Fig. 5

Profiling bivalent chromatin dynamics in DPPA2/4 knockout mouse embryonic stem cells. A Schematic depicting how DPPA2/4 maintain both H3K4me3 and H3K27me3 at a subset of bivalent genes, priming them for future activation. Loss of DPPA2/4 leads results in loss of both H3K4me3 and H3K27me3 and gain of repressive DNA methylation (black circles). B Genome browser view of wild type (WT, dark) and DPPA2/4 double knockout (DKO, light) embryonic stem cell clones. Two clones of each genotype are shown. In-line total H3K4me3 (green), in-line total H3K27me3 (red) and bivalent K4-K27 (purple) and K27-K4 (blue) reChIP data tracks are shown. Dppa2/4-dependent promoters (lose bivalency when DPPA2/4 absent) are denoted by orange bars. Stable promoters are denoted by blue bars. C Scatterplots showing enrichment (log₂ CPM/bp) for K4-K27 (top) and K27-K4 (bottom) reChIPs between wild type (x-axis) and Dppa2/4 double knockout (DKO) (y-axis) across all bivalent peaks (grey). Highlighted are those differentially enriched in the K4-K27 (purple), K27-K4 (blue) or both (orange) reChIP datasets. D box plot showing normalised enrichment (CPM/bp) of previously annotated Dppa2/4-dependent gene promoters (light orange) and novel Dppa2/4-dependent gene promoters (dark orange) across the different datasets and clones. As a comparison a subset of stable Dppa2/4-independent gene promoters (bivalent promoters that do not change) are shown (blue). E Enrichment heatmaps showing normalised enrichment of previously annotated Dppa2/4-dependent genes (top, light orange) and novel DPPA2/4-dependent genes (middle, dark orange) across the different datasets averaging across clones. As a comparison a subset of Dppa2/4-independent genes (bivalent promoters that do not change) are shown (bottom, blue). F Log₂ RPKM expression levels of original (light orange), novel (dark orange) Dppa2/4-dependent genes and high confidence but not differentially enriched (blue) genes across the different datasets between wild type (WT) and DPPA2/4 double knockout (DKO) cells. As a comparison the bottom 20% (light grey) and top 20% (dark grey) expressed genes are shown. G log₂ normalised expression levels of previously annotated (light orange, top), novel (dark orange, middle) Dppa2/4-dependent genes compared to stable bivalent genes (blue, bottom) during 9 days of mouse embryoid body differentiation in wild type cells (left) and DPPA2/4 double knockout cells (right). Each gene has been normalized separately across the time series to aid visualisation of expression patterns, genes are grouped using correlation based clustering