The centromeric nucleosome in yeast: Interaction with the inner kinetochore and assembly by the chaperone Yta7/ ATAD2

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The faithful transmission of genetic information from mother to daughter cells during mitosis requires that the sister chromatids of each chromosome are properly segregated to the daughter cells. For this, the kinetochore attaches on one side to the centromeric chromatin and on the other side to the microtubule and thus provides a physical link that allows the chromatids to be pulled towards opposite poles of the cell during chromosome segregation. In the last grant period, we have characterized the attachment of the inner kinetochore to the centromeric nucleosome in the yeast Saccharomyces cerevisiae. Specifically, we identified a physical interaction between the heterodimer Okp1 CENP Q/ Ame1 CENP-U and the extended amino-terminus of the yeast CENP-A homolog Cse4, and we found this interaction to be modulated by posttranslational modifications on the Cse4 CENP-A N-terminus. Okp1 CENP Q/ Ame1 CENP-U is part of the CTF19 complex of the inner kinetochore, the yeast equivalent of human CCAN. Furthermore, we have identified the histone chaperone Yta7 ATAD2 as a novel regulator of centromeric Cse4 CENP-A levels in yeast. We postulate that Yta7, a bromodomain-containing AAA+ ATPase with homology to unfoldases, functions to unfold Cse4/ H4 and to hand it to the CENP-A chaperone Scm3 HJURP for incorporation into the centromeric nucleosome. The current project proposes to build on this work and to obtain molecular insights into how Okp1 CENP Q/ Ame1 CENP-U interacts with the Cse4 N-terminus. Specifically, we will use proximity information obtained using biorthogonal amino acids and cross-linking/ mass spectrometry as well as structural analysis to determine the molecular details of the interaction between the centromeric nucleosome of yeast and Okp1/ Ame1 of the inner kinetochore. This is important in order to understand how physical attachment of the kinetochore to chromatin is achieved. Furthermore, we will elucidate in mechanistic detail how Yta7 ATAD2 regulates centromeric chromatin. We will characterize its interaction with Cse4/ H4 and the Cse4 chaperone Scm3 and its role in chromatin assembly using biochemical and structural approaches. We will combine this with an in vivo analysis to determine functionally relevant residues and domains of Yta7. Altogether, this project will provide mechanistic insights into regulation at the centromere that are important to understand how defects in this process contribute to chromosome missegregation.