-Seq cleavage activity for all TUs, LETU, and HETU sets. Cleavage is shown strand-specifically. f Logo of EcTopoI-binding motif identified in sequences under the ChIP-Seq peaks. A motif is shown in both orientations. g EcTopoI cleavage motif identified by alignment of TCSs. The cleavage site between nucleotides −1 and 1 is indicated by a dashed line. The cleavage signal (N3E) is plotted below. h Affinity of purified EcTopoI to oligonucleotides measured with EMSA. The binding of forward (left) and reverse-complement (right) oligonucleotides is shown. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected (Supplementary Table ). i Affinity of purified EcTopoI to oligonucleotides measured with MST. Data were represented as mean values ± SEM, minimum of three independent MST experiments were performed. For source data, see Table in the Source Data file. j Cleavage of oligonucleotides by purified EcTopoI. A control with EcTopoI inactivated by a high temperature is indicated with a boxed + sign. Cleavage products are marked with a blue arrow. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Binding Assay, Activity Assay, Control, ChIP-qPCR, Affinity Purification, Amplification, Concentration Assay, Gel Shift, Purification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Growth curves for E. coli DY330 topA -SPA harboring pCA24 GFP (i), pCA24 14 kDa CTD (ii), or pCA24 topA (iii) plasmids. Data for induced (+IPTG 1 mM) and non-induced (−IPTG) cultures are shown. Shade represents a 0.95 confidential interval of the mean of three biological replicates. Gray lines mark aliquots collection for plasmid extraction. b Quantification of cell length in CTD or GFP producing cultures (left). Cells >10 µm are collected into an overflowing bin. The number of cells is indicated in parentheses. Representative fields are shown on the right. c Graphical representation of truncated versions of a topA gene constructed by recombineering in E. coli BW25113. d Growth curves of E. coli BW25113 strains with truncated versions of topA and the wild-type control (left). Shade represents 0.95 confidential intervals of the mean. Quantification of doubling time for exponential regions of growth curves (right). e Quantification of cell length for E. coli BW25113 strains with truncated versions of topA and the wild-type (left). Vertical dashed line marks 2*mean cell length for wild-type. Representative fields are shown on the right. f Mutations in gyrase genes ( gyrA , gyrB ) found in E. coli BW25113 topAΔ30 clones. An asterisk indicates amplification of a chromosomal region containing TopoIV genes; clones lacking compensatory mutations are highlighted in green. g Supercoiling of pCA24 GFP (i), pCA24 topA (ii), and pCA24 14 kDa CTD (iii) plasmids extracted from exponentially growing E. coli DY330 topA -SPA. Time-points correspond to panel a . Supercoiling of pCA24 GFP (iv) plasmid extracted from exponentially growing E. coli BW25113 topAΔ 30 (time-course, on the left) or E. coli BW25113 wt (two rightmost lanes). (v) Supercoiling level of the pCA24 GFP plasmid extracted from overnight cultures of different clones of E. coli BW25113 topA mutants and from the wild-type control. Clone numbers correspond to panel f . Nic - nicked plasmid, L - linear plasmid, −sc - negatively supercoiled plasmid, HCF - hypercompacted plasmid. h Metagene plots of normalized strand-specific read coverage depth obtained in DRIP-Seq experiments for E. coli DY330 topA -SPA for HETU (upper panel, rRNA operons were excluded) and LETU (lower panel) sets. Schematic TUs are shown below. Data for CTD-/Rif- condition are shown with a dashed line, coverage depths for the coding and template strands are indicated by dark-red and dark-blue fillings, respectively. Data for CTD+/Rif− condition are shown with a solid line, coverage depths for the coding, and template strands are indicated by light-red and light-blue fillings, respectively. i DRIP-Seq data for pCA24 14 kDa CTD for CTD+/Rif− and CTD+/Rif+ conditions and corresponding RNase HI-treated controls. Coverage depths for “−” and “+” strands are shown in light-blue and light-red, respectively. A linearized map of the plasmid is shown below. Source data are provided as a Source Data file.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: Plasmid Preparation, Extraction, Construct, Control, Clone Assay, Amplification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Average normalized enrichment of TopoI, DNA gyrase, and RNAP over transcription units of E. coli ( a , “open” model) and Mycobacterium ( b , “closed” model). Graphical representations of twin-domain sub-models are shown below. ChIP-Seq data for M. tuberculosis MtbRNAP, MtbGyrase, and M. smegmatis MsmTopoI was taken from publicly available datasets , , . c Other “semi-open” hypothetical variations of the twin-domain model, based on the interaction of key topoisomerases (TopoI, DNA gyrase) with RNAP and their activity within a complex. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema for analysis of BM primitive SLAM LSK HSPCs. Murine femurs and tibias were harvested, flushed, and crushed, to collect maximal yield of bone and marrow cells. BM labeled cells were flow sorted for the SLAM LSK markers: Live\Ter-119 neg \Lineage neg \Sca-1 + \c-Kit + \CD150 + \CD48 neg . Next, combined multiome single-nuclei RNA/ATAC (snRNA/ATAC) sequencing analysis was performed. b, Weighted nearest neighbor (WNN) UMAP with hematopoietic stem and progenitor cell type annotation for hematopoietic stem cell (HSC), multi-potent progenitor (MPP), megakaryocyte progenitor (MkP), and erythrocyte progenitor (EryP) sub-cluster representation. c, Engrafting LTR-HSC transcriptional signature (from Rodriguez-Fraiticelli et al . 2020) assigned on a WNN UMAP space. d, Heatmap representation of differential transcriptional nuclei output from distinct HSPC sub-clusters by averaged Z-score, with selected genes presented. e, Heatmap representation of differential ChromVAR motif activity in distinct HSPC sub-clusters by averaged Z-score, with selected TF motifs presented.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Labeling, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded as described in Fig. S5A. Harvested cells were analyzed by flow cytometry and/or flow sorted for LSK HSPCs which were competitively co-transplanted with congenic SJL BM cells into lethally irradiated congenic SJL recipient mice. a, Representative images of co-cultures at the end point before harvest. Yellow arrows indicate megakaryocytes. Note expansion of round hematopoietic cells in Fli-1 ROSAΔ N1-IC iOE co-cultures without any appearance of megakaryocytes. Bar = 100 µM. b, Frequency of LSK HSPCs was determined by flow cytometry and fold expansion was calculated. One-way ANOVA multiple comparisons was used; n=4 BM donor mice per genotype, with 2 technical replicates per donor. c, Frequency of chimerism indicating engraftment levels as determined by flow cytometry. One-way ANOVA multiple comparisons was used; n=8 recipient mice per genotype. d-g, WT, Fli-1 ROSAΔ , and Fli-1 ROSAΔ with conditionally inducible Notch1 internal component overexpression transgene (Fli-1 ROSAΔ N1-IC iOE ) BM LSK HSPCs were isolated and expanded. After 48 hours of expansion in co-culture, cells were harvested and hematopoietic LSK HSPCs were sorted and applied for single cell RNA-seq analysis; n=4 per genotype “pooled” together. d, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted LSK HSPCs. e. Dot plot for the E-SLAM HSC markers EPCR, CD34, and CD150 per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. f. Distribution of clusters 7 and 15 in UMAP for HSC identified populations among LSK HSPC clusters based on Garnette and the Dot plot from panel (e). g, Dot plot for cell cycle scores (S phase and G2/M phases) per cluster identity. Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. h, Cell cycle status classification in UMAP for clusters 7 and 15: WT (left UMAP), Fli-1 ROSAΔ (middle UMAP) and Fli1 ROSAΔ N1-IC iOE (right UMAP). i-l, BM cells were harvested, 16 weeks post engraftment of WT and “rescued” Fli-1 ROSAΔ N1-IC iOE long term repopulating HSCs and pooled from n=4 recipient mice (from 4 different donors) per genotype. CD45.2 + /Lin − cells were flow sorted and applied for single cell RNA-seq analysis. (HSC: hematopoietic stem cells, MPP: multi-potent progenitors, LPP: lymphoid-primed progenitors, MEP: megakaryocyte/erythroid progenitors). i, Dimensionality reduction by UMAP of single cell transcriptomes from donor-derived lineage-negative BM samples (CD45.2 + /Lin − ) following transplantation of WT or Fli1 ROSAΔ N1-IC iOE LSK HSPC. j, Cell type classification of hematopoietic sub-populations using Garnette: WT (left panel) and Fli1 ROSAΔ N1-IC iOE (right panel) samples in UMAP, and distribution of cell types between samples (bar plot). k, Distribution of WT and Fli1 ROSAΔ N1-IC iOE sample cells in UMAP among populations classified as multi-lineage stem/progenitor cell types (HSC, MPP, or LPP) using Garnette, and distribution of cell types between samples (bar plot). See also Table S5. l , Heatmaps of gene-set scores for the subset of multi-lineage stem and progenitor cell types (HSC, MPP, LPP) in UMAP (left panels), and violin plots of gene-set scores between samples (WT vs Fli1 ROSAΔ N1-IC iOE ) (right panels), for HSC molecular overlap signature genes (from Wilson et al . 2015) and engrafting long term HSC signature genes (from Rodriguez-Fraiticelli et al . 2020). Gene-set scores in violin plots are shown only for the subset of cells classified as HSC cell type. Wilcoxon Rank Sum Test was used to determine p-Values.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Over Expression, Isolation, Flow Cytometry, Irradiation, Co-Culture Assay, RNA Sequencing, Cell Culture, Expressing, Derivative Assay, Transplantation Assay

