Centromere Identity and the Nature of the Cenp-A-Containing Nucleosome

The centromere is an essential chromosomal locus that serves as the site of kinetochore formation, ensuring accurate chromosome segregation during mitosis and meiosis. While most centromeres form on repetitive DNA, the underlying DNA sequence is neither necessary nor sufficient to support centromere function, suggesting that this locus is epigenetically defined. The histone H3 variant centromere protein A (CENP-A) replaces H3 in nucleosomes at the centromere and is the best candidate to provide this epigenetic mark. This thesis aims to understand the features of the CENP-A nucleosome that impart its ability to mark and stabilize functional centromeres. In the first part of the thesis, our work provides an in-depth study on the structure of CENP-A-containing nucleosomes and shows that CENP-A nucleosomes adopt an unconventional conformation in solution that results in both an altered histone core and wrap of DNA. Upon binding of the nonhistone protein CENP-C, the histone core and path of DNA wrapping it revert back to a canonical shape, but DNA termini flexibility becomes enhanced. These structural transitions imparted by CENP-C have important functional consequences, as endogenous centromeres lacking CENP-C lose CENP-A and have increased mitotic defects. In the second part of the thesis, I discuss the role that DNA sequence plays in centromere function. While seemingly indispensable, both the quality of DNA sequence and the quantity of DNA repeats are important for maintaining centromeres, and I outline our work that aims to understand both of these phenomena. Taken together, these works greatly increase our understanding of the intrinsic and extrinsic components of the CENP-A nucleosome and its role in centromere identity.