Mechanisms of cohesin regulation in vertebrates

项目摘要/摘要 在脊椎动物细胞中，粘附素蛋白复合体在核结构和功能中起着至关重要的作用。它系住了 DNA复制的相同产物聚集在一起，称为姐妹染色单体，直到细胞分裂，它还 调节染色体内的桥接相互作用，形成染色体环和区域。当凝聚力 姐妹染色单体之间对于准确的染色体分离和某些类型的DNA修复是至关重要的， 将染色体压缩成环和结构域对于正常转录和正常是必不可少的 发展。这些不同类型的凝聚力在分子水平上是如何不同的，确保 每一种结果以及它们之间的重叠量都不是很好地理解。多重的存在 一些粘附素亚基和调节子的同源基因，以及独一无二的索罗林的存在 对于后生动物，暗示了高等真核生物中粘附素调控的复杂性。我们的工作解决了几个关键问题 该领域的挑战：1)DNA复制和粘附素调节在脊椎动物中是如何正确整合的 细胞，2)脊椎动物特有的凝聚力机制是如何对功能做出贡献的，以及3)如何 粘附素是否可以局部重塑以确保特定的结果，如基因表达或访问的变化 和DNA修复机制的功能。像以前一样，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，来自青蛙卵的无细胞裂解物，青蛙胚胎，以及 体外纯化的蛋白质。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 在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，请访问 在下一个资助期结束时，我们希望充分了解ESCO1和ESCO2是如何 粘附素修饰物通过其惊人的无结构结构域，在特定于上下文的 举止。我们还将定义DNA复制分叉上的蛋白质，特别是启动因子TICRR， 影响粘附素的稳定性，以及这一过程在早期发育过程中的调节。最后，我们将利用 描述脊椎动物粘附素贡献的一种易处理的定点DNA损伤模型 调节和修饰损伤诱导的局部粘连蛋白重塑，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.