Mechanisms of cohesin regulation in vertebrates

项目概要/摘要 在脊椎动物细胞中，粘附蛋白复合物在核结构和功能中起关键作用。它束缚着 DNA复制的相同产物，称为姐妹染色单体，直到细胞分裂，它也 介导染色体内桥接相互作用，形成染色体环和结构域。虽然凝聚力 姐妹染色单体之间的相互作用对于精确的染色体分离和某些类型的DNA修复至关重要， 染色体压缩成环和结构域对于正确的转录和正常的转录是必需的。 发展这些不同种类的凝聚力在分子水平上是如何不同的， 每一个结果，以及它们之间的重叠量都没有得到很好的理解。存在多 许多粘附素亚基和调节子的直系同源物，以及Sororin的存在，这是独特的 后生动物，表明在高等真核生物中的复杂性的粘附调节。我们的工作解决了几个关键问题 该领域的挑战：1）DNA复制和粘着蛋白调节如何在脊椎动物中正确整合 细胞，2）脊椎动物特有的内聚器如何有助于功能，以及3）如何 粘着蛋白是否可以局部重塑以确保特定的结果，例如基因表达或通路的变化 和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，来自青蛙卵的无细胞裂解物，青蛙胚胎， 体外纯化蛋白质。我们的优势在于使用分子遗传学方法， 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， 在下一个资助期结束时，我们希望能够完全了解ESCO 1和ESCO 2脊椎动物 内聚蛋白修饰剂，通过其惊人的非结构化结构域，稳定在特定的上下文中的内聚， 方式我们还将定义DNA复制叉处的蛋白质，特别是起始因子TICRR， 影响粘着蛋白的稳定性，以及在早期发育过程中对该过程的调节。最后，我们将利用 脊椎动物粘着蛋白对位点特异性DNA损伤的作用 调节剂和修饰损伤诱导的局部粘连蛋白重塑，从而基因组的维护。

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.