Dissecting Molecular Mechanisms of Centromeric CENP-A Assembly

The centromere facilitates the assembly of the kinetochore ensuring the accurate chromosome segregation. Active centromeres are specified epigenetically by nucleosomes containing the histone H3 variant, CENP-A, which is in place of histone H3. CENP-A is deposited by an assembly factor called HJURP in humans and Scm3 in yeast. However, homologs of HJURP/Scm3 are only present in a subset of eukaryotes. How CENP-A is deposited exclusively at centromeres in organisms that lack CENP-A chaperones remains unknown. This thesis addresses the above-mentioned gaps in the understanding of CENP-A assembly and identifies CAL1 as an essential recruiter for Drosophila CENP-A, which fulfills the function of HJURP/Scm3 in the dipteran lineage. Mis-targeting CAL1 is sufficient to trigger the formation of a functional centromere at the ectopic site, where the recruitment of centromeric proteins and microtubule attachments are detected. This additional centromere can be propagated epigenetically to the next generation and thus contributes to chromosome segregation defects. In addition, this thesis also provides evidence for chromatin requirements of CENP-A deposition, showing that transcription-coupled chromatin reorganization enables CENP-A incorporation. Together, this work demonstrates a mechanism of centromere establishment by an alternative CENP-A chaperone, CAL1