Evolutionarily conserved Cse4 and its chaperone Scm3 (HJURP in humans) which are essential for chromosome segregation have been shown to be overexpressed in many cancers. Overexpression and mis-localization of HJURP has been reported in lung and breast cancer cells and patients with elevated HJURP expression show reduced survival rate. Whether HJURP overexpression induces tumorigenesis is not understood. We showed that imbalanced stoichiometry of HJURP and SCM3 lead to defects in chromosome segregation and kinetochore integrity in human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers. Genome wide screens will now allow us to identify genes/pathways that suppress or enhance phenotypes associated with overexpression of SCM3/HJURP for possible extrapolation to cancers. In continuation of these studies we have shown that Pat1 (Protein associated with topoisomerase II) interacts with Scm3. We determined that structural integrity of centromeric chromatin and faithful chromosome segregation requires Pat1. In collaboration with Kerry Bloom we used a pat1 null strain to define the number of Cse4 molecules at the yeast kinetochore. Studies with Pat1 have provided important insights into how topological structure of centromeric chromatin regulates chromosome segregation an area of research that is largely unexplored at the present time. Faithful chromosome segregation is also regulated by post-translational modifications (PTM) of centromeric histones and kinetochore proteins. We investigated the nature and role of PTM of centromeric histone H4 and Cse4 in budding yeast with the long-term objective of targeting PTM of histones for anti-cancer therapy. We showed for the first time that budding yeast centromeres contain hypoacetylated histone H4 and also that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation. We also discovered that a balance in H4K16 acetyltransferase, Sas2, and Histone Deacetylase (HDAC), Sir2, is required for chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. We are now investigating if acetylation pattern of H4 is cell cycle regulated, if altered H4 acetylation affect the structure of centromeric chromatin and the role of HDAC's in chromosome segregation. HDAC inhibitors are used for treatment of certain cancers, however, we do not fully understand the molecular targets of these inhibitors. We propose that combining HDAC inhibitors with drugs that compromise kinetochore function may be more effective for cancer treatment with minimal effect on normal cells. In order to investigate the role of PTM of Cse4 we devised an innovative approach for biochemical purification of Cse4 and this facilitated the first comprehensive analysis of PTMs of Cse4. Conserved sites for acetylation, methylation and phosphorylation in Cse4 were identified. We generated a phospho-specific antibody and showed the association of phosphorylated Cse4 with centromeres and determined that Ipl1 phosphorylates Cse4 in vivo and in vitro to regulate chromosome segregation. In continuation of our studies with PTM, we have shown that the N-terminus of Cse4 is ubiquitinated to regulate its proteolysis and localization. We established the cause and effect of Cse4 mis-localization by showing that altered histone dosage and mis-localization of Cse4 to non-centromeric loci correlates with chromosome loss. In collaboration with Charlie Boone we have used genome-wide screens to identify several new regulators for Cse4 proteolysis that are evolutionarily conserved. Overexpression and mis-localization of human homolog of Cse4 (CENP-A) is observed in colorectal cancers and leads to aneuploidy in flies. The long-term objective of our research is to identify pathways that will specifically lead to killing of cancer cells overexpressing CENP-A. Given the evolutionary conservation of pathways for genome stability we decided to use budding yeastinvestigate the role of haploinsufficiency (HI). HI is a condition where a single functional copy of a gene is insufficient to sustain normal activity and leads to a mutant phenotype. HI leads to higher incidences of tumorigenesis and many tumors display aneuploidy. We designed a novel genome-wide screen using the hemizygous yeast deletion library representing nearly all genes (6500) to identify and characterize genes that are HI for genome stability. We defined novel roles for BCY1 and the evolutionarily conserved Gamma Tubulin complex as HI for chromosome segregation. Taken together, our studies with budding yeast and its human homologs are providing critical insights into the causes and consequences of errors in chromosome segregation that are frequently observed in many cancers.
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