s12  (Developmental Studies Hybridoma Bank)

Journal: Nature Communications

Article Title: Addressing the specific roles of histone modifications in transcriptional repression

doi: 10.1038/s41467-025-66426-z

Figure Lengend Snippet: a Schematic illustration of PRC2 composition. SUZ12 acts as a scaffold for the complex, containing a C-terminal VEFS domain binding to core-subunits EZH1/2 and EED and an N-terminal region (S12 N ) interacting with the PRC2 non-core subunits. The VEFS domain of SUZ12 is required for catalytic activity, while chromatin binding depends on the S12 N domain and the associated non-core subunits. b Mean SUZ12 ChIP-seq signals (RPKM) for WT mESCs and Suz12 KO mESCs with ectopic expression of S12 N or WT SUZ12 within a 40 kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs ( n = 1). c Representative western blot probing for global H3K27me3 and the expression of fusion constructs using an anti-SUZ12 antibody ( n = 3). d Pearson correlation for the expression phenotypes of the indicated cell lines in three independent biological replicates. e Schematic drawing of the experimental strategy to recruit H3K9me3 and H3K36me3 to PRC2 target genes, using the S12 N -PRC2 as a recruiter. f Mean SUZ12 ChIP-seq signals (RPKM) ( n = 1) of extracts prepared from Suz12 KO mESCs with ectopic expression of fusion constructs within a 40 kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs.

Article Snippet: For KDM4 inhibitor experiments, Suz12 KO mESCs expressing S12 N :SUV2 were treated with 50 nM or 100 nM of the KDM4C inhibitor QC6352 (MedChemExpress) for 96 h with media renewal every 24 h. An overview of cell lines can be found in Supplementary Table .

Techniques: Binding Assay, Activity Assay, ChIP-sequencing, Expressing, Western Blot, Construct

Journal: Nature Communications

Article Title: Addressing the specific roles of histone modifications in transcriptional repression

doi: 10.1038/s41467-025-66426-z

Figure Lengend Snippet: a Schematic drawing of the domain architecture of the fusions S12 N :SD2 and S12 N :SD2* with indications of boundaries (amino acid numbers referring to the sequence in WT SUZ12 and SETD2). Asterisk indicates a point mutation introduced to generate catalytic inactive S12 N :SD2*. b – e CUT&RUN tracks showing H3K27me3, H3K4me3, and H3K36me3 signals (RPKM) from the indicated cell lines within a representative genomic region that includes the HoxA cluster ( b ), PRC2 target genes Bmi1 , Spag6 , and Carlr ( c ), PRC2 target genes Fgf3 and Fgf15 and the endogenously H3K36me3 positive Fgf4 gene ( d ), and PRC2 target gene Ntng2 and the endogenously H3K36me3 positive Setx gene ( e ). The tracks are representative from one biological replicate, performed in biological duplicates ( n = 2). CGI annotations (red) are shown below the track. f Mean H3K36me3 CUT&RUN signals (RPKM) in indicated cell lines within a 40 kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs. g Plot of H3K36me3 CUT&RUN signals (RPM) enrichment within peak boundaries of all H3K27me3 peaks identified in WT mESCs (promoter and non-promoter; n = 22307) for the indicated cell lines quantified using BedTools Multicov. Smoothed lines represent generalized additive model (GAM) fits of mean H3K36me3 signal intensity (RPM) from two independent biological replicates ( n = 2), as a function of H3K27me3 CUT&RUN signal intensity (RPM) in WT mESCs ( n = 1); shaded ribbons show the 95% confidence interval of the fitted mean (not variability between biological replicates).

Article Snippet: For KDM4 inhibitor experiments, Suz12 KO mESCs expressing S12 N :SUV2 were treated with 50 nM or 100 nM of the KDM4C inhibitor QC6352 (MedChemExpress) for 96 h with media renewal every 24 h. An overview of cell lines can be found in Supplementary Table .