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a-d, Sorted CD45 + /CD34 + HSPC from cord blood (CB) and adult mobilized peripheral blood (mPB) sources, were co-cultured on top of a vascular niche for 48h to encourage HSPC activation. Next, cells were harvested and CD45 + /CD34 + HSPC were sorted and applied for single cell RNA-seq analysis. a-c, Dimensionality reduction by UMAP of single cell transcriptomes from co-cultured and sorted CD45 + /CD34 + HSPC, displaying cell distribution by CB and mPB source (a), cluster identity (b), and single cell cycle phase (c). d, Dot plot for cell cycle scores (S phase and G2/M phases) per cellular source (CB or mPB), for total cells in analysis (upper panel) and for HSC\MPP cluster (lower panel). Dot plot size indicates percent expression in cluster and color intensity indicates average expression score. e, To highly enrich for HSCs, the mononuclear fraction from CB (n=3) and mPB (n=4) sources was isolated, labeled, and sorted for CD45 + /CD34 + /CD38 − /CD45RA − /CD90 + /CD49f + . Sorted cells were lysed and processed for RNAseq analysis. GSEA analysis plots for HSC activation signatures (left panels) and for HSC quiescence signatures (right panels) acquired from Venezia et al., 2004 (upper panels) and from Roy et al., 2021 (lower panels), showing a positive enrichment for the activation signatures in CB HSCs and positive enrichment for quiescence signatures in mPB HSCs. f, g, UMAP projections of the single cell ATAC-seq enrichment analysis for sorted HSPCs of all 10 signatures identified in Takayama et al., 2021. Colors indicate the degree of enrichment in each cell (blue, depleted; red, enriched). Scale bar indicates enrichment Z-score. f, Enrichment for activated HSPCs (upper panel) and quiescent HSPCs (lower panel) signatures defined in in Takayama et al., 2021., as calculated by chromVAR. g, Enrichment for called peaks from the FLI-1 HSPC ChIP-seq (Fli-1 signature) data set (Beck et al., 2013) overlayed on single cell ATAC-seq HSPC data set (Takayama et al., 2021), as analyzed by chromVAR.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Cell Culture, Activation Assay, RNA Sequencing, Expressing, Isolation, Labeling, ChIP-sequencing

