We have used chromosome transmission fidelity (ctf) mutants and the deletion strain collections of S. cerevisiae to identify and characterize genes required for kinetochore function and checkpoint function. Studies with the ctf mutants led to the identification and characterization of the roles of SPT4 and NUP170 in chromosome segregation and spindle assembly checkpoint (SAC) function. We established a novel role for Spt4p in heterochromatic silencing. Using a cross-species approach, we showed that the yeast spt4 strains are complemented by human SPT4. Most importantly, we showed that S. cerevisiae SPT4 contributes to the proper localization of evolutionarily conserved centromeric histone H3 variant (CenH3) Cse4p. The major research goal of our laboratory is to investigate the molecular mechanisms that regulate the function of Cse4p and its interacting partners (Scm3p and Histone H4) to mediate faithful chromosome segregation. We investigated the mechanism of Cse4p localization and have established that mislocalization of Cse4p and altered histone stoichiometry lead to defects in chromosome transmission. Our studies have also shown that overexpression Scm3p and its human homolog HJURP leads to genome instability in yeast and human systems. We examined the effect of chromatin modifiers and post-translational modification of kinetochore proteins on the assembly/function of CenH3 chromatin. Our results showed that hypoacetylation state of centromeric histone H4 is critical for faithful chromosome segregation. Our recent results with Cse4p localization and histone dosage in S. cerevisiae are consistent with those in S. pombe, suggesting conservation of the underlying mechanisms. Thus, studies in S. cerevisiae that elucidate a mechanism for Cse4p localization and the role of chromatin modifications in centromere function may help us understand analogous pathways in humans and other systems. Our research on the molecular determinants of faithful chromosome transmission in S. cerevisiae will help us understand analogous processes in humans and their implications in human disease. Our laboratory is uniquely poised to utilize conventional genetic, biochemical, and cell biology approaches, as well as high-throughput genomic analysis for our research projects. We use an array of gene-deletion strains and a colony-picking robot for the identification of possible cancer drug targets and also for genetic screens by synthetic genome analysis (SGA), developed in the laboratory of Charlie Boone (University of Toronto).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010822-05
Application #
8349186
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$1,290,445
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Mishra, Prashant K; Ciftci-Yilmaz, Sultan; Reynolds, David et al. (2016) Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis. Mol Biol Cell 27:2286-300
Ohkuni, Kentaro; Takahashi, Yoshimitsu; Fulp, Alyona et al. (2016) SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin. Mol Biol Cell :
Mishra, Prashant K; Guo, Jiasheng; Dittman, Lauren E et al. (2015) Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination. Mol Biol Cell 26:2067-79
Ohkuni, Kentaro; Takahashi, Yoshimitsu; Basrai, Munira A (2015) Protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins Ndc10 and Ndc80. J Vis Exp :e52482
Lai, Xianning; Beilharz, Traude; Au, Wei-Chun et al. (2013) Yeast hEST1A/B (SMG5/6)-Like Proteins Contribute to Environment-Sensing Adaptive Gene Expression Responses. G3 (Bethesda) 3:1649-59
Haase, Julian; Mishra, Prashant K; Stephens, Andrew et al. (2013) A 3D map of the yeast kinetochore reveals the presence of core and accessory centromere-specific histone. Curr Biol 23:1939-44
Mishra, Prashant K; Ottmann, Alicia R; Basrai, Munira A (2013) Structural integrity of centromeric chromatin and faithful chromosome segregation requires pat1. Genetics 195:369-79
Boeckmann, Lars; Takahashi, Yoshimitsu; Au, Wei-Chun et al. (2013) Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae. Mol Biol Cell 24:2034-44
Au, Wei Chun; Dawson, Anthony R; Rawson, David W et al. (2013) A novel role of the N terminus of budding yeast histone H3 variant Cse4 in ubiquitin-mediated proteolysis. Genetics 194:513-8
Choy, John S; O'Toole, Eileen; Schuster, Breanna M et al. (2013) Genome-wide haploinsufficiency screen reveals a novel role for ?-TuSC in spindle organization and genome stability. Mol Biol Cell 24:2753-63

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