All organisms have mechanisms to ensure that cells produced from mitotic and meiotic divisions contain the proper number of chromosomes. The cell monitors that chromosomes are copied exactly once and then distributed correctly to daughter cells. This is critical since errors result in an incomplete chromosome complement which is highly correlated with cancer and causes spontaneous miscarriage, Downs, and other developmental disorders in humans. Our long term goal is to understand the molecular mechanisms that contribute to the fidelity of chromosome distribution. Many chromosome segregation mechanisms are conserved from budding yeast to man. Due to the ease of genetic manipulations in budding yeast, we use S. cerevisiae as our model organism. In particular, this proposal will explore the molecular mechanisms of chromosome segregation with particular attention to centromeres and kinetochores. Centromeres are cis acting sequences on chromosomes that are required for their correct segregation. Although centromere sequence is highly variable between organisms, centromeres are universally marked by a histone H3 variant, known as Cse4/CENP-A. This histone variant is incorporated into nucleosomes with centromere sequence which is critical to direct the formation of the kinetochore, a multi-protein structure essential for microtubule attachment and therefore chromosome segregation. The goal of this proposal is to understand in molecular detail how Cse4/CENP-A- containing nucleosomes are established and maintained in the genome. In particular we will characterize the role of a novel Cse4-associated factor on Cse4-deposition, kinetochore function and chromosome segregation. Our ultimate goal is to reconstruct centromeric chromatin and the inner kinetochore in vitro. These types of experiments will help us build the first detailed molecular understanding of centromeric chromatin and inner kinetochore formation in any organism. Our studies will help us evaluate current models for the function of centromeres and kinetochores in chromosome segregation, and may help elucidate the etiology of cancer and new avenues for therapy.

Public Health Relevance

Each cell in the body must maintain the correct number of chromosomes. When chromosomes are not accurately divided between cells, aneuploidy results, which is the state of having too many or too few chromosomes. Aneuploidy is highly correlated with cancer and causes spontaneous miscarriage and developmental disorders such as Downs syndrome. In particular, the topic of this proposal, Cse4/CENP-A, is a protein that is essential for the formation of centromeres and kinetochores, features of chromosomes that are required for their accurate segregation. Cse4/CENP-A has been shown to be overexpressed and mistargeted in human primary colorectal cancers [1]. An associated protein, CENP-H, induces aneuploidy when overexpressed in human cells [2]. Overexpression of CID, the fly homolog of CENP-A, promotes formation of ectopic centromeres and multicentric chromosomes which causes chromosome missegregation, aneuploidy, and growth defects [3]. These results demonstrate that Cse4/CENP-A significantly contributes to genome maintenance. A better understanding of the molecular requirements for localization of Cse4/CENP-A to centromeres and its participation in kinetochore function is crucial to our understanding of basic mechanisms that contribute to accurate chromosome segregation. 1. Tomonaga, T., et al., Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res, 2003. 63: p. 3511-6. 2. Tomonaga, T., et al., Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res, 2005. 65: p. 4683-9. 3. Heun, P., et al., Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell, 2006. 10: p. 303-15.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Carter, Anthony D
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Stowers Institute for Medical Research
Kansas City
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Hewawasam, Geetha S; Mattingly, Mark; Venkatesh, Swaminathan et al. (2014) Phosphorylation by casein kinase 2 facilitates Psh1 protein-assisted degradation of Cse4 protein. J Biol Chem 289:29297-309
Hewawasam, Geetha S; Gerton, Jennifer L (2011) Cse4 gets a kiss-of-death from Psh1. Cell Cycle 10:566-7
Shivaraju, Manjunatha; Camahort, Raymond; Mattingly, Mark et al. (2011) Scm3 is a centromeric nucleosome assembly factor. J Biol Chem 286:12016-23
Hewawasam, Geetha; Shivaraju, Manjunatha; Mattingly, Mark et al. (2010) Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4. Mol Cell 40:444-54
Camahort, Raymond; Shivaraju, Manjunatha; Mattingly, Mark et al. (2009) Cse4 is part of an octameric nucleosome in budding yeast. Mol Cell 35:794-805