Journal: bioRxiv

Article Title: Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing

doi: 10.1101/2023.03.27.534417

Figure Lengend Snippet: a, Schema of experimental design. Sorted human CD34 + HSPCs from cord blood (CB) or mobilized peripheral blood (mPB) sources were transduced by electroporation with FLI-1 modified-RNA molecules (2 µg FLI-1 modRNA per 10 5 cells) and expanded for 1 week on top of E4orf1 vascular niche cells in a sub-optimal ratio of 1:3 (HSPCs:ECs). Next, expansion co-cultures were analyzed and transplanted into immunodeficient NSG KitW41 mice without myeloablative preconditioning. b , Fold expansion of CB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 CB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. c , Fold expansion of mPB derived hematopoietic subtypes after 1 week in co-culture following transduction with FLI-1 (red) or control (blue) modified-RNA. Unpaired two tailed t-test was used; n = 4 mPB donors. Each mark represents the averaged triplicate (n = 3 technical repeats) per donor. d, Representative flow dot plots of mPB HSPC analysis post 1 week of expansion in co-culture following transduction with FLI-1 or control modified-RNA. The population of CD34 + \CD38 neg HSPCs is colored in red. e, Human CD45 chimerism analysis in peripheral blood (PB), spleen, and bone marrow (BM) of NSG KitW41 mice. Tissues were harvested and analyzed by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. f, Frequency of BM engrafted human HSPCs as determined by flow cytometry 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. g, Representative flow dot plots for BM engrafted human CD34 + and CD34 + \CD38 neg HSPCs acquired 16 weeks post transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors. h, Total BM from primary NSG KitW41 recipient mice was transplanted into secondary NSG KitW41 recipient mice without myeloablative preconditioning. Human CD45 chimerism in the BM of recipient mice was determined by flow cytometry 16 weeks post secondary transplantation. Unpaired two tailed t-test was used; n = 6 mPB donors.

Article Snippet: Human hematopoietic CD34 + HSPCs from either cord blood (CB) or mobilized peripheral blood (mPB) donors, were isolated using human CD34 microbead kit (Miltenyi Biotec) and by passing twice through LS columns (Miltenyi Biotec) attached to a magnetic stand, achieving a purity >95%.

