Supplementary Figure 2: MLL2 knockout leads to increased PRC2 and PRC1 at bivalent promoters.

a, Heat map of MLL2, H3K4me3 and H3K27me3 ChIP-seq centered at the TSS (±5 kb) of 1,669 HD bivalent genes, in Mll2^WT and Mll2^KO ESCs. Genes are ranked by the degree of loss of H3K4me3 upon Mll2 depletion. Numbers show the correspondence between values and the color scale. b, ChIP-seq of H3K4me3 (left) or H3K27me3 (right) around the TSS of the indicated groups of genes (Methods and Supplementary Table 1). c, TSS (±5 kb) plots of histone modifications, SUZ12 or RING1B in Mll2^WT and Mll2^KO cells, at 1,669 HD bivalent genes (top) and 3,868 bona fide bivalent genes (bottom). ChIP-seq signals were normalized to Drosophila spike-in. d, Western blots of whole-cell extracts in Mll2^WT and Mll2^KO mESCs. e, Western blots of acid-extracted histones, with total histone H3 as loading control. Numbers correspond to the ratio of band intensities in Mll2^KO relative to Mll2^WT. f, Western blot of cellular fractionations of Mll2^WT and Mll2^KO mESCs into total, cytoplasm (cyto), total nuclear (Nuc), nucleoplasm (Np), or chromatin (Chr) fractions. Equal volumes of each fraction were loaded for comparison of band intensities. The Western blot experiments in d–f were repeated for n = 3 independent cell cultures with similar results. g, Scatterplot of significantly differentially expressed genes after Mll2 knockout (n = 2; adjusted P < 0.1). Genes significantly upregulated (orange) and downregulated (blue) are shown. h, Bar plot showing the proportion of genes deregulated after MLL2 depletion that are MLL2 targets by ChIP-seq. i, GO analysis of 744 genes downregulated upon Mll2 knockout. j, Boxplots of expression levels of the indicated groups of genes in Mll2^WT and Mll2^KO. A pseudocount of 0.1 was added to the total RNA RPKM values. The bottom and top of the boxes correspond to the first and third quartiles, and the line within represents the median (n = 2 independent cell cultures). Whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. P values were computed using the Wilcoxon test (two-sided). k, Metagene plot of RPB1 ChIP-seq at 1,669 HD bivalent genes in Mll2^WT and Mll2^KO. l, As in k for RNAPII phosphorylated at Ser5. m, Boxplot of the RNAPII-Ser5P level ±500 bp around the TSS of 1,669 HD bivalent genes in Mll2^WT and Mll2^KO ESCs. P values were computed using the Wilcoxon test (two-sided).