Chromosome structure and function is modulated by Cohesin and its associated regulatory proteins

The Structural Maintenance of Chromosomes (SMC) family of proteins form a class of protein complexes that mediate chromosome structure through sister chromatid cohesion, chromosome condensation, DNA repair and transcription. Accessory proteins including Wpl1p, Pds5p and Eco1p regulate the SMC complex, cohesin, temporally and spatially to achieve these different functions. The roles and interactions of these three regulators are complicated. The goal of my project is to parse out the physical and genetic interactions between these regulators to understand how they regulate cohesin’s various functions./nPds5p and Wpl1p, known to form a sub-complex, appear to positively and negatively regulate cohesin. However, it is not known which functions of Wpl1p are mediated through its interaction with Pds5p, and which, if any are independent of Pds5p. I have shown that Wpl1p interacts with a non-essential domain in the N-terminus of Pds5p to promote cohesion and inhibit condensation. There is a discrepancy between the N-terminus of Pds5p acting as an inhibitor of condensation, and the full-length protein acting as a promoter or condensation. Thus, I have proposed a model in which Wpl1p inhibits Pds5p function through its interaction with the N-terminal regulatory domain of Pds5p. Additionally, this interaction is necessary but not sufficient for Wpl1p function./nI have also expanded our knowledge of an underappreciated role for Wpl1p in the DNA damage response. I have shown that Wpl1p function is important for efficient repair of S-phase DNA damage. Additionally, I have shown that this role in DNA repair is independent of Pds5p. Thus there are likely two forms of cohesin, a Pds5p-bound form that promotes cohesion and condensation, and a form not bound to Pds5p that mediates DNA damage-induced cohesion./nEco1p is known to critically acetylate the Smc3p sub-unit of cohesin at K112 K113 to promote cohesion. Aside from inhibiting Wpl1p function, it is unknown how this acetylation promotes cohesion. The smc3-D1189H allele located in the head domain of cohesin is able to compensate for loss of acetylation at K112 K113 to promote cohesion in a Wpl1p-independent manner. This characterization, along with other alleles in the Smc3p ATP binding pocket, show that cohesion activation through this pathway down-regulates ATPase activity, implicating ATPase function in a step past DNA binding for the first time. Additionally, in the absence of Eco1p function, smc3-D1189H only modestly promotes cohesion and fails to support viability, indicating that additional targets of Eco1p are needed to fully promote cohesion, viability and condensation./nTogether, the work presented here shows how both positive and negative regulation influence cohesin function. Additionally, put together, this work shows that balance between the positive and negative regulation is important for proper cohesin functions in cohesion, condensation and DNA repair. Wpl1p must promote cohesin to be dynamic by destabilizing cohesin’s interaction with DNA, while Eco1p and Pds5p promote stabilization of cohesin. Being able to promote a dynamic form of cohesin allows DNA tethering to change under each context. There still remains much to parse out, such as how Pds5p promotes condensation through cohesin, as well as how Wpl1p promotes destabilization of cohesin. Finally, identification of additional targets of Eco1p, and understanding how they modulate cohesin function will help to understand how cohesin-mediated tethering of DNA can structure the genome to perform diverse functions.

Advisor: Koshland, Douglas