Techniques: Electroporation, Modification, Derivative Assay, Co-Culture Assay, Transduction, Control, Two Tailed Test, Flow Cytometry, Transplantation Assay

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: The identification of bona fide TEAD1 target genes. (A) Venn graph showing potential TEAD1 targets (59 genes) by cross-referencing TKO RNA-seq (955 upregulated genes) and TEAD1-ChIP-seq (392 genes) datasets. (B) Narrowing down to nine TEAD1 direct target genes (TTG) based on regulatory sequence proximity to transcription start sites (TSS) (<1000 bp). (C) TEAD1-ChIP-seq in pancreatic progenitor cells showing strong TEAD1-bound signals that are close to TSS of the candidate TTGs. (D) TEAD1-ChIP-seq in wild-type mouse islets showing TEAD1-bound signals that are close to TSS on WWC2, NR4A3, Amotl2, and LATS2 genes. (E) Quantitative PCR showing the expression of TTGs in TKO and control islets isolated from 12-week-old male mice. (F) Illustration of human CTGF promoter (hCTGF) driven luciferase reporter and mutant human CTGF promoter (ΔhCTGF) driven luciferase reporter on which the MCAT motif was moved to the reverse strand. A luciferase assay showing no difference in activity between ΔhCTGF and hCTGF promoters with co-transfection of YAP5SA and TEAD1. (G) Luciferase assays using mouse YAP1 promoter reporter (mYAP1r) show that TEAD1 and TEAD1 + VGLL4 repress YAP1 transcription, while YAP5SA promotes YAP1 transcription. (H) Split-GFP system showing no binding as indicated by GFP signal between ΔTEAD1 (truncated TEAD1) and YAP1/TAZ/VGLL4. mCherry signal indicate transfection efficiency. Nuclei were counterstained with diamidino-2-phenylindole (DAPI) (blue). (I) mYAP1r-promoter luciferase assays show both TEAD1 and ΔTEAD1 repress YAP1 transcription. (J) ΔmYAP1r (MCATs mutant)-promoter luciferase assays show absent repression of YAP1 transcription by TEAD1 or ΔTEAD1 whereas YAP5SA transcriptional activation of YAP1 is impaired. (K) Human YAP1 promoter and (L) Human TEAD3 promoter luciferase assays show TEAD1 and ΔTEAD1 repression and YAP5SA activation of transcription. (M) ΔTEAD1 inhibit HeLa cell growth. GFP positivity demonstrate ΔTEAD1 or backbone (empty vector) lentiviral transduction. (N) YAP1 protein expression by Western blotting after TEAD1 and ΔTEAD1 overexpression in Hela cells. (O) Human TTGs promoter luciferase assay show TEAD1 and ΔTEAD1 repress the transcription of most TTGs except KNTC1, while YAP5SA promotes the transcription of all TTGs. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: In vivo tumorigenesis using J:NU nude mice Upon reaching ∼80% confluence, TEAD1-overexpressed or vehicle lentiviral transduced HeLa cells were rinsed twice with phosphate buffered saline and detached from the culture dishes with 0.25% trypsin–EDTA solution (#25200056; Thermo Fisher Scientific).

Techniques: RNA Sequencing, ChIP-sequencing, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Isolation, Luciferase, Mutagenesis, Activity Assay, Cotransfection, Binding Assay, Transfection, Activation Assay, Plasmid Preparation, Transduction, Western Blot, Over Expression

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: Function validation of TTGs. (A) HOPFLASH reporter luciferase assay show WWC2, AMOTL2, WTIP and YAP5SA regulation of Hippo signaling activity. (B) Human Ki67-promoter reporter (hKi67r) construct. (C) hKi67r reporter activities suggest differential regulatory effects of proliferation by TTGs. (D) Flow cytometry analysis of proliferative (EDU+) HeLa cells after TTGs overexpression (GFP+); double positives are indicated by grey dots. (E) Quantification of flow cytometry analysis in TTGs overexpressed INS2 cells. (F) Schematic representation of NR4A3 isoforms: NR4A3L, full-length NR4A3; NR4A3S, short form of NR4A3. (G) Quantitative PCR showing NR4A3L and NR4A3S mRNA expression in TKO islets. (H) Quantitative PCR showing Ins1, Ins2, Ki67, NR4A3L and NR4A3S mRNA expression in INS2 cells after TEAD1 overexpression (TEAD1OV). (I) Immunostaining and quantification of Ki67 + Insulin + cells in NR4A3L- and NR4A3S-overexpressing mouse islets. (J) Quantitative PCR showing Ki67, Ins1, Ins2, PDX1, MAFA and UCN3 mRNA expression in NR4A3L- and NR4A3S-overexpressed INS2 cells. (K) hKi67r reporter activity show WTIP, WWC2, AMTOL2, PKCiota and NR4A3 antagonize the proliferation effect of YAP5SA while co-transfecting with YAP5SA at a 1:1 molar ratio in HeLa cells. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: In vivo tumorigenesis using J:NU nude mice Upon reaching ∼80% confluence, TEAD1-overexpressed or vehicle lentiviral transduced HeLa cells were rinsed twice with phosphate buffered saline and detached from the culture dishes with 0.25% trypsin–EDTA solution (#25200056; Thermo Fisher Scientific).

