Mechanisms of cohesin regulation in vertebrates

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.