mRNA-seq in DNA methylation deficient mouse embryonic fibroblasts. Mus musculus

DNA methylation and the Polycomb Repression System are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. By genome-wide mapping of the Polycomb Repressive Complex 2 (PRC2)-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and PRC2 from Polycomb-target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining PRC2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease. Overall design: comparison of Dnmt1+/+ vs Dnmt1-/- mouse embryonic fibroblasts by strand-specific mRNA-seq

DNA甲基化（DNA methylation）与多梳蛋白抑制系统（Polycomb Repression System）均为重要的表观遗传调控机制，在维持基因转录抑制状态中发挥关键作用。现有研究证据表明，DNA甲基化可减弱多梳蛋白组分与染色质的结合能力，进而参与调控其基因组靶向定位。然而，DNA甲基化的这一功能在转录调控语境下是否具有实质重要性，目前仍不明确。本研究通过在严重DNA低甲基化的小鼠体细胞中，对多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）的特征性组蛋白修饰H3K27me3进行全基因组定位分析，证实低甲基化会引发H3K27me3的广泛重分布，其分布模式与野生型细胞中的局部DNA甲基化状态高度相关。出乎意料的是，我们在DNA低甲基化细胞中观察到，包括Hox基因簇在内的多梳蛋白靶向基因启动子区域出现了H3K27me3与PRC2的显著丢失。重要的是，我们证实这类基因中有许多在DNA低甲基化细胞中发生异位表达，这与多梳蛋白介导的转录抑制丧失的结论一致。完整的DNA甲基化组可通过约束PRC2的靶向定位，保障多梳蛋白介导的基因抑制功能正常发挥。本研究揭示了DNA甲基化在基因调控中此前未被关注的作用，因此将改变我们对这一表观遗传机制如何参与正常发育与疾病发生的认知。整体实验设计：通过链特异性mRNA测序，对比Dnmt1+/+与Dnmt1-/-小鼠胚胎成纤维细胞的转录组差异。