Fig. 3

ASH2L transcriptionally regulates cell cycle via direct promoter interactions with G2/M-checkpoint and E2F target gene sets. A Schematic of RNAseq and greenCUT&RUN experiments. B Volcano plot of RNAseq data showing differentially expressed genes (DEGs) in ASH2L depleted U373 cells compared to controls on the 14 day post-transduction. C Normalized enrichment score results of gene set enrichment analysis (GSEA) for all gene sets available from MSigDB v7.5. Top 5 biological processes enriched in downregulated or upregulated genes upon ASH2L knockout are shown. P values were calculated by hypergeometric test. D greenCUT&RUN analysis in U373 cells revealing genomic localization of ASH2L at differentially expressed gene promoters. E Venn diagram and volcano plot of overlapped RNAseq and greenCUT&RUN data to show proportion of differentially expressed genes bound by ASH2L in U373 cells. Genes bound by ASH2L and differentially expressed upon ASH2L depletion are now called as critical genes, denoted by blue (for downregulated) and yellow (for upregulated) dots on volcano plot. F Normalized enrichment score and FDR-qval results of GSEA on critical genes for all gene sets available from MSigDB v7.5. Some of the negatively enriched pathways related to cell cycle are highlighted. G Top 10 biological processes enriched in regulatory genes upon ASH2L knockout. H RNAseq heatmaps of critical genes. I greenCUT&RUN representative igv plots of critical genes. J Flow cytometric cell cycle analysis of ASH2L-depleted U373 by PI staining on post-transduction day 14 and its statistical analysis. P values determined by two-tailed Student’s t-test *P < 0.05, **P < 0.01, ***P < 0.001