Techniques: Sequencing, Mutagenesis

Journal: Nature Communications

Article Title: Addressing the specific roles of histone modifications in transcriptional repression

doi: 10.1038/s41467-025-66426-z

Figure Lengend Snippet: a Cluster heatmap (k-means, 4) of RNA-seq data analyzed with Deseq2 for differential expression (FDR < 0.05). Heatmap shows differentially expressed PRC2 target genes identified by H3K27me3 positive promoters in WT mESCs ( n = 4986) in the indicated cell lines based on three independent biological replicates. b MA plots showing mean changes in gene expression from Deseq2 analysis based on three independent biological replicates ( n = 3). Indicated with red dots are significantly upregulated genes in Suz12 KO mESCs filtered with log2 expression foldchange > 1 and FDR < 0.05. The mean expression of the upregulated genes in Suz12 KO mESCs are traced in the indicated cell lines. c Bar plot showing the fraction of the 1326 upregulated PRC2 target genes in Suz12 KO mESCs that are fully or partially rescued in the indicated cell lines (see methods for filtering criteria). Statistical significance was calculated using Fisher’s exact test, two-tailed (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001). Source data are provided as a Source Data file. d Bar plot showing the percentage of beating clusters identified on day 10 after induction of embryoid body differentiation in the indicated cell lines. The plot is representative of at least three independent experiments. e Mean H3K4me3 CUT&RUN signals (RPKM) ( n = 2) in indicated cell lines within a 40 Kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs. f Custom annotation based DMR methylation (%, see methods) at CpG islands for indicated cell lines ( n = 3). The horizontal lines mark the median; the boxes mark the interquartile range (IQR) and whiskers extend up to 1.5 times the IQR; individual data points beyond this range are plotted. Statistical significance was calculated for the means using an unpaired two-sample t test, two-tailed (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: For KDM4 inhibitor experiments, Suz12 KO mESCs expressing S12 N :SUV2 were treated with 50 nM or 100 nM of the KDM4C inhibitor QC6352 (MedChemExpress) for 96 h with media renewal every 24 h. An overview of cell lines can be found in Supplementary Table .

Techniques: RNA Sequencing, Quantitative Proteomics, Gene Expression, Expressing, Two Tailed Test, Methylation

Journal: Nature Communications

Article Title: Addressing the specific roles of histone modifications in transcriptional repression

doi: 10.1038/s41467-025-66426-z

Figure Lengend Snippet: a Schematic drawing of the domain architecture of S12 N :SUV2 and S12 N :SUV2* with indications of boundaries (amino acid numbers referring to the sequence in WT SUZ12 and SUV39H2). Asterisks indicate point mutations introduced to generate the methyltransferase dead S12 N :SUV2*. b – e CUT&RUN track showing H3K27me3, H3K4me3, and H3K9me3 signals (RPKM and peak-normalization) from indicated cell lines within a representative genomic region that includes the PRC2 target genes Prdm12 , Fibcd1, Lamc3 , and Aif1l ( b ), PRC2 target gene Ntng2 with adjacent endogenous H3K9me3 peak ( c ), the PRC2 target gene Thbs2 with adjacent endogenous H3K9me3 peaks ( d ) and the Hox A cluster ( e ). Representative tracks from one biological replicate, performed in biological duplicates ( n = 2). CGI annotations (red) are shown below the tracks. f Mean H3K9me3 CUT&RUN signals (peak-normalization) in indicated cell lines within a 40 Kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs, scale on the right side. WT mESC H3K4me3 CUT&RUN signal (RPKM) is included for reference with the scale on the left. g Plot of H3K9me3 CUT&RUN signals (RPM) enrichment within peak boundaries of all H3K27me3 peaks identified in WT mESCs (promoter and non-promoter; n = 22307) for the indicated cell lines, quantified using BedTools Multicov. Smoothed lines represent generalized additive model (GAM) fits of mean H3K9me3 signal intensity (RPM) from two independent biological replicates ( n = 2), as a function of H3K27me3 CUT&RUN signal intensity (RPM) in WT mESCs ( n = 1); shaded ribbons show the 95% confidence interval of the fitted mean (not variability between biological replicates).

