Investigating the Function of the ORC-associated Protein LRWD1 in Linking DNA Replication and Chromatin Folding

遗传物质从一个细胞到另一个细胞的忠实遗传对于所有生物体的发育和生存至关重要。在这个过程中，细胞必须确保它们的DNA在每个细胞周期只复制一次，并且所产生的基因组拷贝在母细胞和子细胞之间均匀分离。在高等真核生物中，DNA合成起始于许多复制起点，这些复制起点在所谓的复制结构域中共同调节。有人建议，复制域内的选定起点通过染色质成环在空间上接近，并以协调的方式同时激活。然而，导致这一结论的证据仍然只是间接的，起源被选择和协调的分子机制是未知的。本研究的目的是研究DNA复制和染色质拓扑结构之间的联系，起源被称为起源识别复合物（ORC）的多亚基蛋白复合物所结合，该复合物还包含ORC相关蛋白LRWD 1/ORCA。LRWD 1被证明在将ORC募集到染色质中起作用。为了研究LRWD 1在起源形成中的功能，我们鉴定了与ORC一起的粘附素亚基Smc 3作为LRWD 1相互作用蛋白。鉴定LRWD 1的基因组结合位点的ChIP-seq分析显示，大多数基因组LRWD 1结合位点被Smc 3共占据。除了它在姐妹染色单体凝聚中的作用外，凝聚素还作为一个环因子介导远距离基因组位点之间的染色质相互作用。我们的LRWD 1 ChIP-seq数据与染色体构象捕获研究的比较表明，LRWD 1标记了指定短程染色质环的粘着蛋白结合位点，并且与粘着蛋白与边界因子CTCF一起指定的结构域边界之间的长程接触不同。因此，我们的研究结果，指向LRWD 1，ORC复合物，和cohesin之间的功能关系，从而直接连接DNA复制起始和染色质topology.Based上这些发现，我们提出了一个详细的机制调查LRWD 1的功能连接的起源形成和染色质折叠。我们将为LRWD 1建立一个基于组织培养的dCas 9募集系统，以便将LRWD 1引导到特定的染色质位点，以及可诱导的LRWD 1和粘附素耗尽模型，这些模型允许在特定的细胞周期阶段去除这些因子。我们将结合联合收割机这些实验系统与染色体构象捕获，DNA复制，和ChIP测定ORC和复制前复合物亚基，LRWD 1，和cohesin测试如何拴系或去除LRWD 1影响原点形成和染色质折叠。这种有针对性的方法将使我们能够以前所未有的细节描述起源形成和染色体拓扑结构之间的关系，并将为这些过程如何在分子水平上相互交织提供新的见解。

Faithful inheritance of the genetic material from one cell to another is essential for the development and survival of all organisms. During this process cells must ensure that their DNA is duplicated only once per cell cycle and that the resulting copies of the genome are evenly segregated between the mother and daughter cells. In higher eukaryotes DNA synthesis is initiated at numerous origins of replication that are co-regulated in so-called replication domains. It was suggested that selected origins within a replication domain are brought into close spatial proximity through chromatin looping and activated simultaneously in a coordinated fashion. However, the evidence leading to this conclusion is still only indirect, and the molecular mechanisms by which origins are selected and coordinated are unknown. The goal of this proposal is to investigate this link between DNA replication and chromatin topology.Origins are bound by a multi-subunit protein complex termed the origin recognition complex (ORC) that also contains the ORC-associated protein LRWD1/ORCA. LRWD1 was shown to play a role in recruiting ORC to chromatin. In an effort to investigate the function of LRWD1 in origin formation we identified the cohesin subunit Smc3 alongside ORC as a LRWD1 interacting protein. ChIP-seq analyses to identify genomic binding sites of LRWD1 revealed that most genomic LRWD1 binding sites are co-occupied by Smc3. In addition to its role in sister chromatid cohesion cohesin functions as a looping factor that mediates chromatin interactions between distant genomic loci. Comparison of our LRWD1 ChIP-seq data with chromosome conformation capture studies indicated that LRWD1 marks cohesin binding sites that specify short-range chromatin loops, and that are distinct from the long-range contacts between domain boundaries that cohesin specifies together with the boundary factor CTCF. Our results, therefore, point towards a functional relationship between LRWD1, the ORC complex, and cohesin and thus a direct link between DNA replication initiation and chromatin topology.Based on these findings we propose a detailed mechanistic investigation of the function of LRWD1 in linking origin formation and chromatin folding. We will establish a tissue culture-based dCas9 recruitment system for LRWD1 in order to direct LRWD1 to specific chromatin sites, and inducible LRWD1 and cohesin depletion models that allow removal of these factors during specific cell cycle stages. We will combine these experimental systems with chromosome conformation capture, DNA replication, and ChIP assays for ORC and pre-replication complex subunits, LRWD1, and cohesin to test how tethering or removal of LRWD1 influences origin formation and chromatin folding. This targeted approach will allow us to describe the relationship between origin formation and chromosome topology in unprecedented detail, and will provide novel insights into how these processes are intertwined at the molecular level.