Accurate H3K27 methylation patterns can be established de novo by SUZ12-directed PRC2. Mus musculus

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).

多梳抑制复合体2（Polycomb repressive complex 2, PRC2）催化H3K27甲基化，对维持转录模式与细胞身份不可或缺，但基因组中PRC2结合模式及H3K27甲基化模式的设定与维持机制仍未完全阐明。已有表观遗传调控机制被提出，即预先存在的H3K27甲基化可招募PRC2并调控其催化活性，以维持自身的甲基化状态。本研究旨在探究此类机制是否为小鼠胚胎干细胞中H3K27甲基化模式的建立所必需。通过对PRC2亚基的基因敲除实验，我们发现PRC2负责所有水平的H3K27甲基化；且在缺乏预先存在的H3K27甲基化与PRC2染色质结合的条件下，重新导入PRC2亚基时，甲基化模式可被精准从头建立。此外，我们发现核心亚基SUZ12可引导PRC2的基因组结合，这对建立正确的甲基化模式至关重要。实验整体设计如下：对野生型、Ezh1与Ezh2双敲除（Ezh1/Ezh2 dKO）以及重新表达野生型EZH2的小鼠胚胎干细胞，进行H3K27me1、H3K27me2、H3K27me3富集谱的染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq）分析，共包含10个样本，其中1个为Ezh1/Ezh2 dKO的ChIP input样本。对野生型、Suz12敲除以及分别重新表达野生型SUZ12或两种SUZ12片段之一的小鼠胚胎干细胞，进行H3K27me1、H3K27me2、H3K27me3富集谱的ChIP-seq分析，共15个样本。此外，对经Ezh2抑制剂处理的E14小鼠胚胎干细胞，在抑制剂洗脱后开展时间梯度的ChIP-seq分析，以检测H3K27me1、H3K27me2、H3K27me3的富集谱，共24个样本。针对不同基因型（野生型、敲除型或突变型）的PRC2相关小鼠胚胎干细胞，通过Suz12、Ezh2及Flag抗体进行ChIP-seq，以检测PRC2亚基的富集谱，其中包含9个Suz12 ChIP样本、2个Flag ChIP样本及2个Ezh2 ChIP样本。对野生型（3个生物学重复）、Ezh1/Ezh2双敲除（3个生物学重复）以及重新表达野生型EZH2的小鼠胚胎干细胞，开展RNA测序（RNA Sequencing, RNA-seq）以分析差异基因表达，共9个样本。