Our ongoing research is focused on the following: 1) Centromeric association of evolutionarily conserved Pat1 (Protein associated with topoisomerase II) and polo kinase Cdc5 regulate faithful chromosome segregation. Cse4 and its chaperone Scm3 (HJURP in humans), both of which are essential for chromosome segregation, are overexpressed and mis-localized in many cancers. Patients with elevated HJURP expression show a reduced survival rate. The role of HJURP overexpression in tumorigenesis is not yet understood. We are investigating the molecular mechanisms that regulate expression and localization of Cse4/CENP-A and its interacting proteins Scm3/HJURP for faithful chromosome segregation. We have shown that the imbalanced stoichiometry of HJURP and Scm3 lead to chromosome mis-segregation in both human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers (Mishra et al., 2011). Future studies will utilize genome-wide screens to identify genes/pathways that show lethality with overexpression of HJURP for possible treatment of cancers with deregulated HJURP expression. Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and we have uncovered a role for Pat1 in the topology of centromeric chromatin and chromosome segregation (Mishra et al., 2013). We used a pat1 deletion strain to define the number of Cse4 molecules at the yeast kinetochore (Hasse, Mishra 2013, Mishra et al., 2015). Our results show that Pat1 regulates the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. Ongoing research is aimed at understanding how topology of centromeric chromatin affects chromosome segregation an area of research that is largely unexplored at the present time. In addition to kinetochore proteins, association of cohesins with centromeres and along the length of the chromosomes ensures faithful segregation of sister chromatids during mitosis. Our studies have shown that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016). Future studies will allow us to understand the mechanism by which Cdc5 regulates removal of centromeric cohesins. 2) Post-translational modifications (PTMs) of centromeric histones affect chromosome segregation. Distinctive acetylation pattern of centromeric histone H4 has been previously reported in other systems, however, the physiological role for this pattern is not fully understood. Using budding yeast with a single nucleosome we determined that the acetylation pattern of centromeric histone H4 affects chromosome segregation. We provide the first evidence that yeast centromeres contain hypoacetylated histone H4 and that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation (Choy et al., 2011). Even though HDAC inhibitors (HDACi) are used in clinical trials we do not fully understand their mode of action. Hence, we performed a genome-wide screen with an HDACi to identify pathways that are vulnerable to altered histone acetylation. Our results showed that chromosome segregation mutants are more sensitive to HDACi (Choy et al., 2015). Future studies will examine if combining HDACi with drugs that affect chromosome segregation are more effective for cancer treatment with a minimal effect on normal cells. An innovative approach for the biochemical purification of Cse4, allowed us to provide the first comprehensive analysis of PTMs of Cse4 (Boeckmann et al., 2013). 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 evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation. Future studies will allow us to understand the molecular role of Cse4 phosphorylation and methylation in chromosome segregation and determine if these PTMs are conserved in human CENP-A. 3) Stringent regulation of cellular levels of Cse4 prevents its mislocalization for genome stability. We showed previously that S. cerevisiae spt4 mutants show mislocalization of Cse4 and chromosome segregation defects that are complemented by human SPT4 (Basrai et al, 1996 and Crotti and Basrai 2004). We established the cause and effect of Cse4 mislocalization by showing that altered histone dosage and mislocalization of Cse4 to non-centromeric chromatin correlate with chromosome loss (Au et al., 2008). One mechanism that prevents mislocalization of Cse4 is ubiquitin-mediated proteolysis of Cse4 by E3 ligase Psh1. We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013). We recently reported that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016, 2018). We have undertaken genome-wide approaches to identify regulators that prevent mislocalization of Cse4 to euchromatin. Our studies have revealed a role for histone chaperones (Ciftci-Yilmaz et al., 2018) and other E3 Ub ligases in Cse4 proteolysis. Our ongoing studies are aimed at in-depth analysis of the yeast genes identified in the screen to understand the molecular mechanisms that prevent mislocalization of Cse4 for genome stability. 4) Mislocalization of CENP-A contributes to CIN in human cells. Given the clinical significance of high CENP-A expression and its correlation with cancer, it is critical to understand how CENP-A overexpression contributes to tumorigenesis and whether CENP-A expression can be exploited for prognosis, diagnosis and targeted treatment of CENP-A overexpressing cancers. We established cell lines and optimized cell biology based assays to address a long-standing question of whether mislocalization of overexpressed CENP-A contributes to CIN. We determined that constitutive or inducible expression of CENP-A in HeLa and stable diploid RPE1 cells results in mislocalization of CENP-A to non-centromeric regions. Comprehensive analysis for mitotic effects showed a dose-dependent effect of CENP-A overexpression on chromosome segregation defects and higher incidence of micronuclei. Altered localization of kinetochore proteins contributes to a weakening of the native kinetochore in CENP-A overexpressing cells. Depletion of the histone chaperone DAXX prevents CENP-A mislocalization and rescues the CIN phenotype in CENP-A overexpressing cells. These results show that mislocalization of CENP-A is one of the major contributors for CIN in CENP-A overexpressing cells. Our studies provide the first evidence for how mislocalization of CENP-A to non-centromeric chromatin contributes to CIN in human cells and provide mechanistic insights into how CENP-A overexpression may contribute to aneuploidy in CENP-A overexpressing cancers (Shrestha et al., 2017). We are pursuing studies with human homologs of the yeast genes identified in genome wide screens and using other approaches to identify and characterize pathways that prevent mislocalization of CENP-A for genome stability.
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