During cell division chromosomes must be faithfully replicated and segregated to daughter cells. Errors in chromosome segregation are known to result in human genetic diseases and promote the progression of cancer. Therefore, understanding the mechanisms that control chromosome segregation will provide insight into the etiology of human diseases. Each eukaryotic chromosome possesses a single site called the centromere that is essential for chromosome segregation. During mitosis centromeres template the assembly of the mitotic kinetochore that attaches chromosomes to the microtubule spindle for segregation. Our long-term goal is to understand how centromeres and kinetochores are assembled to generate a microtubule-binding site that mediates chromosome segregation. The centromere is epigenetically determined by the replacement of histone H3 with the H3 variant centromere protein A (CENP-A). In the absence of CENP-A, centromeres and kinetochores do not assemble and chromosome segregation fails. The goals of this proposal are threefold. First, we will use human cell and cell extract based assays to determine the mechanism that couples centromere assembly to the cell cycle by studying the function of APC dependent proteolysis in the targeting of centromere assembly factors to the chromosome to assemble new CENP-A nucleosomes. Second, using reconstituted biochemical systems we will study how CENP-A nucleosomes are recognized by other centromere proteins to assemble the centromere and how the binding of other centromere proteins to CENP-A chromatin regulates chromatin structure. Third, using in vitro replication and assembly systems in cell extracts we will determine how CENP-A nucleosomes are distributed during replication and how the sites of new CENP- A assembly are determined. Our experimental plan should provide new insight into the problems of how centromeric chromatin is assembled and how that chromatin is recognized to assemble the centromere and kinetochore.

Public Health Relevance

Accurate chromosome segregation during cell division is essential for growth and development of all organisms. Chromosome segregation errors cause genetic disease and promote the progression of cancer in humans. Our proposal is focused on understanding the molecular mechanisms that ensure accurate chromosome segregation to inform how chromosome missegregation arises and contributes to human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074728-08
Application #
8323831
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Carter, Anthony D
Project Start
2005-09-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
8
Fiscal Year
2012
Total Cost
$325,737
Indirect Cost
$122,787
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Shang, Wei-Hao; Hori, Tetsuya; Westhorpe, Frederick G et al. (2016) Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres. Nat Commun 7:13465
Miell, Matthew D D; Straight, Aaron F (2016) In Vitro Kinetochore Assembly. Methods Mol Biol 1413:111-33
Westhorpe, Frederick G; Fuller, Colin J; Straight, Aaron F (2015) A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance. J Cell Biol 209:789-801
Westhorpe, Frederick G; Straight, Aaron F (2014) The centromere: epigenetic control of chromosome segregation during mitosis. Cold Spring Harb Perspect Biol 7:a015818
Lee, Carolyn S; Bhaduri, Aparna; Mah, Angela et al. (2014) Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma. Nat Genet 46:1060-2
Miell, Matthew D D; Fuller, Colin J; Guse, Annika et al. (2013) CENP-A confers a reduction in height on octameric nucleosomes. Nat Struct Mol Biol 20:763-5
Westhorpe, Frederick G; Straight, Aaron F (2013) Functions of the centromere and kinetochore in chromosome segregation. Curr Opin Cell Biol 25:334-40
Kato, Hidenori; Jiang, Jiansheng; Zhou, Bing-Rui et al. (2013) A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. Science 340:1110-3
Earnshaw, W C; Allshire, R C; Black, B E et al. (2013) Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21:101-6
Westhorpe, Frederick G; Straight, Aaron F (2012) The split personality of CENP-A nucleosomes. Cell 150:245-7

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