Fig. 7: Interplay of H3K27me3, DNA methylation and MLL2 and its impact on MLL2 KO differentiation defects.

(a) Box-and-whisker plot quantifying changes in CpG methylation around TSS (± 1 kb) for cluster1 that was sub-clustered into genes with more or less than 25% increase in DNA methylation in MLL2 KO vs WT (n = 3 biological replicates). P values were calculated using a Brown-Forsythe and Welch ANOVA test assuming unequal variances with the Dunnett T3 correction for multiple comparisons (two-sided). The boxplots indicate the median (middle line), the third and first quartiles (box) and the first and fourth quartiles (error bars). (b) Boxplots quantifying H3K27me3 ChIP-seq signal around TSS (± 3 kb) are shown (n = 2). (c) Heatmap of gene expression in WT, MLL2 KO, MLL2 KO 5dAza-treated and MLL2/SUZ12 double KO EBs at day 6. Differentially expressed genes were selected for genes down-regulation in MLL2 KO EBs compared to WT EBs and rescued upon 5dAza treatment as well as SUZ12 KO. (d) GSEA of genes deregulated between WT vs MLL2 KO EBs, MLL2 KO vs MLL2 KO 5dAza-treated and MLL2 KO vs MLL2/SUZ12 double KO at day 6 (normalized enrichment score (NES); false discovery rate-adjusted P value (FDR q)), n = 434. P values were computed using GSEA that utilizes an empirical phenotype-based permutation test procedure (two-sided).