ABSTRACT

The ten-eleven translocation (TET) family (TET1/2/3) initiates conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thereby orchestrating the DNA demethylation process and changes in epigenetic marks during early embryogenesis. In this study, CRISPR/Cas9 technology and a TET-specific inhibitor were applied to elucidate the role of TET family in regulating pluripotency in preimplantation embryos using porcine embryos as a model. Disruption of TET1 unexpectedly resulted in the upregulation of NANOG and ESRRB transcripts, although there was no change to the level of DNA methylation in the promoter of NANOG. Surprisingly, a threefold increase in the transcript level of TET3 was observed in blastocysts carrying modified TET1, which may explain the upregulation of NANOG and ESRRB. When the activity of TET enzymes was inhibited by dimethyloxalylglycine (DMOG) treatment, a dioxygenase inhibitor, to investigate the role of TET1 while eliminating the potential compensatory activation of TET3, reduced level of pluripotency genes including NANOG and ESRRB, and increased level of DNA methylation in the NANOG promoter was detected. Blastocysts treated with DMOG also presented a lower inner cell mass/TE ratio, implying the involvement of TET family in lineage specification in blastocysts. Our results indicate that the TET family modulates proper expression of NANOG, a key pluripotency marker, by controlling its DNA methylation profile in the promoter during embryogenesis. This study suggests that TET family is a critical component in pluripotency network of porcine embryos by regulating gene expression involved in pluripotency and early lineage specification.

摘要

十一十一易位（TET）家族（TET1 / 2/3）启动了5-甲基胞嘧啶向5-羟甲基胞嘧啶的转化，从而协调了DNA脱甲基过程以及早期胚胎发生过程中表观遗传标记的变化。在这项研究中，使用猪胚胎作为模型，使用了CRISPR / Cas9技术和TET特异性抑制剂来阐明TET家族在调节植入前胚胎多能性中的作用。中断TET1意外导致的上调NANOG和ESRRB成绩单，虽然是DNA甲基化的启动子的水平没有变化NANOG。令人惊讶的是，TET3的转录水平增加了三倍在携带修饰的TET1的胚泡中观察到这种现象，这可能解释了NANOG和ESRRB的上调。当双氧合酶抑制剂二甲基草酰甘氨酸（DMOG）处理抑制TET酶的活性时，研究TET1的作用，同时消除TET3的潜在补偿性激活，降低多能性基因（包括NANOG和ESRRB）的水平，并增加DNA甲基化的水平在NANOG启动子中被检测到。用DMOG处理的胚泡还表现出较低的内部细胞质量/ TE比，这意味着TET家族参与了胚泡的谱系规格。我们的结果表明，TET家族调节NANOG的正确表达是关键的多能性标记，可以通过控制胚胎发生过程中启动子中的DNA甲基化分布来实现。这项研究表明，TET家族是通过调节涉及多能性和早期谱系规范的基因表达而成为猪胚胎多能性网络中的关键组成部分。