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An in vitro CENP-A assembly assay reveals a role for CENP-C in CENP-A deposition [electronic resource]

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule-binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant centromere protein A (CENP-A). CENP-A nucleosomes are believed to epigenetically specify centromere identity, thus understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. HJURP binds to pre-nucleosomal CENP-A and facilitates the deposition of new CENP-A nucleosomes into centromeric chromatin. The mechanistic roles of the Mis18 complex in CENP-A assembly and maintenance are not well understood. The Mis18 complex and HJURP localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The molecular interactions that control their targeting are unknown. Several constitutive centromere proteins that remain associated with CENP-A chromatin throughout the cell cycle have also been implicated in CENP-A assembly, including CENP-C. Their functions in CENP-A assembly are unknown. The first chapter of this thesis work describes the development of an in vitro system for centromeric chromatin assembly in Xenopus laevis egg extracts. We show that CENP-A assembly in extract recapitulates the cell cycle dependence and HJURP requirement of CENP-A assembly in somatic cells. We then use this in vitro system to identify a novel role for CENP-C in recruiting CENP-A assembly factors to the centromere to promote CENP-A assembly, described in Chapter 2. We show that CENP-C is required to target the Mis18 complex protein M18BP1 to Xenopus sperm centromeres in metaphase. In the absence of CENP-C, M18BP1 and HJURP targeting to centromeres is disrupted and new CENP-A assembly into centromeric chromatin is inhibited. We find that CENP-C interacts directly with M18BP1 through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly. Overall, this work extends our mechanistic understanding of how the pre-existing centromere directs the local assembly of new CENP-A nucleosomes to ensure stable propagation of the centromere. Furthermore, the ability to assemble centromeric chromatin in vitro will provide a valuable tool for dissecting the biochemical and cell cycle regulatory mechanisms that control new CENP-A assembly and ensure faithful centromere propagation.