BACKGROUND The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. RESULTS We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. CONCLUSIONS We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

中文翻译：

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删除 Tet 蛋白会导致 ESC 分化期间的数量差异，部分原因是基因表达的改变。

背景 Tet 蛋白家族（Tet1、Tet2 和 Tet3）通过将 5-甲基胞嘧啶转化为 5-羟甲基胞嘧啶来调节 DNA 甲基化，最终导致 DNA 去甲基化并在哺乳动物早期发育和多能性中发挥关键作用。虽然多个组产生了结合小鼠胚胎干细胞 (ESC) 中不同 Tet 蛋白丢失的敲除，但遗传背景和方法的差异使得直接比较结果和辨别 Tet 蛋白调节转录组的直接机制变得困难。为了解决这个问题，我们在等基因多能性背景中利用基因组编辑，这允许结合基因表达和 DNA 甲基化变化的全基因组评估进行定量的、基于流式细胞术的多能性测量。我们的最终目标是生成大规模数据集资源，以进行假设生成实验。结果我们证明了 Tet 蛋白缺失之间分化能力的数量差异，其中 Tet2 单敲除表现出最严重的缺陷，而单独的 Tet1 或 Tet 基因组合的缺失显示出数量上的中间表型。使用转录组学和表观基因组学方法的组合，我们证明了 DNA 高甲基化的增加和 Tet 缺失中多能性转录谱的差异，其中 Tet2 的缺失对未分化的 ESC 具有最深远的影响。结论我们得出结论，与其他基因型相比，Tet2 的缺失对 ESC 的表型和转录组的影响最为显着。虽然 Tet 蛋白的缺失增加了 DNA 超甲基化，尤其是在基因启动子中，但 DNA 甲基化的这些变化与基因表达变化无关。因此，虽然不同 Tet 蛋白的丢失会改变 DNA 甲基化，但这种变化似乎并不是转录组变化的直接原因。因此，Tet 蛋白的缺失可能通过改变 5mC 和其他机制在表观遗传上调节转录组。尽管如此，与野生型相比，多能 Tet2-/- ESC 的转录组变化意味着分化的差异可部分归因于基因表达的基线改变。这种变化似乎不是转录组变化的直接原因。因此，Tet 蛋白的缺失可能通过改变 5mC 和其他机制在表观遗传上调节转录组。尽管如此，与野生型相比，多能 Tet2-/- ESC 的转录组变化意味着分化的差异可部分归因于基因表达的基线改变。这种变化似乎不是转录组变化的直接原因。因此，Tet 蛋白的缺失可能通过改变 5mC 和其他机制在表观遗传上调节转录组。尽管如此，与野生型相比，多能 Tet2-/- ESC 的转录组变化意味着分化的差异可部分归因于基因表达的基线改变。