The role of M18BP1 in the spatiotemporal regulation of CENP-A nucleosome assembly and centromere inheritance [electronic resource]

Faithful chromosome transmission during cell division underlies organismal reproduction and multicellular development in all kingdoms of life. In eukaryotes, attachment of chromosomes to microtubules of the mitotic spindle via a specialized region of chromatin called the centromere ensures the fidelity of this process. Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. The goal of my thesis work has been to understand how the CENP-A assembly machinery is recruited to the proper chromosomal locus and how the process of assembly is coupled to cell cycle progression to ensure proper establishment of the centromere before each cell division. New CENP-A assembly takes place during G1 to counterbalance the equitable redistribution of CENP-A to sister chromatids during DNA replication. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin and how its activity is regulated is unknown. Using a combination of in vitro biochemistry, cell-free reconstitution in Xenopus egg extract, and mutant analysis in cultured human cells, we have investigated several aspects of how the Mis18 complex component M18BP1 contributes to the spatiotemporal regulation of new CENP-A nucleosome assembly. First, we found that M18BP1 directly binds pre-existing CENP-A nucleosomes via a widely conserved motif that it shares with the constitutive centromere protein CENP-C. CENP-A nucleosome binding is inhibited in mitosis and is required for interphase M18BP1 localization and new CENP-A assembly. Second, we found that during metaphase, when CENP-A assembly is inhibited by high cyclin-dependent kinase (Cdk) activity, M18BP1 localizes to centromeres by binding CENP-C. The interaction between M18BP1 and CENP-C is mediated by a conserved SANTA domain in M18BP1 and requires M18BP1 phosphorylation by Cdk. In metaphase, M18BP1 binds the region of CENP-C that binds HJURP during interphase, suggesting a novel mechanism by which CENP-A assembly is inhibited during mitosis. Third, we found that M18BP1 is a substrate of the anaphase-promoting complex (APC). M18BP1 appears to be protected from premature degradation by associating with chromatin, and mutations that prevent M18BP1 recognition by the APC cause its accumulation at G1 centromeres. Mutations that disrupt both APC recognition and Cdk phosphorylation cause persistent M18BP1 localization throughout the cell cycle, indicating the presence of complementary mechanisms to limit M18BP1 localization and, likely, CENP-A assembly. In sum, we have elucidated key mechanisms targeting CENP-A assembly machinery to centromeric chromatin in interphase to promote its propagation and inheritance. This work identifies key roles for M18BP1 both in recognizing and assembling CENP-A nucleosomes, helping to define the 'epigenetic loop' of centromere inheritance. In addition, we have identified Cdk- and APC-mediated regulation of M18BP1 that govern its proper localization, ensuring faithful coupling of CENP-A assembly to cell cycle progression. We propose that dual Cdk/APC of M18BP1 may constitute a licensing mechanism that regulates the amount of new CENP-A assembly to ensure the faithful replication and inheritance of centromeric chromatin across cell divisions.

Keywords: Biochemistry.
Advisor: Straight Aaron
Krasnow Mark
Theriot Julie advisor. Thesis advisor