The role of M18BP1 in the propagation of centromeric chromatin
Abstract/Contents
- Abstract
- 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.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Moree, Carl Benton III |
|---|---|
| Associated with | Stanford University, Department of Biochemistry |
| Primary advisor | Straight, Aaron, 1966- |
| Thesis advisor | Straight, Aaron, 1966- |
| Thesis advisor | Herschlag, Daniel |
| Thesis advisor | Theriot, Julie |
| Advisor | Herschlag, Daniel |
| Advisor | Theriot, Julie |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Carl Benton Moree, III. |
|---|---|
| Note | Submitted to the Department of Biochemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Carl Benton Moree
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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