Polycomb-like proteins link the PRC2 complex to CpG islands. Mus musculus

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo. Overall design: MTF2, SUZ12 and H3K27me3 ChIP-Seq in mouse ES cells, in Control, MTF2 KO, MTF2 wildtype rescue, MTF2 K339A rescue cells. RNA-Seq was performed in Control and MTF2 knockout cells. Experiments were performed in three biological replicates.

多梳抑制复合体2（Polycomb repressive complex 2, PRC2）主要介导转录抑制，在维持细胞身份、正常分化等多种生物学过程中发挥核心作用。多梳样（Polycomb-like, PCL）蛋白（如PHF1、MTF2与PHF19）是PRC2的结合辅因子，可与PRC2核心组分形成亚复合体，既往研究提示其可调控PRC2的酶活性，或介导PRC2招募至特定基因组位点。哺乳动物PRC2结合位点富含CG序列，这与DNA甲基化水平较低的CpG岛（CpG islands）具有相关性。然而，PRC2招募至CpG岛的具体机制尚未完全明确。
本研究解析了在含组蛋白H3第36位赖氨酸三甲基化（H3K36me3）的组蛋白肽存在条件下，结合含CpG DNA的PHF1与MTF2的N端结构域的晶体结构。研究表明，两种蛋白的延伸同源区域折叠为翼螺旋（winged-helix）结构，该结构可特异性识别未甲基化的CpG基序，但其识别方式与经典翼螺旋DNA识别基序完全不同。本研究还证实，PCL蛋白的延伸同源结构域对于小鼠胚胎干细胞（mouse embryonic stem cells）中PRC2有效招募至含CpG岛的启动子区域是必需的。据我们所知，本研究首次提供直接证据，证明PCL蛋白在PRC2招募至CpG岛的过程中发挥关键作用，并进一步阐明了这些蛋白在体内转录调控中的功能。
整体实验设计：在对照组、MTF2敲除组、MTF2野生型挽救组、MTF2 K339A突变型挽救组的小鼠胚胎干细胞中，分别开展MTF2、SUZ12及组蛋白H3第27位赖氨酸三甲基化（H3K27me3）的染色质免疫共沉淀测序（ChIP-Seq）；在对照组与MTF2敲除细胞中开展RNA测序（RNA-Seq）。所有实验均设置3次生物学重复。