Investigation of the role of Pds5 and Wapl in sister chromatid separation in mammalian cells

The key element of sister chromatid cohesion (SCC) is the cohesin complex composed of Smc1, Smc3, Scc1 and Scc3 (SA1 and SA2 in vertebrates). However, additional proteins such as Pds5 and Wapl are also known to regulate SCC. Most of our knowledge of Pds5 function derives from studies in yeasts and humans, and its role in SCC, although explored, remains unclear. Previous studies have implicated Pds5 in maintaining SCC in budding yeast and fungi (Sordaria). In Xenopus egg extracts, however, Pds5 is reported to be required for sister chromatid resolution during prophase. Since the role of Pds5 in regulating SCC in mammalian cells remains unclear, the aim of this project was to characterize the function of the two human Pds5 homologues, Pds5A and Pds5B and human Wapl in the regulation of sister chromatid cohesion. Here using mammalian cells, I show that the Wapl protein level is cell cycle regulated and the protein dissociates from chromatin during prophase. In contrast, Pds5A and Pds5B protein levels are constant throughout the cell cycle and dissociate from chromatin in a consecutive manner during mitosis. siRNA-mediated depletion of Pds5A, Pds5B and Wapl individually prevented mitotic chromosome separation and activated the spindle assembly checkpoint (SAC), although some cells circumvented the SAC and exhibited mitotic defects. However, unlike Wapl, depletion of Pds5 proteins was also found to slow S-phase progression and induce apoptosis through activation of the DNA damage checkpoint. I also found that the association of Wapl with Pds5A increased after S-phase and continued until Wapl degradation after mitosis. Inhibition of either Cdk1/cyclin B1 or Plk1 abolished the association between Wapl and Pds5A. The current model of vertebrate cohesin dissociation from chromosomes indicates a role for Sororin and SA2 phosphorylation in the removal of the cohesin complexes from chromosomal arms. Interestingly, I show that Wapl is a new substrate for Cdk1/cyclin B1 and Plk1. My data imply that Wapl phosphorylation at prophase is also required for the removal of cohesin complexes from chromosomal arms. Taken together, my results suggest that a reconsideration of the current model may be required to explain the mechanism of cohesion removal from chromosomes at mitosis.