Znhit1在小鼠胚胎干细胞中调控机理的研究

SRCAP is one ATP-dependent chromatin-remodeling complex which utilizes the energy of ATP hydrolysis to catalyze the incorporation of H2A.Z into H2A-containing nucleosomes. As one subunit of SRCAP complex, Znhit1 is involved in several processes such as DNA damage response, cell apoptosis, cell cycle and muscle differentiation. However, the function of Znhit1 during early cell fate decision and lineage differentiation has never been reported. Although H2A.Z is necessary for the proper execution of developmental programs during embryonic stem (ES) cell differentiation, the molecular mechanism how H2A.Z is recruited to specific targets remain unknown. In this study, we combined CRISPR/Cas9 with Cre-LoxP techniques and generated Znhit1 conditional knockout ES cells. Our preliminary data indicate that knockout of Znhit1 significantly inhibits the differentiation of ES cells to mesoderm, endoderm and trophectoderm. Next, we will use ChIP-Seq and RNA-Seq techniques to investigate the molecular mechanism of Znhit1 in regulating ES cell differentiation and further define whether Znhit1 functions through recruiting H2A.Z to the chromatin. This study provides insights into our understanding of the regulatory mechanism of ES cell self-renewal and differentiation as well as the development of stem cell therapy and translational medicine.

SRCAP是一类ATP依赖的染色质重塑复合物，利用ATP水解产生的能量，介导组蛋白变异体H2A.Z替换经典组蛋白H2A，从而调控基因的表达。Znhit1作为SRCAP复合物的重要成员，参与调控DNA损伤修复、细胞凋亡和肌肉分化等过程。但是，其在早期命运决定和谱系分化的研究尚属空白。尽管报道表明H2A.Z参与调控胚胎干细胞的分化，但其招募机制尚未明确。本项目前期结合CRISPR/Cas9和Cre-LoxP技术，通过一步法成功构建Znhit1条件敲除的小鼠胚胎干细胞，并发现Znhit1缺失抑制中、内胚层和滋养外胚层的分化。本项目将采用ChIP-Seq和RNA-Seq等手段揭示Znhit1调控胚胎干细胞分化的分子机制，并进一步验证Znhit1是否通过招募H2A.Z调控胚胎干细胞的分化。本项目从表观遗传学层次进一步揭示了胚胎干细胞维持和分化的调控机理，对细胞治疗和再生医学的发展具有重要的借鉴意义。

Znhit1作为染色质重塑复合物SRCAP中的关键组分，在小鼠胚胎干细胞干性维持和三胚层分化中的研究尚属空白。组蛋白变异体H2A.Z尽管被报道参与胚胎干细胞的分化，但是其如何被招募至染色体的分子机制尚未明确。本项目通过构建Znhit1条件性敲除细胞系，发现Znhit1缺失影响小鼠胚胎干细胞的干性维持和增殖活性，导致周期异常，并引起凋亡现象。通过拟胚体实验证实，Znhit1缺失抑制胚胎干细胞的中、内胚层分化，并且该表型能被外源ZNHIT1恢复。进一步的机制研究表明，Znhit1通过影响核心转录因子Nanog等的表达参与干性维持，并通过影响ERK信号通路的活性调控中、内胚层分化。并且，我们构建H2A.Z KO细胞系，观察到类似的中、内胚层分化缺陷。通过ChIP-Seq实验，我们发现Znhit1缺失影响H2A.Z在基因组靶位点上的结合，并引起组蛋白H3K27Ac等修饰的改变，从而调控下游靶基因的表达。与此一致的是，我们在Znhit1 KO细胞系中过表达H2A.Z，Znhit1缺失造成的分化表型并不能被恢复，进一步证实Znhit1是通过影响H2A.Z在染色质上的结合，而非调控H2A.Z的表达来影响胚胎干细胞分化。更多的测序数据包括RNA-Seq，ChIP-Seq和ATAC-Seq还在进行细致的生物信息学挖掘，致力于从染色质构象、组蛋白表观遗传修饰和关键转录因子结合等不同的层次上，揭示Znhit1和H2A.Z协同调控胚胎干细胞维持和分化的分子机理。本项目的实施在表观遗传层次上丰富了小鼠胚胎干细胞干性维持和分化的调控机理，为再生医学的发展提供了一定的理论基础。