Fig. 4

An artificial H3K27me3-H3K4me1 transition regulates the ESCs differentiation. A Left panel: western blotting analysis of H3K27me3 modification in WT and Eed^−/− OG2 mESCs. H3 as the loading control; Right panel: heatmap showing H3K27me3 patterns of promoter CGIs and their shores in WT and Eed^−/− OG2 mESCs. OG2: WT OG2 mESCs; OG2 Eed^−/−: Eed^−/− OG2 mESCs. Each line represents a single CpG island. B Heatmaps and average line plots showing H3K4me1, H3K4me3 and H3K27me3 patterns at promoter CGIs and their shores of two different groups (H3K4me1 bimodal-loss group and bimodal-gain group) in WT and Eed^−/− OG2 mESCs. Each line represents a single CpG island. H3K4me1 Biloss: bimodal-loss group; H3K4me1 Bigain: bimodal-gain group. C Boxplot showing expression alteration (log2FoldChange) of bivalent genes in H3K4me1 biloss group and H3K4me1 bigain group in Eed^−/− OG2 mESCs compared with WT OG2 mESCs. H3K4me1 Biloss: bimodal-loss group; H3K4me1 Bigain: bimodal-gain group. Significance was examined with Wilcoxon rank-sum test, ****p value < 0.0001). D Histogram showing the number of up-regulated (Up) and down-regulated (Down) bivalent genes in Eed^−/− OG2 mESCs. E Histogram showing the percentage of H3K4me1 biloss group genes and H3K4me1 bigain group genes in the up-regulated (Up) or down-regulated (Down) genes in Eed^−/− OG2 mESCs compared with WT OG2 mESCs, respectively. H3K4me1 Biloss: bimodal-loss group; H3K4me1 Bigain: bimodal-gain group. Significance level was determined using χ2 tests. p value  = 1.75e−09. F Average line plots showing H3K4me1 pattern of promoter CGIs and their shores in WT and Eed^−/− OG2 mESCs between up-regulated (Up) and down-regulated (Down) bivalent genes in H3K4me1 bimodal-loss group. Boxplot showing the differences of normalized H3K4me1 coverage at promoter CGIs and their shores between WT and Eed^−/− OG2 mESCs of up-regulated (Up) and down-regulated (Down) bivalent genes in H3K4me1 bimodal-loss group. OG2: WT OG2 mESCs; OG2 Eed^−/−: Eed^−/− OG2 mESCs. Significance was examined with t-test, p value = 5.1e-10, 0.43, 0.0038. G GO enrichment analysis of up-regulated and down-regulated bivalent genes in H3K4me1 bimodal-loss group. H Images showing the morphology of mESC cells after RA treatment. Right panel: qRT-PCR analysis of lineage markers. WT mESCs is OG2 mESCs with GFP expression controlled by Oct4 promoter (Oct4: GFP). Scale bar, 100 μm. Significance was examined with t-test, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001. The data represent mean ± SD from three repeats