Dynamics and regulation of Shugoshin and other pericentromeric proteins in budding yeast

Abstract

Accurate distribution of genetic material is critical for the formation of functional cells and their proliferation. During cell division, sister chromatids separate from each other and segregate to opposite poles. To ensure accurate chromosome segregation all sister chromatids should be attached to microtubules from opposite spindle poles, known as bi-orientation. Cohesin is a protein complex that holds sister chromatids together from the time of its replication in S phase until anaphase onset, and it is required for proper chromosome segregation both in mitosis and in meiosis. It is distributed intermittently along the full length of chromosomes with significant enrichment in the region surrounding the centromere, known as the pericentromere. This chromosome domain was shown to be crucial for chromosome bi-orientation. In my PhD I studied how the establishment of tension between sister chromatids in the process of bi-orientation affects the distribution of different pericentromeric proteins on budding yeast chromosomes. It was known that levels of cohesin at the pericentromere are decreased in response to the establishment of tension. I demonstrate that other proteins, such as subunits of condensin, members of the Chromosome Passenger Complex (CPC) and others, exhibit similar dynamics, and suggest a model to explain this phenomenon. Out of all studied proteins, Shugoshin (Sgo1) was the only one that was completely removed from the pericentromere in response to spindle tension establishment. There is evidence that Sgo1 plays a role in sensing spindle tension and halting the cell cycle until this has been achieved but how it does so is not known. Therefore, removal of Shugoshin from the pericentromere might be a signal for the cell that bi-orientation occurred. I then found that spindle tension itself is not sufficient for Sgo1 re-localization from the pericentromere, and there are other factors that affect it. I showed that deletion of RTS1, a highly conserved regulatory subunit of Protein Phosphatase 2A (PP2A), results in substantial enrichment of Shugoshin at the pericentromere in the situation when spindle tension is absent. In addition, Bub1 kinase, a protein that is required for Sgo1 localization, was found to be removed from the centromere in response to spindle tension as well as Sgo1. The role of Bub1 the in localization of Shugoshin is to phosphorylate histone H2A, which then becomes a mark for Sgo1 loading. Therefore, we assume that Sgo1 dynamics and, potentially, its role in sensing bi-orientation, are regulated through the array of phosphorylation and de-phosphorylation events at the pericentromeric area. Finally, I have also found that budding yeast Sgo1 undergoes the posttranslational modification as sumoylation. I showed that sumoylation of Shugoshin is not required for its removal from the pericentromere during biorientation. However, it might be important for the regulation of Sgo1 degradation and its role in the metaphase to anaphase transition in mitosis.