Fig. 7

ZMYND8-mediated chromatin repression is associated with H4(K16)ac. A Heatmap of clustered, normalized feature emission probabilities, and associated functional annotation of the new 12-feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See “Methods” for details on optimal model selection. SE: super-enhancer. B Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C Modeled chromatin states among reference gene promoter-proximal regions (±3 kb around annotated TSS). Left, proportion of promoter-proximal chromatin for siARID1A DE genes (DESeq2, FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter-proximal chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter-proximal chromatin states associated with ARID1A transcriptional repression (i.e., siARID1A upregulation) compared to activation (i.e., siARID1A downregulation). D Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac− (gray) promoter-proximal super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E Principal component analysis (PCA) of RNA-seq expression log₂FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls (n = 3). In total, 1974 genes with _slog₂FC variance >0.1 were used for PCA. F Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated (DESeq2, FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G Clustered heatmap of expression log₂FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter-proximal region or gene body. H Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I Example target gene loci, PLAU and TRIO, marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, and ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05)