NURF重塑复合物调控iPS细胞重编程作用及表观遗传机制研究

The epigenetic mechanisms of cell reprogramming have recently become a hot research field in the stem cell study. In an our previous study, we found that autophagy is essential for the reprogramming of iPSC. During the first 1-2 days of iPSC induction, Sox2 recruits the NuRD complex to the promoter of mTOR to repress its expression. After three days’ induction, the levels of mTOR and autophagy return to a basal level. We further found that the NURF complex binds to the promoter of mTOR. Silencing of the components of NURF complex reduces the expression level of mTOR and inhibits the formation of iPSC, indicating that the NURF complex is required for cell reprogramming. Based on our previous studies, we will reveal the regulation role of NURF complex in the induction of cell reprogramming, veryfy the relationship between the NURF complex and mTOR expression, and explore the epigenetic mechanisms involved in the cell reprogramming. By this project, we will reveal a noval molecular mechanism underlying iPS reprogramming, and provide theoretical basis to improve the efficiency and quality of iPSC generation. This project conforms to the reserch requirement of “The epigenetic mechanisms of cell reprogramming” proposed by 2014 Major Program of National Natural Science Foundation of China.

细胞重编程的表观遗传机制研究是干细胞领域研究的热点。我们前期研究发现，自噬过程对于iPS细胞重编程至关重要。自噬发生在iPS诱导的第1-2天，并发现Sox2通过招募NuRD复合物结合到mTOR基因的启动子区下调mTOR的表达。自iPS诱导的第3天后，自噬和mTOR恢复至稳态水平。我们进一步研究发现，在iPS诱导后期mTOR启动子区结合NURF复合物。沉默NURF复合物组分的表达均下调mTOR的表达，并且阻止了iPS细胞的形成，表明NURF复合物在细胞重编程中发挥着重要的调节作用。在此基础上，本项目将探明NURF复合物对iPS细胞重编程的调控作用，揭示NURF复合物对mTOR基因表达的调节作用及与细胞重编程诱导的关系，阐明NURF复合物调控细胞重编程的表观遗传机制。以期揭示iPS细胞诱导形成的分子机制，为提高iPS细胞的诱导效率和质量提供理论基础。

本课题经过2年的集成研究，我们证明了iPS诱导后期mTOR启动子区结合NURF复合物，沉默NURF复合物表达均下调mTOR的表达，从而阻止了iPS细胞的形成。我们发现了新转录因子Zic2在肝癌干细胞中特异性高表达。Zic2是肝癌干细胞自我更新所必需的，Zic2的缺失能够明显抑制肝癌干细胞体外的小球形成和体内成瘤。证实了Zic2能够招募NURF复合物到多能因子Oct4的启动子区，进而促进Oct4的转录，驱动肿瘤干细胞的重编程。还发现长非编码RNA lncKdm2b在造血细胞中特异性敲除会导致ILC3细胞的减少以及效应功能受损。LncKdm2b能够招募NURF复合物到转录因子Zfp292的启动子区，诱导Zfp292的表达，在天然免疫细胞ILC3的稳态维持中发挥重要作用。我们还证明了多聚谷氨酸化修饰对于细胞重编程、干细胞多能性维持和早期胚胎发育过程是必须的。发现敲除CCP1或CCP6显著促进了iPS细胞的形成。在重编程过程中，核心转录因子Klf4在其第381位谷氨酸上被TTLL1或TTLL4催化进行谷氨酸化修饰。这一修饰抑制了Klf4的泛素化降解过程从而维持了其蛋白稳定性。Klf4第381位谷氨酸突变为丙氨酸的基因敲入小鼠因无法发育正常囊胚而早期胚胎致死。敲除TTLL1或TTLL4则显著抑制了细胞重编程效率及早期胚胎发育过程。通过该项目的完成揭示了iPS诱导形成的表观遗传机制，为认识细胞重编程机制提供了理论基础。