Article Snippet: For KDM4 inhibitor experiments, Suz12 KO mESCs expressing S12 N :SUV2 were treated with 50 nM or 100 nM of the KDM4C inhibitor QC6352 (MedChemExpress) for 96 h with media renewal every 24 h. An overview of cell lines can be found in Supplementary Table .

Techniques: Sequencing

Journal: Nature Communications

Article Title: Addressing the specific roles of histone modifications in transcriptional repression

doi: 10.1038/s41467-025-66426-z

Figure Lengend Snippet: a Cluster heatmap (k-means, 4) of RNA-seq data analyzed with Deseq2 for differential expression (FDR < 0.05) for the indicated cell lines. Heatmap shows z-score normalized counts of differentially expressed PRC2 target genes identified by H3K27me3 positive promoters in WT mESCs ( n = 4986). Based on three independent biological replicates ( n = 3). b MA plots showing mean changes in gene expression from Deseq2 analysis based on three independent biological replicates ( n = 3). Indicated with red dots are significantly upregulated genes in Suz12 KO mESCs filtered with log2 expression fold change> 1 and FDR < 0.05. The mean expression of the upregulated genes in Suz12 KO mESCs are traced in the indicated cell lines. c Bar plot showing the fraction of the 1326 upregulated PRC2 target genes in Suz12 KO mESCs that are fully or partially rescued in the indicated cell lines (see methods for filtering criteria). Statistical significance was calculated using Fisher’s exact test, two-tailed (* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001). Source data are provided as a Source Data file. d Bar plot showing the percentage of beating cluster identified on day 10 after induction of embryoid body differentiation in the indicated cell lines. The plot is representative of at least three independent experiments. e Mean H3K4me3 CUT&RUN signals (RPKM) ( n = 2) in indicated cell lines within a 40 Kb window centered on 7732 H3K27me3 positive promoter peaks identified in WT mESCs. f Mean H3K4me3 CUT&RUN signals (RPKM) within 40 Kb window centered on PRC2 target promoters for the H3K9me3-rescued versus non-rescued genes in Suz12 KO mESCs expressing S12 N :SUV2. g Mean H3K9me3 CUT&RUN signal (peak-normalized) within 40 Kb window centered on PRC2 target promoters for the H3K9me3-rescued versus non-rescued genes in Suz12 KO mESCs expressing S12 N :SUV2 ( n = 2). h Heatmaps of sequence depth normalized H3K4me3 CUT&RUN data (blue), SUZ12 ChIP-seq data (S12 N fusion proteins) (red), and peak-normalized H3K9me3 CUT&RUN data (black) at PRC2 target promoters in RBBP5-FKBP12 F36V + Suz12 KO mESCs expressing S12 N :SUV2 treated with dTAG-13 or DMSO for 24hrs. Top: Average plots of the mean signal for the region displayed in heatmaps. i Bar plot of relative gene rescue (%) in RBBP5-FKBP12 F36V + Suz12 KO mESCs expressing S12 N :SUV2 treated with dTAG-13 for 24 h with DMSO treatment for 24 hrs as reference ( n = 3).

Article Snippet: For KDM4 inhibitor experiments, Suz12 KO mESCs expressing S12 N :SUV2 were treated with 50 nM or 100 nM of the KDM4C inhibitor QC6352 (MedChemExpress) for 96 h with media renewal every 24 h. An overview of cell lines can be found in Supplementary Table .

Techniques: RNA Sequencing, Quantitative Proteomics, Gene Expression, Expressing, Two Tailed Test, Sequencing, ChIP-sequencing