In mammals, 5-methylcytosine (5mC) and Polycomb repressive complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is massive gain of 5mC concomitantly with restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. Instead, this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip (encoding EZH inhibitory protein). At the subset of regions where 5mC appears to genuinely supplant H3K27me3, we identified 163 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Using site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. Our study also implies a noncanonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.

在哺乳动物中，5-甲基胞嘧啶 （5mC） 和多梳抑制复合物 2 （PRC2） 沉积的组蛋白 3 赖氨酸 27 三甲基化 （H3K27me3） 通常在富含 CpG 的区域是互斥的。当小鼠胚胎干细胞退出初始多能状态时，伴随着 5mC 的广泛 H3K27me3 限制在无 5mC、富含 CpG 的区域，同时出现 5mC 的大幅增加。为了正式评估 5mC 如何塑造 H3K27me3 景观，我们分析了在存在和不存在 DNA 甲基化机制的情况下幼稚细胞和分化细胞的表观基因组。令人惊讶的是，我们发现不需要 5mC 积累来限制大多数 H3K27me3 结构域。相反，这种 5mC 非依赖性 H3K27me3 限制是由 PRC2 拮抗剂 Ezhip（编码 EZH 抑制蛋白）的异常表达介导的。在 5mC 似乎真正取代 H3K27me3 的区域子集中，我们鉴定了 163 个候选基因，这些基因似乎需要 5mC 沉积和/或 H3K27me3 耗竭才能在分化细胞中激活。使用定点表观基因组编辑直接调节 5mC 水平，我们证明 5mC 沉积足以拮抗 H3K27me3 沉积并在单个候选基因上赋予基因激活。总而言之，我们在概括早期胚胎动力学的系统中系统地测量了 5mC 和 H3K27me3 之间的拮抗相互作用。我们的结果表明，H3K27me3 约束直接或间接地取决于 5mC。我们的研究还暗示了 5mC 在基因激活中的非经典作用，这不仅对正常发育很重要，而且对癌症进展也很重要，因为致癌细胞经常表现出 H3K27me3 的 5mC 的动态替代，反之亦然。