A Primary Role of TET Proteins in Establishment and Maintenance of De Novo Bivalency at CpG Islands of Developmental Genes

TET protein-catalyzed 5mC oxidation not only creates novel DNA modifications such as 5hmC, but also initiates active or passive DNA demethylation. However, the TETs’ function in crosstalk with specific histone modifications is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domain underlying the methylated DNA CpG islands (CGIs). Overexpression of wild type (WT) but not catalytic inactive mutant (Mut) TET2 in TET-low-expressing cells results in increase of 5hmC level and accompanying DNA demethylation at a subset of CGIs. Importantly, this is sufficient to create de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of key developmental gene promoters, which are often located within CpG islands that are previously hyper-methylated and silenced. On the other hand, depletion of Tet1 and Tet2 in mouse ES cells results in an apparent loss of H3K27me3 at bivalent domains, which are located within CGIs and associated with a particular set of key developmental gene promoters. Collectively, these data suggest that TET proteins have a primary role in charge of regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at a subset of CpG islands associated with developmental genes. We examined H3K4me3,H3K27me3,5mC and 5hmC in 293T and mES cell types,using Illumina Hiseq2500.

TET蛋白（TET protein）催化的5-甲基胞嘧啶（5-methylcytosine, 5mC）氧化反应，不仅可生成5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）等新型DNA修饰产物，还能启动主动或被动DNA去甲基化过程。然而，TET蛋白与特定组蛋白修饰之间的串扰机制，目前尚不完全明确。本研究表明，由TET2介导的DNA去甲基化，在甲基化DNA的CpG岛（CpG islands, CGIs）区域内的组蛋白H3第4位赖氨酸三甲基化（H3K4 trimethylation, H3K4me3）与组蛋白H3第27位赖氨酸三甲基化（H3K27 trimethylation, H3K27me3）二价结构域（bivalent domain）的从头建立与维持过程中发挥核心作用。在低表达TET的细胞中过表达野生型（wild type, WT）而非催化失活突变体（catalytic inactive mutant, Mut）TET2，可提升5hmC水平，并伴随部分CGIs区域发生DNA去甲基化。至关重要的是，这一变化足以在这些基因座上从头构建二价结构域。全基因组分析显示，这些从头形成的二价结构域，主要与一类关键发育基因的启动子区域相关，而这些启动子通常位于此前高度甲基化且处于沉默状态的CpG岛中。另一方面，在小鼠胚胎干细胞（mouse embryonic stem cells, mES）中敲低Tet1与Tet2的表达，会导致位于CGIs区域内、与特定关键发育基因启动子相关的二价结构域上的H3K27me3水平显著降低。综上，本研究数据表明，TET蛋白在调控两种关键表观遗传机制——DNA甲基化与组蛋白甲基化（H3K4me3与H3K27me3）——之间的串扰过程中发挥核心作用，尤其在与发育基因相关的部分CpG岛区域中。本研究使用Illumina HiSeq2500测序平台，对293T细胞与小鼠胚胎干细胞中的H3K4me3、H3K27me3、5mC及5hmC进行了检测。