Mechanisms of cohesin regulation in vertebrates

项目概要/摘要 在脊椎动物细胞中，粘连蛋白复合物在核结构和功能中起着关键作用。它拴着 DNA复制的相同产物（称为姐妹染色单体）聚集在一起，直到细胞分裂，并且它也 介导染色体内桥接相互作用，形成染色体环和结构域。同时凝聚力 姐妹染色单体之间的相互作用对于准确的染色体分离和某些类型的 DNA 修复至关重要， 将染色体压缩成环和结构域对于正确转录和正常转录至关重要 发展。这些不同类型的内聚力在分子水平上有何不同，确保的机制 每个结果以及它们之间的重叠程度尚不清楚。多个的存在 许多粘连蛋白亚基和调节因子的直系同源物，以及 Sororin 的存在，这是独一无二的 对于后生动物，表明高等真核生物中粘连蛋白调节的复杂性。我们的工作解决了几个关键问题 该领域面临的挑战：1）DNA复制和粘连蛋白调节如何在脊椎动物中正确整合 细胞，2）脊椎动物特有的凝聚装置如何促进功能，以及 3）如何 粘连蛋白能否在局部进行重塑以确保特定的结果，例如基因表达或访问的变化 以及DNA修复机制的功能。和以前一样，我们将继续在多个系统中工作 适当的，包括基因组修饰的培养细胞、青蛙卵、青蛙胚胎的无细胞裂解物，以及 体外纯化蛋白质。我们的优势在于使用分子遗传学方法，使我们能够直接进行测试 所有这些实验系统中特定相互作用的影响。通过跟进我们最近的工作， 在下一个资助期结束时，我们希望充分了解 ESCO1 和 ESCO2 脊椎动物如何 粘合素修饰剂通过其惊人的非结构化域，在特定的上下文中稳定粘合力 方式。我们还将定义 DNA 复制叉上的蛋白质，特别是起始因子 TICRR，如何 影响粘连蛋白的稳定性，以及早期发育过程中对该过程的调节。最后，我们将利用 一个易于处理的位点特异性 DNA 损伤模型，用于表征脊椎动物粘连蛋白的贡献 损伤诱导的局部粘连蛋白重塑的调节剂和修饰，从而维持基因组。

Project Summary/Abstract In vertebrate cells the cohesin protein complex plays critical roles in nuclear structure and function. It tethers together the identical products of DNA replication, called sister chromatids, until cell division and it also mediates intra-chromosomal bridging interactions, forming chromosome loops and domains. While cohesion between sister chromatids is critical for accurate chromosome segregation and certain kinds of DNA repair, the compaction of chromosomes into loops and domains is essential for proper transcription and normal development. How these different kinds of cohesion differ at the molecular level, the mechanisms that ensure each outcome, and the amount of overlap between them are not well understood. The existence of multiple orthologs of a number of cohesin subunits and regulators, as well as the presence of Sororin, which is unique to metazoans, suggest complexity of cohesin regulation in higher eukaryotes. Our work addresses several key challenges in the field: 1) how are DNA replication and cohesin regulation properly integrated in vertebrate cells, 2) how do vertebrate-specific elaborations of the cohesion apparatus contribute to function, and 3) how can cohesin be remodeled locally to ensure specific outcomes, such as changes in gene expression or access and function of DNA repair machinery. As previously, we will continue to work in multiple systems as appropriate, including genome-modified cultured cells, cell free lysates from frog eggs, frog embryos, and purified proteins in vitro. Our strength lies in using molecular genetic approaches that allow us to test directly the impacts of specific interactions, in all of these experimental systems. By following up on our recent work, at the end of this next funding period we hope to fully understand how the ESCO1 and ESCO2 vertebrate cohesin modifiers, through their strikingly unstructured domains, stabilize cohesion in a context-specific manner. We will also define how proteins at the DNA replication fork, particularly the initiation factor TICRR, impact cohesin stabilization, and the regulation of this process during early development. Finally, we will exploit a tractable model for site-specific DNA damage to characterize the contributions of vertebrate cohesin regulators and modifications to damage-induced local cohesin remodeling and thus genome maintenance.