TET-deficient knockout cells transit between pluripotent states and exhibit precocious germ cell differentiation

TET1, TET2 and TET3 are DNA demethylases with critical roles during early embryonic development and cell differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. ESCs lacking all alleles of Tet1 and Tet2, or all alleles of Tet1, Tet2 and Tet3 form epiblast-like cells and epiblast stem cells in culture, indicating that TET proteins are not required to transit between naïve, formative and primed pluripotency. Moreover, in differentiation protocols, ESCs with double-knockouts of Tet1 and Tet2 or triple-knockouts of Tet1, Tet2 and Tet3 do not differentiate normally , fail to activate somatic gene expression and retain expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly, ESCs with double knockouts of Tet1 and Tet2, or triple knockouts of Tet1, Tet2 and Tet3 cells do differentiate into primordial germ cell-like cells (PGCLCs). Furthermore, PGCLC differentiation of Tet1, Tet2 double knockouts, or Tet1, Tet2, Tet3 triple knockouts occurs with high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in the absence of TET proteins. These results establish TET proteins as key regulators enabling epiblast cells to choose between somatic and germline fates. The functions of Tet1 and Tet2, but not Tet3 are required to enable epiblast cells to undergo somatic differentiation and in the absence of TET protein function, epiblast cell differentiation defaults to the germline. Wild type and TET tripple knockout (TET-TKO) cells were differentiated into first EpiLCs and then PGCLC aggregates in the presence or absence of cytokines. Cells were collected at day 0 (EpiLCs), day 2 and day 6 of differentiation, total RNA was extracted, depleted for rRNA and sequenced using the NextSeq 2000 platform.

TET1、TET2与TET3是一类在早期胚胎发育及细胞分化过程中发挥关键作用的DNA去甲基化酶（DNA demethylase）。为探究TET蛋白在早期发育过程中对细胞功能的调控贡献，研究人员在小鼠多能胚胎干细胞（pluripotent embryonic stem cells, ESCs）中构建了Tet基因的单敲除与复合敲除模型。携带Tet1与Tet2双等位基因敲除，或Tet1、Tet2与Tet3三等位基因敲除的ESCs在体外培养时可形成上胚层样细胞与上胚层干细胞，这表明TET蛋白并非多能状态从初始态、形成态到始发态转换所必需。此外，在分化实验体系中，Tet1与Tet2双敲除或Tet1、Tet2与Tet3三敲除的ESCs无法正常分化，不能激活体细胞基因表达，且持续表达多能性转录因子。由此可见，TET1与TET2（而非TET3）可通过功能冗余的方式促进体细胞分化。值得注意的是，Tet1与Tet2双敲除或Tet1、Tet2与Tet3三敲除的ESCs仍可分化为原始生殖细胞样细胞（primordial germ cell-like cells, PGCLCs）。进一步研究发现，无论是否添加促进原始生殖细胞生成的细胞因子，Tet1与Tet2双敲除或Tet1、Tet2与Tet3三敲除的细胞均可高效分化为PGCLCs。此外，在缺失TET蛋白的情况下，PGCLC的转录程序激活速度更快。上述实验结果证实，TET蛋白是调控上胚层细胞在体细胞与生殖细胞命运间进行选择的关键因子。Tet1与Tet2（而非Tet3）的功能是上胚层细胞进行体细胞分化的必要条件；当TET蛋白功能缺失时，上胚层细胞的分化将默认走向生殖细胞谱系。野生型与TET三敲除（TET triple knockout, TET-TKO）细胞在添加或不添加细胞因子的条件下，先分化为上胚层样细胞（EpiLCs），再形成PGCLC聚集体。分别在分化第0天（即上胚层样细胞阶段）、第2天与第6天收集细胞，提取总RNA并去除核糖体RNA（rRNA），随后利用NextSeq 2000测序平台进行转录组测序。