Techniques: Biomarker Discovery, Luciferase, Activity Assay, Construct, Flow Cytometry, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Immunostaining

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: TEAD1 prevents RNA-polymerase II (POLII) from binding to DNA. (A) Two potential POLII binding sites (checkpoints, CK) in YAP1 reporter vector were chosen to perform POLII-ChIP experiments. (B) ChIP assay showing lower POLII binding on the two CKs after TEAD1 overexpression. (C) ChIP assay showing lower POLII binding on the endogenous YAP1 and NR4A3 promoter region after TEAD1 overexpression in HeLa cells. (D) Western blotting showing POLII expression in TEAD1-overexpressing or empty vector transduced HeLa cells. (E) Schematic representation showing the TEAD1-TTG regulatory loop in pancreatic β cells. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: In vivo tumorigenesis using J:NU nude mice Upon reaching ∼80% confluence, TEAD1-overexpressed or vehicle lentiviral transduced HeLa cells were rinsed twice with phosphate buffered saline and detached from the culture dishes with 0.25% trypsin–EDTA solution (#25200056; Thermo Fisher Scientific).

Techniques: Binding Assay, Plasmid Preparation, Over Expression, Western Blot, Expressing

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: TET3 affects DNA methylation and histone modifications of the MED12, TGFBR2, and TSP1 promoters. a UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative TET3 enrichment over input. n = 3. Red numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red-stripped bars. b Sequences of critical transcription regulatory regions (CTRR) of MED12 , TGFBR2 , and TSP1 . The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. c UtLM cells were transfected with siCon or siTET3 for 48 h, followed by QMSP analysis. n = 3. d UtLM cells were transfected with siCon or siTET3 for 48 h, followed by ChIP-qPCR analysis. Data are presented as mean relative enrichment over input. n = 3. All data are representative of at least two independent experiments and are presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: DNA Methylation Assay, Transfection, ChIP-qPCR, Methylation

Journal: Oncogene

Article Title: H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas

doi: 10.1038/s41388-019-0808-4

Figure Lengend Snippet: H19 and TET3 co-express with fibroid-promoting genes in vivo. a , c RT-qPCR analyses were performed on RNAs extracted from human fibroids and matched myometrium tissues. Spearman’s correlation showed positive correlations between expression of H19 and TET3 ( a , left panel), as well as TET3 and its target genes MED12 , TGFBR2 , and TSP1 ( c ) in a statistically significant manner. No correlation between expression of H19 and HMGA2 at the RNA level was detected ( a , right panel). Spearman’s correlation coefficient, p -values, and sample numbers are presented. b Results of western blotting analysis of HMGA2 in human fibroids and matched myometrium. n = 3. Data are representative of two independent experiments and are presented as mean ± SEM

Article Snippet: Antibodies for TET3 (GeneTex, GTX121453; used at a dilution of 1/500), TGFBR2 (Abcam, ab184948; used at a dilution of 1/1000), TSP1 (Abcam, ab85762; used at a dilution of 1/500), MED12 (Novus Biological, NB100–2357; used at a dilution of 1/500), HMGA2 (Proteintech, 20795–1-AP; used at a dilution of 1/500), GRAF1 (Cell Signaling, 8802; used at a dilution of 1/500), SPARC (Cell Signaling, 8725; used at a dilution of 1/500), COL3A1 (LS-Bio, LS-C159386; used at a dilution of 1/1000), COL4A1 (LS-Bio, LS-C100552; used at a dilution of 1/500), COL5A2 (Origene, TA809611; used at a dilution of 1/500), and GAPDH (Abcam, ab128915; used at a dilution of 1/10000) were purchased.

