真核生物ORC复合体调控表观遗传学修饰的研究

DNA replication is a precisely coordinated process characterized with both organization and complication. In eukaryotes, ORC (Origin Recognition Complex) is in charge of the initial step of DNA replication by recognition of DNA replication origin at specific sites in genome. A huge bunch of studies showed that ORC has multiple functions beyond the DNA replication initiation. Based on the data published previously, we proposed a hypothesis that ORC might be involved in the regulation of epigenetic modifications, as the localization and proper functioning of ORC are always closely correlated with several epigenetic modifications..To provide a reliable answer to this hypothesis, we employed a model organism Neurospora crassa. In this study, by means of a diverse mix of technologies such as biochemistry, molecular biology, and genetics, we aimed to explore the mechanism of ORC in regulating epigenetic modifications, after digging out the internal logic between the localization of ORC and epigenetic modifications..The interpretation of the mechanism will certainly be a great inspiration for explaining the fundamental machinery that how genetic information, especially the epigenetic information, transmitted into the descendants during cell division. . Meanwhile, because of the evolutional and functional conservation of ORC, this study will be significantly important for the research area of both DNA replication and epigenetics.

DNA复制是一个复杂而有序的过程。在真核生物中，复制起始源于复制起始位点识别复合体ORC对基因组上DNA复制起始位点的识别与结合。研究表明，ORC不仅参与DNA复制的起始，还具有多种生物学功能。本课题对已有研究综合分析后发现，ORC在基因组中的定位及其功能与多种表观遗传学修饰密切相关，并由此提出ORC参与调控表观遗传学修饰建成的假设。.针对这一假设，本课题拟选取模式生物粗糙脉孢菌为研究材料，采用生物化学、分子生物学和遗传学等相互结合的方法，在发掘ORC定位与表观遗传学修饰之间内在联系的基础上，探索ORC影响表观遗传学修饰建成的分子机制。.对这一机理的阐释，将有助于解析遗传信息，特别是表观遗传学信息，在细胞分裂过程中传递的重要机制。同时，鉴于ORC在进化上和功能上的高度保守性，本课题对复制和表观遗传学相关领域的研究具有重要的参考价值和现实意义。

DNA复制起始识别复合体ORC（Origin Recognition Complex）是真核生物DNA复制机器的重要组分。它作为染色质上的重要结合蛋白，不仅能够负责DNA复制的起始，还参与多种表观遗传学修饰的建立，是一类多功能的保守复合体。CRL4型E3泛素连接酶（cullin 4-RING ubiquitin ligase）也是真核生物细胞中一类对DNA复制和表观遗传学修饰均具有调控功能的保守复合体。CRL4与ORC这两者之间在上述功能调控中，特别是在表观遗传学修饰建立方面是否存在关联，仍不清楚。.本课题首先通过染色质免疫共沉淀并结合高通量测序的方法，发现Cul4和Orc4共定位于基因组上多个位点。蛋白质免疫共沉淀实验还发现，Cul4与ORC复合体组分之间存在相互作用。通过将Cul4和Orc2蛋白强制定位到qa基因簇基因启动子区，发现无论是Cul4还是Orc2的强制定位，都可以引导另一复合体的蛋白在基因组的新位点定位。以上结果证明CRL4与ORC之间能够相互作用，并且在染色体上共定位。.本课题进一步研究了CRL4和ORC共定位对表观遗传学修饰的影响。这些修饰酶系统的定位与所在位点的表观遗传学修饰状态却并不存在一致性。作者还发现，Cul4-QF-DBD和Orc2-QF-DBD可以募集这些表观遗传学修饰系统到染色质上，当CRL4存在缺陷或是细胞处在DNA复制胁迫条件下时，这些表观遗传学修饰系统在CDS位点富集量下调，说明了CRL4和ORC负责调控这些表观遗传学修饰系统在CDS定位。.以上实验结果表明，CRL4和ORC复合体在细胞内相互作用，并且富集在染色质上的特定位置CDS，为多种表观遗传学修饰系统提供结合位点。但是这些系统在染色质上的共定位对表观遗传学修饰建立的影响还有待进一步探索。这些发现为解析表观遗传学修饰建立的调控机制提供新的思路和实验证据。