干细胞分化过程中异染色质区域H3.3动态组装及其调控机制的研究

Histone H3.3 is a conserved histone variant of Histone H3, which has only five amino acid different from H3.1. H3.3 is incorporated into chromatin in both a replication-dependent and replication-independent manner and plays an important role in gene transcription and maintaining the stability of centromeres and telomeres. H3.3 enrichments at tolemeres in mouse embryonic stem(ES) cells, but not in non-pluripotent cells or differented ES cells.The enrichment of H3.3 in telomeres changed during the mouse embryonic stem cells (ES) differentiation process. DAXX (the death domain-associated protein) is a histone chaperone which can interact with H3.3 specifically, it is required for H3.3 accumulation at telomeres and pericentric heterochromatin in mouse ES cells and mouse embryonic fibroblasts (MEF), respectively. In this study, we will mainly focus on the dynamic change of H3.3 in the telomeres and pericentromeric heterochromatin during the ES differentiation. Furthermore, we will examine the molecular mechanism of how DAXX post-translational modifications regulate H3.3 incorporation. Through this study, we are going to illuminate the molecular mechanism of H3.3 dynamic change in the pericentromeres and telomeres heterochromatin during embryonic stem cells differentiation, to further increase the awareness of H3.3 function in stem cells renewal and differentiation processes.

H3.3 是组蛋白H3的一种保守的变体蛋白，它与H3.1只有5个氨基酸差异，能够通过"DNA复制依赖"和"DNA复制不依赖"两种方式组装到染色质中，在调节基因转录以及维持着丝粒和端粒异染色质稳定过程中发挥重要作用。H3.3在小鼠胚胎干细胞的端粒染色质区域富集，但是分化过程中H3.3在端粒分布发生变化。DAXX是一种能够特异性识别H3.3的组蛋白伴侣，并介导MEF细胞近着丝粒以及胚胎干细胞端粒区域H3.3的组装。本项目主要研究小鼠胚胎干细胞神经定向分化过程中H3.3在端粒和近着丝粒区域的动态变化及调控机制，特别针对DAXX 翻译后修饰在调节H3.3动态变化过程中的作用机制进行研究。通过本项目的研究，我们将从分子水平上阐明干细胞分化过程中H3.3在端粒和近着丝粒异染色质区域动态变化的调控机制，揭示H3.3在胚胎干细胞自我更新和定向分化过程中可能的生物学功能。

H3.3（Histone H3.3）是组蛋白H3的一种保守的变体蛋白，它与H3.1只有5个氨基酸差异，能够通过“DNA复制依赖”和“DNA复制不依赖”两种方式组装到染色质中，在调节基因转录以及维持近着丝粒区域和端粒等异染色质稳定过程中发挥重要作用。组蛋白在染色质上的分布受其特异性组蛋白伴侣调控，H3.3特异性组蛋白伴侣主要有DAXX-ATRX复合体和HIRA复合体，而目前研究者对HIRA复合体识别H3.3的分子机制尚不清楚。本项目主要研究HIRA复合体识别H3.3分子机制及HIRA复合体对H3.3染色质特异性定位的调控作用，并对H3.3在小鼠胚胎干细胞向神经定向分化过程中染色质定位的动态变化以及生物学功能进行研究。通过本项目的研究工作我们取得以下几方面成果：首先，我们阐明了HIRA复合体特异性识别H3.3的分子机制，HIRA复合体中UBN1/2亚基直接与H3.3结合，并且介导HIRA亚基与H3.3之间相互作用，而HIRA亚基能够进一步增强UBN1/2与H3.3的结合作用；H3.3蛋白Ala87和Gly90两个氨基酸在H3.3与HIRA复合体结合过程中发挥关键作用；其次，利用染色质免疫共沉淀及高通量测序方法（Chromatin Immunoprecipitation sequencing,ChIP-seq）我们揭示了HIRA复合体主要介导H3.3在基因启动子及增强子区域特异性定位及组装；最后通过体外诱导分化体系研究发现阻断染色质上H3.3的正确组装能够削弱干细胞定向分化能力，揭示了H3.3在小鼠胚胎干细胞定向分化过程中具有重要的生物学功能。