细胞内代谢产物⍺-酮戊二酸在原始生殖细胞特化过程中的作用及其分子机制研究

The specialization of primordial germ cells is the first step in the development of the mammalian germ lineage of mammals. In mice implanted early embryos, BMP signaling stimulates a few cells in the epiblast to express the three core transcription factors Prdm1, Prdm14 and Tcfap2c, thereby establishing the fate of primordial germ cells. However, the mechanism by which activation of these core factors is regulated is unclear. In our laboratory, we focused on this scientific issue and systematically analyzed the dynamic changes of intracellular transcriptome during the activation of core transcription factors by using in vitro differentiation system. It was found that the expression of intracellular α-ketoglutarate-producing enzymes increased during this process. Decreasing or increasing the amount of intracellular α-ketoglutarate could significantly inhibit or promote the expression of PGC core transcription factors, which in turn affect the specialization of PGC, suggesting that intracellular α-ketoglutarate may be involved in the process of PGC specialization. This project intends to combine in vitro PGCLC-induced differentiation system and knockout mouse model to elucidate α-ketoglutarate as a cofactor of histone demethylase and directly regulate the specialization of primordial germ cells. Reveal the role of metabolites cooperate with epigenetic reprogramming in cell fate determination.

原始生殖细胞的特化是哺乳动物生殖细胞谱系发育的第一步。在小鼠着床后的早期胚胎中，BMP信号分子刺激表胚层的细胞表达Prdm1, Prdm14以及Tcfap2c这三个核心转录因子从而确立原始生殖细胞命运，但调控这些核心因子激活的机制并不清楚。本实验室前期围绕这一科学问题，利用体外分化体系，系统分析了核心转录因子激活过程中，细胞内转录组的动态变化。发现与细胞内α-酮戊二酸生成相关酶的表达在这一过程中会增加，降低或提高细胞内α-酮戊二酸的量，可以显著抑制或促进PGC 核心转录因子的表达，进而影响PGC的特化，提示细胞内α-酮戊二酸可能参与PGC特化过程。本项目拟结合体外PGCLC诱导分化系统和基因敲除小鼠模型，阐明α-酮戊二酸作为组蛋白去甲基化酶的辅助因子，直接调控原始生殖细胞的特化。揭示代谢产物与表观遗传重编程偶联在细胞命运决定过程中的作用。

胚胎干细胞向原始生殖细胞体外分化过程中，胚胎干细胞首先退出多能性状态，再通过细胞外信号分子如BMP4诱导原始生殖细胞标志基因表达，形成原始生殖细胞，这一过程涉及细胞信号传导，细胞代谢转换、表观修饰以及转录因子表达等复杂的调控机制。本项目聚焦揭示原始生殖细胞命运决定调控机制这一关键科学问题，分析了在体外PGC特化过程中细胞中代谢物的改变，发现-酮戊二酸能够通过调节细胞内H3K27me3的去甲基化促进PGC的特化。本项目深入探究了BMP4诱导PGC特化的分子机制，发现BMP4能够调控转录因子TBX3的表达, TBX3敲除后会阻碍PGC的特化, 提示TBX3在BMP4信号通路下游正调控PGC的特化.另外本项目研究发现FGF-MEK 信号通路能够限制PGC的特化, 抑制该信号通路能在体外和体内条件下促进PGC的特化，通过磷酸化蛋白质组学技术，我们发现MEK能够磷酸化USP36调控OTX2蛋白的稳定性从而限制PGC的特化。在胚胎干细胞体外分化的研究中，我们发现18S rRNA特异的m6A甲基转移酶METTL5，能够选择性的调控mRNA翻译效率, 当METTL5敲除后会造成FBXW7 E3泛素连接酶蛋白水平降低，使其底物蛋白C-MYC不能及时降解,从而阻碍胚胎干细胞的分化. 本项目从代谢调控、信号传导、表观修饰以及转录调控等多个维度系统解析了小鼠原始生殖细胞特化的调控机制。