着丝粒染色质建立和维持的表观遗传调控

Centromere is a structurally and functionally specialized chromatin region that directs kinetochore assembly and chromosome segregation, and ensures faithful and equal distribution of genetic materials into daughter cells. The establishment and maintenance of centromere function do not rely on the underlying centromeric DNA sequences, but are epigenetically controlled. However, up to date, the epigenetic regulation and its underlying molecular mechanisms of the establishment and maintenance of centromeric chromatin are still unclear. A unique histone H3 variant known as CENP-A, which is therefore proposed as the epigenetic mark of the centromere, is responsible for centromere identity. Although so far the function and regulatory mechanisms of CENP-A have been systematically investigated for several years, the mechanism by which the centromeric deposition of CENP-A could occur accurately at the right time is still obscure. Our previous results reveal that the dynamic reversible phosphorylation of CENP-A at Ser68 regulates the recognition of CENP-A by HJURP and plays critical roles in directing the deposition of newly-synthesized CENP-A into centromeres at the right time during the cell cycle. In this project, employing a combination of biophysical, biochemical, molecular and cell biology techniques, we mainly focus on studying the epigenetic regulation and its underlying molecular mechanisms of the establishment and maintenance of centromeric chromatin. Following our previous studies, we are going to study the regulatory function and its underlying mechanisms of Ser68 phosphorylation in controlling the ubiquitin-dependent proteasome mediated CENP-A degradation and the dynamic assembly of CENP-A at centromeres throughout the cell cycle. Especially, we are going to demonstrate the biological function and its molecular mechanism of a new identified E3 ligase, which can specifically recognize and bind to Ser68 phosphorylated CENP-A，in regulating the dynamic deposition and maintenance of CENP-A at centromeres during the cell cycle. In addition, we also going to reveal the regulatory role and its underlying mechanisms of a novel CENP-A histone chaperone, which specifically bind to Ser68 phosphorylated CENP-A, in the dynamic deposition and maintenance of CENP-A at centromeres during the cell cycle. The incorporation of CENP-A at centromeres promotes the chromatin folding into of a specific higher-order structure, which will regulate the dynamic binding of CENP-N to centromeres during the cell cylce. Employing cryo-EM, single-molecular magnetic tweezers and an in vitro chromatin reconstitution system, we aim to demonstrate the epigenetic regulation of CENP-A on the higher order structure and function of centromeric chromatin. Through this project, we will elucidate the molecular mechanism of epigenetic regulation of chromatin structure and functions at centromeres at molecular level, which will provides important mechanistic basis for underlying the accurate chromosomal segregation at the normal cell division and the abnormal chromosomal segregation due to dysfunction of centromeres in diseases.

着丝粒是基因组中确保细胞分裂过程中染色体正常分离的一段重要的染色质区域，对生物个体基因的稳定遗传起着非常重要的作用。但是到目前为止，着丝粒染色质建立和维持的表观遗传调控及其分子机理，特别是细胞周期中组蛋白变体 CENP-A是如何正确、精准、忠实地定位到着丝粒区域上的，还不是十分清楚。本项目将围绕着丝粒染色质建立与维持的表观遗传调控及其分子机理，进一步研究Ser68磷酸化对细胞周期中CENP-A蛋白稳定性的调控作用；同时，我们还进一步研究Ser68磷酸化调控细胞周期中CENP-A动态装配的分子机制；此外，运用体外重构和结构生物学技术研究CENP-A装配后对着丝粒染色质高级结构与功能的调控及作用机理。通过本项目的实施，我们将从分子水平上阐明着丝粒染色质建立与维持的表观遗传调控和分子机制，为理解细胞分裂过程中染色质的正常分离和疾病发生过程中由于着丝粒异常导致的染色质分离异常等提供了理论基础。

本项目以三种具有重要功能的组蛋白变体CENP-A、H2A.Z及H3.3为研究对象，从分子机制的水平阐明了它们如何介导细胞分裂与基因转录调控等重要的染色质事件。着丝粒是一段结构和功能都高度特化的染色体区域，对遗传物质在细胞分裂过程中的精准分配具有决定性作用。CENP-A是构成活跃的具有生物学功能的着丝粒的充分且必要基本要素，是着丝粒区域的重要表观遗传标识物。着丝粒属性的精准继承呈现高度的细胞周期依赖性。与常规组蛋白不同的是，在DNA复制的时期，CENP-A在细胞内并不表达。在姐妹染色单体成功分离后的有丝分裂末期及下一个细胞周期开始的G1早期，新合成的CENP-A才开始补充到着丝粒区域。这一DNA复制解偶联的CENP-A装配模式是由它的特异性分子伴侣/装配因子HJURP来介导完成的。然而，CENP-A在有丝分裂期就已经表达，此时细胞内的HJURP却不能够与CENP-A结合并装配CENP-A。我们前期的研究证实CENP-A在有丝分裂期会被CDK1-CyclinB激酶符合物催化发生68位丝氨酸的磷酸化修饰。这一位点的修饰直接影响了HJURP对CENP-A的识别。到了有丝分裂末期，姐妹染色单体成功分离之后，CDK1的活性由于CyclinB在细胞内含量的减少而下降，从而使得PP1去除了CENP-A Ser68的磷酸化修饰，保证HJURP可以及时的识别CENP-A并装配CENP-A到着丝粒区域。以此为研究基础，在本项目中我们结合生物化学、细胞生物学及蛋白组学的手段，纯化并鉴定了可以特异性识别并结合Ser68磷酸化修饰CENP-A的E3受体分子DCAF11。DCAF11在有丝分裂期介导CENP-A在Lys49和Lys124位的多泛素化及蛋白酶体依赖的降解。这种降解调控对于防止多余的CENP-A在CENP-A装配的G1期错误的被装配到非着丝粒区域具有重要的贡献，有效的防止了染色体分离和细胞增值的缺陷。本项目对于CENP-A稳定性调控的分子机制的揭示对于以CENP-A装配和着丝粒功能为靶点的药物设计具有重要的意义。本项目另外研究了组蛋白变体分子H2A.Z及H3.3对重要的基因转录调控标识分子H3K27三甲基化修饰及基因转录进行调控的分子机制。