Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.

尽管表达正（DNMT3A / B）和负（TET1-3）调节剂，哺乳动物细胞仍稳定地维持高水平的DNA甲基化。在这里，我们使用一组基因敲除的人类胚胎干细胞（ESC）系分析了这些调节剂对DNA甲基化景观的独立和组合影响。在缺乏DNMT3的细胞中观察到了对总体甲基化水平的最大影响，包括在成千上万个正常甲基化基因座上可再现的局部去甲基化。去甲基化取决于TET表达，并且仅在两个DNMT3都不存在时发生。动态基因座富含羟甲基胞嘧啶，并与假定的体细胞增强子在ESC中甲基化并可以在分化时激活的子集重叠。我们在小鼠胚胎干细胞中观察到相似的动力学，这在表皮干细胞（EpiSCs）中较少见，而在体细胞组织中则稀少，表明了保守的多能性相关机制。综上所述，我们的数据揭示了在多能细胞内成千上万的体细胞调节序列上，DNMT3和TET之间的竞争受到严格调控。