Techniques: In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) X-gal staining to detect Rcor2 expression patterns. Whole-embryo staining at E11.5 stage shows Rcor2 is mainly expressed in the CNS. Scale bar, 1 mm. ( b ) Western blot analysis of Rcor2 expression levels during brain development. The decreased expression of Rcor2 with embryonic development is noteworthy. β-Actin is used as an endogenous control. ( c ) In situ hybridization to detect endogenous Rcor2 mRNA expression patterns in cortical development at E11.5, E13.5, E15.5 and E17.5. Insets show high-magnification image of Rcor2 expression in the neocortex at E13.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 100 μm. ( d ) Confocal images of immunofluorescence to detect cellular localization of Rcor2 in the neocortex at E13.5. Rcor2 localized mainly in the nucleus at interphase and metaphase, and localized between separated chromosomes in anaphase of dividing cells in VZ. Dotted lines circle the shape of nuclei. Scale bar, 5 μm. ( e ) Western blot analysis of Rcor2 expression level in Rcor2 fl/fl and Rcor2 cko brains at E13.5 and E15.5, respectively. Rcor2 expression was depleted in Rcor2 cko brains. β-Actin is used as an endogenous control. ( f ) Representative images of Rcor2 fl/fl and Rcor2 cko brain size at different stages of development. Rcor2 cko mice show severe brain growth retardation at E13.5 and E15.5. Scale bar, 1 mm. ( g ) Representative images of Rcor2 fl/fl and Rcor2 cko cortex at E15.5 by Nissl staining. Structural abnormalities of lamination with reduced cortical thickness are observed in Rcor2 cko cortex. Scale bar, 200 μm. ( h ) RT–qPCR analysis of knockdown efficiencies of the two shRNAs targeting Rcor2. Transcripts were normalized to the control group. Data are shown as mean±s.e.m., t -test, **** P <0.0001, n =3. ( i ) Confocal images of E16.5 cortical sections electroporated with shControl (red), shRcor2-a (red) and shRcor2-b (red) plasmids at E13.5. Knockdown of Rcor2 results in impaired cortical development. IZ, intermediate zone. Scale bar, 20 μm. ( j ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after electroporation as shown in i . proportion of RFP + cells in different zones ( y axis). Data are shown as mean±s.e.m., t -test, * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001, n =3 individual experiments.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Staining, Expressing, Western Blot, Control, In Situ Hybridization, Immunofluorescence, Quantitative RT-PCR, Knockdown, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Immunostaining images of Sox2 at E13.5. Sox2 is dramatically reduced on Rcor2 depletion. VZ, ventricular zone; SVZ, subventricular zone. Scale bar, 20 μm. ( b ) Quantification of Sox2 + cell ratios in VZ/SVZ regions shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Confocal images of Tbr2 expression at E13.5. Tbr2 is dramatically reduced on Rcor2 depletion. Scale bar, 20 μm. ( d ) Quantification of Tbr2 + cell ratios in VZ/SVZ regions shown in c , respectively. Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Immunostaining images of Nestin, Sox2 and Tbr2 in cultured Rcor2 fl/fl and Rcor2 cko NPCs, all of which exhibit significantly reduced expression in the Rcor2 cko NPCs. Scale bar, 20 μm. ( f ) Confocal images of immunofluorescence for Ki67 and PHH3 in Rcor2 fl/fl and Rcor2 cko cortex at E13.5 and E15.5. Ki67 signals (red), but not PHH3 signals (green), are dramatically reduced in Rcor2 cko developing brains. Scale bar, 20 μm. ( g ) Quantification of Ki67 + cells in the VZ/SVZ regions of the developing neocortex as shown in f . Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 separate stainings from three independent brains. ( h ) Confocal images of BrdU (green) and Ki67 (red) staining in Rcor2 fl/fl and Rcor2 cko cortex 24 and 48 h after BrdU incorporation. Scale bar, 100 μm. ( i ) Quantification of the cell cycle exit by percentage of BrdU + and Ki67 + NPCs divided by BrdU + cells shown in h . Data are shown as mean±s.e.m., t -test, *** P <0.001 and **** P <0.0001, n =3 individual experiments. ( j , k ) Representative images ( j ) and quantification of ( k ) of Rcor2 fl/fl and Rcor2 cko neurosphere sizes. The neurospheres'radius of Rcor2 cko are much smaller than those of Rcor2 fl/fl , t -test, **** P <0.0001, n =12. Scale bar, 50 μm. ( l ) Representative time-lapse imaging of the RGC dividing process in the sections of the cerebral cortex electroporated with RFP-shControl (upper panels) and RFP-shRcor2 (lower panels). The radial glial dividing pattern is abnormal on Rcor2 knockdown, resulting in cell death. Arrows, mother RGCs. Arrowheads, two daughter cells. Scale bar, 50 μm. ( m ) Representative time-lapse images of Rcor2 fl/fl cortex sections electroporated with EGFP-Control (upper panels) and EGFP-Cre (lower panels). Loss of cells is observed with Rcor2 knockout by Cre recombinase electroporation. Scale bar, 50 μm.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Immunostaining, Expressing, Cell Culture, Immunofluorescence, Staining, BrdU Incorporation Assay, Imaging, Knockdown, Control, Knock-Out, Electroporation

