During mitosis, all 23 human chromosomes must be faithfully transmitted to progeny cells. Errors in this process result in aneuploidy, a form of genetic instability associated with tumorigenesis. Thus, mechanisms controlling chromosome segregation are increasingly viewed as significant to cell homeostasis and human health. Centromeric regions of chromosomes are essential for chromosome segregation because they mediate attachment to the mitotic spindle and act as cohesion sites linking sister chromatids. The central hypothesis underlying this proposal is that the ubiquitin-related Smt3/SUMO-1 protein modifier is a key regulator of these two aspects of centromere function. We have found the yeast Smt3/SUMO-1 deconjugase Smt4 interacts with the Mif2 kinetochore protein and smt4 mutants display a defect in centromere chromatin organization. Based on these data, our first aim tests the hypothesis that Smt3/SUMO-1 modification regulates a pathway connecting Mif2 and centromere/kinetochore integrity. We will analyze this pathway by isolating mif2 mutants defective for Smt4 interaction and suppressors of the smt4 defect in CEN structure. Our data also indicate that smt4 mutants fail to maintain cohesion at centromeric regions. Thus, our second aim tests the hypothesis that Smt3/SUMO-1 regulates a mechanism that specifically controls cohesion at centromeres. It has been proposed that centromeric cohesion is controlled by a tensiometer, a chromatin domain that modulates cohesion in response to tension exerted by the mitotic spindle. To examine whether Smt3/SUMO-1 modification regulates a tensiometer-like activity we will visualize centromere cohesion dynamics in Smt3/SUMO-1 conjugation mutants and develop a chromatin affinity purification technique to identify Smt3/SUMO-1 substrates that may be tensiometer components. Finally, we have isolated DNA topoisomerase II (Top2) as a Smt3/SUMO-1 substrate that can suppress the Smt4 cohesion defect.
Our third aim tests the hypothesis that Top2 Smt3/SUMO-1 modification coordinates chromatid topology with centromere cohesion dynamics. We will determine whether chromatid intertwining is associated with centromeric cohesion and examine the function of Top2 Smt3/SUMO-1 modification by isolating factors requiring this modification for survival. These studies will elucidate potentially conserved mechanisms controlling chromosome segregation and identify additional Smt3/SUMO-1 substrates regulating centromere function.
