化学小分子促进小鼠多能干细胞primed态向naïve态转化的表观遗传机理研究

Chemical control of cell fate transition precisely is one of most significant ways to manipulate life activities. Besides, histone methylation plays critical roles in epigenetic modulation through chromatin accessibility and gene transcription regulation. However, mechanisms of chemical control of cell fate transition through histone methylation and chromatin accessibility were still largely unknown. And chemical novel functions in distinct cell fate transition system need to be discovered. We previously found that histone H3K27 and H3K79 methyltransferase Ezh2 and Dot1L inhibitors could boost conversion of mouse primed pluripotent stem cells into naïve state, and established a robust system with over 80% efficiency in 8 days after optimization, providing an ideal model to study mechanisms of epigenetic regulation of cell fate transition by chemicals. Meanwhile, we found that knockdown Ezh2 gene via shRNA behaved differently from Ezh2 inhibitor during primed-naïve transition, indicating it might have novel function. In this project, we attempt to further investigate mechanisms of Ezh2 inhibitor and Dot1L inhibitor boosting mouse primed pluripotent stem cells into naïve state through H3K27 and H3K79 methylation and chromatin accessibility and discover their novel biological functions in cell fate transition based on chemical library screening and ATAC-seq technology. This project would provide an example for investigating how chemicals influence cell fate transition through epigenetic regulation, which would enrich theory of small molecular regulation.

化学小分子精细操纵细胞命运变化是调控生命活动的根本途径，组蛋白甲基化通过影响染色质可及性变化调控基因转录是表观遗传学研究的重要内容。目前，化学小分子通过组蛋白甲基化影响染色质可及性调控细胞命运转化分子机理尚未明确以及在不同细胞命运转化中的新功能有待发掘。本课题组前期工作发现组蛋白H3K27甲基化酶Ezh2和H3K79甲基化酶Dot1L抑制剂显著促进小鼠多能干细胞primed-naïve转化，基于该发现建立了8天>80%的高效转化体系，同时发现敲降Ezh2基因与其抑制剂功能表型不同，为研究化学小分子调控细胞命运转化机理和新功能提供了理想突破口。本研究拟通过化学小分子库筛选比较不同Ezh2和Dot1L抑制剂在primed-naïve转化模型作用异同，借助ATAC-seq等技术，深入研究两个抑制剂经组蛋白甲基化影响染色质可及性变化调控细胞命运转化机理，挖掘其新的生物学功能，以丰富小分子调控理论。

化学小分子诱导细胞命运转变是安全的新一代再生技术，化学小分子精细操纵细胞命运变化是调控生命活动的根本途径。而naïve态多能干细胞是异种器官再造的关键种子细胞，化学小分子诱导获取naive多能干细胞具有高度的转化应用价值。然而，化学小分子如何调控和诱导naive多能干细胞分子机理尚未明确。因此，建立高效快速的化学小分子诱导naive多能干细胞技术体系，阐明其分子机制，促进其在国民健康和经济方面的转化应用。..本项目建立了BMP4驱动的高效的化学小分子诱导小鼠多能干细胞primed态向naïve态转化体系（BMP4-induced PNT，BiPNT），分为2个诱导阶段，8天内诱导效率超过80%。基于该体系，本项目发现BMP4，Ezh2和Dot1l抑制剂显著促进PNT。利用ATAC-seq等技术，揭示了BiPNT过程染色质可及性的动态变化规律，并阐明BMP4通过新的下游靶点Zbtb7a和Zbtb7b调控naive多能性基因染色质位点的开放介导BiPNT的发生。同时，比较描绘与验证了BiPNT和转录因子介导PNT细胞命运转变过程染色质可及性动态变化的异同规律。另外，结合单细胞RNA-seq等技术深入研究发现，在BiPNT过程中，细胞命运主要分为原始生殖样细胞（PGCLC）介导的naive多能性分支和胎盘分支。同时，阐明化学小分子Dot1l抑制剂EPZ5676/SGC0946通过抑制Gata3/Gata6等胚层谱系因子，以及联合Ezh2抑制剂EPZ6438通过动态调控H3K9me2，H3K27me3和H3K79me2促进PGCLC 而抑制胎盘细胞命运转变，进而促进BiPNT发生。..因此，本项目建立的高效快速的naive多能干细胞技术体系对于技术推广和未来转化应用具有重要的应用创新意义。基于该体系研究揭示了naive多能性建立的新分子机制，拓展了我们对多能性转变的理解和认识，为探索化学小分子诱导人和其他物种naive多能性建立的普适性规律提供了新的切入点，同时也为体外研究生殖细胞特化的表观遗传机制建立了新的研究平台。

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