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Confocal images of Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5, which exhibit significant reduction on Rcor2 knockout. Scale bar, 50 μm. ( b ) Quantification of Satb2 + and Tbr1 + cells in Rcor2 fl/fl and Rcor2 cko cortex at E15.5 in a indicates Satb2 and Tbr1 expressions are decreased on Rcor2 depletion during development. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3 individual experiments. ( c ) Western blot to analyse Dcx, Satb2 and Tbr1 expressions in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control. ( d ) Representative images of Map2 and Tuj1 immunostaining in cultured neurons directly dissociated from Rcor2 fl/fl and Rcor2 cko brain cortex at E15.5. Decreased expression of both markers and reduced neurofilaments can be observed in Rcor2 cko cultured neurons. Scale bar, 20 μm. ( e ) Confocal images of in-vitro cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation using neuronal marker Map2 and Tuj1 antibodies, both of which are significantly reduced in the differentiated Rcor2 cko NPCs. Scale bar, 20 μm. ( f , g ) RT–qPCR analysis of neuronal markers expression in both Rcor2 fl/fl and Rcor2 cko neocortex at E15.5 stage ( f ) and in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs 5 days post spontaneous differentiation ( g ). Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-Out, Western Blot, Control, Immunostaining, Cell Culture, Expressing, In Vitro, Marker, Quantitative RT-PCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Schematic overview of strategy to generate an Rcor2 Flag knock-in allele by CRISPR/Cas9. The sgRNA sequence site is shown as a green arrowhead. The start codon of Rcor2 is indicated and capitalized. The oligo donor contained 50 bp homologies on both sides flanking the DSB, in which 3 × Flag sequences are labelled as a red box. ( b ) Western blot analysis to validate FLAG, RCOR2 and LSD1 expressions in Rcor2 Flag knock-in neocortex using Flag-M2 antibody. β-Actin was used as an endogenous control. ( c ) Pie chart depicts distribution of Rcor2 occupancies in genome-wide scale in FLAG ChIP-seq results using Rcor2 Flag knock-in neocortex at E13.5. ( d ) WebLogos of consensus binding motifs of Rcor2 generated by Multiple EM for Motif Elicitation (MEME) motif analysis tool. ( e ) GO analysis for Rcor2-binding regions in genome-wide scale revealed by Flag ChIP-seq results using Rcor2 Flag brain. ( f ) GO analysis for LSD1 occupancy in genome-wide scale revealed by LSD1 ChIP-seq results using Rcor2 Flag brain. ( g ) Density plots analysis of H3K4me1 signal change in promoter regions (−2- to ∼0.5 kb from TSS) on Rcor2 depletion. Compared with all genes, the change of H3K4me1 signal is significantly ( P <0.0005, Kolmogorov–Smirnov test) enhanced in the promoter regions of Shh pathway-related genes on Rcor2 depletion. H3K4me1 signal change on Rcor2 depletion ( x axis); H3K4me1 signal density ( y axis). ( h ) Gene tracks of Rcor2, LSD1 and H3K4me1 enrichments by ChIP-seq analysis at core promoter regions of Dlx2 and upstream regulatory regions of Shh, which are closely related to Shh signalling. ( i ) ChIP–qPCR analysis of Rcor2 Flag and Rcor2 cko cortex at E13.5 using specific FLAG-M2 antibody. Significant enrichments of the Rcor2 at the regulatory regions of Dlx2 and Shh gene locus detected in g in the Rcor2 flag samples are worth noting. Fold enrichments of Rcor2 occupancy compared with input ( y axis). Data are shown as mean±s.d., t -test, *** P <0.001, n =3.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knock-In, CRISPR, Sequencing, Western Blot, Control, Genome Wide, ChIP-sequencing, Binding Assay, Generated, ChIP-qPCR

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a , b ) Scatter plot analysis of genome-wide expression profiles of Rcor2 cko versus Rcor2 fl/fl samples at E13.5 ( a ) and E15.5 ( b ). Dots above or below the dash line indicate upregulated or downregulated genes on Rcor2 depletion, respectively. Red dots or green dots highlight the significantly differentially expressed genes on Rcor2 depletion. Raw counts ( x axis); gene expression fold changes on Rcor2 depletion ( y axis). ( c ) Venn diagrams of upregulated genes (left) and downregulated genes (right) in Rcor2 cko samples compared with Rcor2 fl/fl samples. ( d ) The profiles of Rcor2 and H3k4me1 enrichments analysed in ChIP-seq results shown in in regulatory regions of genome-wide scale (red) and of the upregulated genes (purple) according to RNA-seq results. ( e ) Correlation network of overlapped upregulated genes in both E13.5 and E15.5 samples. Lines indicate the correlations between two connected genes with R >0.55. Genes were analysed by GO analysis and divided into different categories.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Genome Wide, Expressing, Gene Expression, ChIP-sequencing, RNA Sequencing

