TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

中文翻译：

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TET 蛋白保护二价启动子免于人胚胎干细胞中的从头甲基化。

TET 酶将 5-甲基胞嘧啶 (5mC) 氧化为 5-羟甲基胞嘧啶 (5hmC)，这会导致 DNA 去甲基化。然而，由于在区分全局效应与位点特异性效应方面存在挑战，因此难以建立 TET 介导的 DNA 去甲基化和转录输出之间的直接联系。在这里，我们显示 TET1、TET2 和 TET3 三重敲除 (TKO) 人类胚胎干细胞 (hESC) 表现出显着的二价启动子高甲基化，而未分化状态下的基因表达总体上没有相应降低。关注二价 PAX6 基因座，我们发现 DNMT3B 结合增加与启动子高甲基化有关，这会导致神经分化缺陷和 PAX6 在分化过程中诱导失败。dCas9 介导的基因座特异性去甲基化和 TKO hESC 中 DNMT3B 的全局失活部分逆转了 PAX6 启动子的高甲基化并改善了向神经外胚层的分化。将这些发现与进一步的全基因组甲基化和 TET1 和 DNMT3B ChIP-seq 分析相结合，我们得出结论，TET 蛋白保护二价启动子免于从头甲基化，以确保分化时强大的谱系特异性转录。