Yu Minli, Li Dongfeng, Cao Wanyan, Chen Xiaolu, Du Wenxing
Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095 Jiangsu Province, PR China.
Reprod Fertil Dev. 2019 Mar;31(3):509-520. doi: 10.1071/RD18145.
Ten-eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5-E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.
十一易位蛋白1（Tet1）参与原始生殖细胞（PGC）的DNA去甲基化过程；然而，其精确的调控机制仍不清楚。在本研究中，分析了发育中的PGC中5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）的动态变化以及Tet1在PGC去甲基化中的作用。结果显示，胚胎第4天（E4）后5mC水平显著下降，5hmC水平在E5至E5.5时升高并达到峰值。有趣的是，TET1蛋白在E5至E5.5期间高表达，这与5hmC呈现一致趋势。多能性相关基因（Nanog、PouV和SRY盒2（Sox2））以及生殖细胞特异性基因（小窝蛋白1（Cav1）、piwi样RNA介导的基因沉默1（Piwi1）和无精子症样缺失基因（Dazl））的表达在E5后上调，而DNA甲基转移酶家族基因的表达则下降。此外，Dazl基因在早期PGC中高度甲基化，随后逐渐去甲基化。敲低Tet1导致PGC的存活和增殖受损，5mC水平升高，5hmC水平降低。进一步分析表明，敲低Tet1导致Dazl的DNA甲基化水平升高，包括Dazl在内的基因表达下调。因此，本研究揭示了鸡PGC在体内的动态表观遗传重编程以及Tet1在PGC发育过程中调控基因组DNA去甲基化和Dazl去甲基化的分子机制。