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) qPCR analysis of the expression of genes related to the Shh signalling pathway in the cortex of Rcor2 fl/fl and Rcor2 cko brains during development. Significant upregulation of these genes on Rcor2 depletion is noteworthy. Transcripts were normalized to Rcor2 fl/fl group. Data are shown as mean±s.d., t -test, * P <0.05, ** P <0.01 and *** P <0.001, n =3. ( b ) Confocal images of Shh and Ptch1 expressions in Rcor2 fl/fl and Rcor2 cko cortex. Enhanced Shh and Ptch1 signals are observed in Rcor2 cko compared with Rcor2 fl/fl neocortex at E13.5 and E15.5. Insets show high-magnification images of the outlined regions. Scale bars, 50 μm. ( c ) Dlx2 expression in Rcor2 fl/fl and Rcor2 cko cortex detected by immunofluorescence analysis at E15.5. Dlx2 + cells were observed in the neocortex on Rcor2 depletion. Scale bars, 50 μm. ( d ) Confocal images of Shh, Ptch1 and Dlx2 expressions in in-vitro -cultured Rcor2 cko NPCs. Scale bar, 20 μm. ( e ) Western blot analysis of expression levels of Dlx2, Shh and Ptch1 in Rcor2 fl/fl and Rcor2 cko cortex at E15.5. β-Actin is used as an endogenous control.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Expressing, Immunofluorescence, In Vitro, Cell Culture, Western Blot, Control

Journal: Nature Communications

Article Title: LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

doi: 10.1038/ncomms10481

Figure Lengend Snippet: ( a ) Knockdown of Rcor2 impairs cortical neurogenesis, which can be partially rescued by knockdown of Shh during cortical development. In-utero electroporation with RFP-shControl (red)/GFP-shControl (green), RFP-shRcor2 (red)/GFP-shControl (green), RFP-shControl (red)/GFP-shShh (green) or RFP-shRcor2 (red)/GFP-shShh (green) plasmids was performed at E13.5. Cerebral sections were fixed and imaged at E16.5. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. Scale bar, 50 μm. ( b ) Quantification of the percentage of RFP + /GFP + cells in different regions of the developing cortex after electroporation shown in a . Data are shown as mean±s.e.m., t -test, ** P <0.01 and *** P <0.001, n =3 individual experiments. ( c ) Inhibition of Shh by Cyclopamine can partially rescue neurogenesis defects caused by Rcor2 downregulation during cortical development. Rcor2 was knocked down at the lateral ventricle in the brain by in-utero electroporation with RFP-shRcor2 plasmids at E13.5. Cerebral sections were collected at E14.5 and then treated with cyclopamine to inhibit Shh activity for 48 h. Scale bar, 50 μm. ( d ) Quantification of the percentage of RFP + cells in different regions of the developing neocortex after knockdown of Rcor2 or inhibition of Shh shown in c . Data are shown as mean±s.e.m., t -test, * P <0.05, n =3 individual experiments. ( e ) Representative images depicting neurosphere size is partially rescued in the in-vitro -cultured Rcor2 cko NPCs after treatment with Cyclopamine. Scale bar, 50 μm. ( f ) Histogram depicting cell numbers of in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment for 48 h. Cells (5 × 10 5 ) are seeded initially. Data are shown as mean±s.e.m., t -test, ** P <0.01, n =3. ( g ) Confocal images of Tuj1 expression in the differentiated cells from in-vitro -cultured Rcor2 fl/fl and Rcor2 cko NPCs with or without Cyclopamine treatment. Tuj1 expressions are partially restored in Cyclopamine-treated Rcor2 cko cells. Scale bar, 20 μm. ( h ) Model of Rcor2 function in neurogenesis in the developing neocortex. Rcor2 safeguards cortical neurogenesis by recruiting LSD1 complex to the regulatory regions of Dlx2 and Shh genes, to inhibit the Shh pathway activation during development. The absence of Rcor2 leads to inhibition release of these genes and thus ectopic activation of Shh signalling in the developing neocortex, resulting in cortical neurogenesis defects.

Article Snippet: Primary antibodies used were as follows: Rcor2 (1:50, catalogue number NBP1-74099, Novus Biological); Nes (1:300, catalogue number Rat-401, DSHB); Pax6 (1:100, DSHB); Sox2 (1:500, catalogue number sc-17319, Santa Cruz); BrdU (1:500, catalogue number ab6326, Abcam); Ki67 (1:300, catalogue number ab9260, Millipore); Satb2 (1:500, catalogue number ab34735, Abcam); Olig2 (1:200, catalogue number ab109186, Abcam); Shh (1:100, catalogue number sc-33943, Santa Cruz); Ptch1 (1:100, catalogue number sc-6149, Santa Cruz); Dlx2 (1:500, catalogue number ab117546, Abcam); Nkx2.1 (1:300, catalogue number ab76013, Abcam); Tbr1 (1:500, catalogue number ab31940, Abcam); Dcx (1:500, catalogue number ab18723, Abcam); Tbr2 (1:500, catalogue number ab23345, Abcam); Flag M2 (1:1,000, catalogue number F1804, Sigma); Map2 (1:500, catalogue number ab32454, Abcam); and Tuj1 (1:1,000, catalogue number mms-435p, Convance).

Techniques: Knockdown, In Utero, Electroporation, Inhibition, Activity Assay, In Vitro, Cell Culture, Expressing, Activation Assay