Regulation of kinetochore function in yeast by modification of the core region of the centromeric nucleosome

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Centromeres are the regions of the chromosome where the kinetochore is assembled, and they establish a physical link between the centromeric chromatin and the microtubule for chromosome segregation in mitosis. In Saccharomyces cerevisiae, centromeric chromatin is defined by a single nucleosome that contains the histone H3 variant CENP-A Cse4 instead of canonical H3. The extended N-terminus of Cse4 interacts with Okp1/ Ame1 (the S. cerevisiae homologs of human CENP-Q/ CENP-U), which are components of the Ctf19 /CCAN complex, and recruits the inner kinetochore proteins to centromeric chromatin. In this proposal, we will investigate the function of the core region of the CENP-A Cse4 nucleosome in kinetochore assembly and centromere function in S. cerevisiae. Specifically, we will investigate a posttranslational modification in the αN-helix of Cse4 that lies close to the entry/ exit site of the DNA into the centromeric nucleosome. This will be complemented by the analysis of selected mutations in neighbouring Cse4 residues, which cause a distinct centromeric defect when mutated. We will isolate suppressor mutations of defects in the Cse4 core region, and the effect of the causative mutation in the context of kinetochore mutations will be evaluated. The respective factors will be investigated using molecular genetic and biochemical approaches in order to dissect their function in the regulation of the centromere and kinetochore. Due to the site of Cse4 modification, such factors are expected to act through positioning and sliding of the centromeric nucleosome, or through interaction of inner kinetochore proteins with the core nucleosome. They will thus identify novel regulators of the centromere and will provide molecular insights into the role of the centromeric nucleosome in kinetochore assembly and function.