SRA

The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate if such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells. Through genetic knockouts of subunits, we find that PRC2 is responsible for all levels of H3K27 methylation, and that methylation patterns can be accurately established de novo, when a subunit is reintroduced in the absence of pre-existing H3K27 methylation and PRC2 chromatin binding. Furthermore, we find that the core-subunit SUZ12 directs genomic binding of PRC2, which is essential for establishing correct methylation patterns. Overall design: H3K27me1, H3K27me2, H3K27me3 enrichment profiles (ChIPseq) analyzed in mouse ES cells which are wildtype, knockout for Ezh1 and Ezh2 (Ezh1/Ezh2 dKO), or re-expressing wildtype EZH2 (10 samples including 1 ChIP input sample for Ezh1/Ezh2 dKO). H3K27me1, H3K27me2, H3K27me3 enrichment profiles (ChIPseq) analyzed in mouse ES cells which are wildtype, knockout for Suz12, or re-expressing either wildtype SUZ12 or one of two SUZ12 fragment (15 samples). In addition, H3K27me1, H3K27me2, H3K27me3 enrichment profiles (ChIPseq) analyzed in E14 mouse ES cells treated with Ezh2 inhibitor and followed in a timecourse study after inhibitor washout (24 samples). PRC2 subunit enrichment profiles through Suz12, Ezh2 and Flag ChIPseq in various PRC2 wildtype, knockout or mutant ES cells (9 Suz12 ChIP samples, 2 Flag ChIP samples, 2 Ezh2 ChIP samples). Differential expression analysis (RNAseq) of mouse ES cells which are wildtype (3 replicates), knockout for Ezh1 and Ezh2 (Ezh1/Ezh2 dKO) (3 replicates), or re-expressing wildtype EZH2 (3 replicates) (9 samples).