Roles of Replication-associated Factors in Cohesin Regulation

Kawasumi, Ryotaro (2020). Roles of Replication-associated Factors in Cohesin Regulation. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00011fc1

Abstract

Cohesin is a ring-shaped protein complex that consists of four core subunits: SMC1 and SMC3 forming a V-shape heterodimer held together at the open ends by the cohesin’s kleisin component, RAD21, and either SA1 or SA2 that associate with RAD21. Functionally, the cohesin ring mediates DNA-DNA interactions between distant loci both in cis and in trans, the latter type being important for sister chromatid cohesion, SCC. The SCC function of cohesin involves topological entrapment of one or two DNA strands into its lumen. Functionally, SCC is important for accurate segregation of chromosomes in mitosis and facilitates error-free recombination repair, overall being critical for genome integrity. Cohesin-DNA interaction is facilitated by the cohesin loader NIPBL-MAU2 and downregulated by WAPL-PDS5 that facilitates unloading of cohesin. The stabilization of cohesin on DNA against WAPL-mediated unloading is favored by cohesin acetylation at two evolutionarily conserved sites on SMC3 that is mediated by Eco1/Eso1 in budding and fission yeast, and by ESCO1 and ESCO2 in vertebrates. In addition, several replisome-associated components are involved in SCC establishment. However, their functions remain incompletely understood, especially in vertebrate cells that contain additional cohesin regulators. Using chicken lymphoma DT40 cells suitable for genetic engineering and cytogenetic studies, I aimed to understand the roles of ESCO1/2 and replication-associated factors in cohesin regulation. Our study revealed previously unknown roles for ESCO1/2 in interphase chromatin organization and both functional separation and cooperation in SCC and proliferation. Moreover, we found that dysfunctions in replisome-associated components can lead to SCC cohesion defects that depend on WAPL but less so on ESCO1/2-mediated SMC3 acetylation. Finally, our IP-MS experiments identified novel cohesin interactors, RUVBL1 and RUVBL2, with roles in SCC. Thus, additional regulations are likely to exist in vertebrate cells to instruct cohesin abundance and its cohesive state.