Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation

Mammalian development begins in transcriptional silence followed by a period of widespread activation of thousands of genes. DNA methylation reprogramming is integral to embryogenesis and linked to Tet enzymes, but their function in early development is not well understood. Here, we generate combined deficiencies of all three Tet enzymes in mouse oocytes using a morpholino-guided knockdown approach and study the impact of acute Tet enzyme deficiencies on preimplantation development. Tet1-3 deficient embryos arrest at the 2-cell stage with the most severe phenotype linked to Tet2. Individual Tet enzymes display non-redundant roles in the consecutive oxidation of 5-methylcytosine to 5-carboxylcytosine. Gene expression analysis uncovers that Tet enzymes are required for completion of embryonic genome activation (EGA) and fine-tuned expression of transposable elements and chimeric transcripts. Whole-genome bisulfite sequencing reveals minor changes of global DNA methylation in Tet deficient 2-cell embryos, suggesting an important role of non-catalytic functions of Tet enzymes in early embryogenesis. Our results demonstrate that Tet enzymes are key components of the clock that regulates the timing and extent of EGA in mammalian embryos.

哺乳动物发育始于转录静默状态，随后进入数千基因广泛激活的阶段。DNA甲基化重编程（DNA methylation reprogramming）是胚胎发生（embryogenesis）不可或缺的组成部分，且与Tet酶（Tet enzymes）密切相关，但学界对其在早期发育中的功能尚缺乏充分认知。本研究采用吗啉代介导的敲低策略，在小鼠卵母细胞中构建了全部三种Tet酶联合缺陷模型，并探究了急性Tet酶缺陷对胚胎植入前发育的影响。Tet1-3缺陷胚胎会阻滞于2细胞阶段，其中表型最严重的情况与Tet2相关。各Tet酶在5-甲基胞嘧啶（5-methylcytosine）向5-羧基胞嘧啶（5-carboxylcytosine）的连续氧化过程中发挥着不可替代的作用。基因表达分析结果显示，Tet酶是完成胚胎基因组激活（embryonic genome activation, EGA）以及精准调控转座元件与嵌合转录本表达所必需的。全基因组亚硫酸氢盐测序（Whole-genome bisulfite sequencing）结果表明，Tet缺陷的2细胞胚胎的全局DNA甲基化水平仅发生微小变化，这提示Tet酶的非催化功能在早期胚胎发生中发挥着重要作用。本研究结果证实，Tet酶是调控哺乳动物胚胎中EGA的时序与程度的核心时钟组分。