The Role of M18BP1 in the propagation of centromeric chromatin
- Carl Benton Moree, III.
- Aug. 2012.
- Physical description
- online resource (x, 170 pages) : illustrations (some color)
- Includes bibliographical references (p. 156-170).
- Accurate chromosome segregation is essential for the viability of all organisms. During mitosis, each chromosome attaches 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). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation. The assembly of CENP-A into centromeric chromatin requires the activities of the Mis18 complex and the CENP-A chaperone HJURP, however how these proteins are recruited to centromeric chromatin is not known. In this work, we use an in vitro CENP-A assembly assay in Xenopus extract to examine the role of Mis18BP1, a component of the Mis18 complex, in CENP-A nucleosome assembly. We show that depletion of CENP-C prevents M18BP1 recruitment to centromeres and inhibits CENP-A chromatin assembly. We find that the recruitment of M18BP1 to centromeres occurs through a direct interaction with conserved domains in CENP-C. These findings suggest that the factors required for CENP-A assembly are recruited by the existing centromeric chromatin to ensure the assembly of new CENP-A nucleosomes at the site of the current centromere. In a separate set of experiments, we use an in vitro degradation assay in Xenopus extract to identify substrates of the Anaphase Promoting Complex or Cyclosome (APC/C) whose degradation regulates CENP-A assembly. We screen a collection of 20 centromere proteins and find that only M18BP1 is an APC/C substrate. We show that depletion of the APC/C from extract or mutation of the conserved destruction motifs prevented the degradation of M18BP1 in vitro. We also show that M18BP1 is degraded in vivo in a manner consistent with APC/C substrates. We find that the expression of a non-degradable M18BP1 mutant does not alter CENP-A assembly over the course of a single cell cycle, suggesting that there are multiple mechanisms in place to ensure the stable propagation of centromeric chromatin. In summary, we have identified a mechanism by which M18BP1 is recruited to centromeres to promote new CENP-A assembly. In addition, we have also demonstrated a novel role for the APC/C in regulating the activity of M18BP1. This work has furthered our understanding of how centromere chromatin is propagated and suggests a possible model in which CENP-C recruits M18BP1 to centromeres to promote new CENP-A assembly and that upon completion, M18BP1 is degraded by the APC/C to restrict CENP-A nucleosome assembly to a specific window in the cell cycle.
- Autoantigens > metabolism
- Chromosomal Proteins, Non-Histone > metabolism
- Xenopus Proteins > metabolism
- Binding Sites
- Centromere > metabolism
- Chromatin Assembly and Disassembly
- Local subject
- Publication date
- Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2012.