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所有生物体的发育和生存是必不可少的。在这个过程中，细胞必须确保它们的DNA在每个细胞周期中只复制一次，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合成是在许多复制起点开始的，这些复制起点是在所谓的复制域中共同调节的。这表明，在一个复制区域内，选择的起源通过染色质环被带到紧密的空间邻近，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。本提案的目的是研究DNA复制和染色质拓扑结构之间的这种联系。起源由一种称为起源识别复合体（ORC）的多亚基蛋白复合体结合，该复合体还含有ORC相关蛋白LRWD1/ORCA。LRWD1在募集ORC到染色质中发挥作用。为了研究LRWD1在起源形成中的功能，我们确定了黏结蛋白亚基Smc3和ORC是LRWD1的相互作用蛋白。对LRWD1基因组结合位点进行ChIP-seq分析发现，大部分基因组LRWD1结合位点被Smc3共占据。除了在姐妹染色单体内聚中发挥作用外，内聚蛋白还作为环因子介导远端基因组位点之间的染色质相互作用。我们的LRWD1 ChIP-seq数据与染色体构象捕获研究的比较表明，LRWD1标记的黏结蛋白结合位点指定了短程染色质环，这与黏结蛋白与边界因子CTCF一起指定的结构域边界之间的远程接触不同。因此，我们的研究结果指向了LRWD1、ORC复合体和内聚蛋白之间的功能关系，从而在DNA复制起始和染色质拓扑结构之间建立了直接联系。基于这些发现，我们建议对LRWD1在连接起源形成和染色质折叠中的功能进行详细的机制研究。我们将建立一个基于组织培养的LRWD1的dCas9募集系统，以引导LRWD1到特定的染色质位点，并诱导LRWD1和内聚蛋白耗尽模型，允许在特定的细胞周期阶段去除这些因子。我们将把这些实验系统与染色体构象捕获、DNA复制和ORC和复制前复合体亚基、LRWD1和内聚蛋白的ChIP分析相结合，以测试LRWD1的系带或去除如何影响起源形成和染色质折叠。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之间的关系，并将为这些过程如何在分子水平上交织在一起提供新的见解。

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.