Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

Division Of Basic Sciences - Nci

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Abstract

We use multi-organismal (yeast, mouse and human cells) and multi-disciplinary (genetic, cell biology, biochemical and genome-wide) approaches to study faithful chromosome segregation, a fundamental process of every living cell. Genetic screens served as a starting point and in-depth mechanistic studies have provided evidence for new roles for kinetochore genes and the identification of new kinetochore genes. We have identified and defined roles for post-translational modifications (acetylation, methylation, phosphorylation, sumoylation and ubiquitination) of Cse4 in chromosome segregation. Our research is focused on understanding the role of Cse4-associated proteins in chromosome segregation and defining pathways that prevent mislocalization of Cse4 to non-centromeric regions. In the first project we defined roles for Scm3, Pat1, Cdc5 and Sgo1 for the assembly of centromeric chromatin and characterized role of post-translational modifications of centromeric histones in faithful chromosome segregation. We determined that imbalanced stoichiometry of a Cse4 chaperone, Scm3 (HJURP in humans) leads 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). Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and Pat1 regulates the topology of centromeric chromatin (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) and provided evidence for a structural role for Pat1 in the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. 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. We reported that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016) and Cdc5-mediated phosphorylation of Cse4 regulates faithful chromosome segregation (Mishra et al., 2019). Furthermore, evolutionarily conserved Sgo1 which protects centromeric cohesion interacts with Cse4 and this is required for faithful chromosome segregation (Mishra et al., 2018). We recently reported that evolutionarily conserved Hpr1 prevents the accumulation of R-loops at centromeric chromatin affects the assembly of kinetochore and leads to chromosomal instability (Mishra et al., 2021). We have done a comprehensive analysis of Post-translational modifications (PTMs) of Cse4 and identified conserved sites for acetylation, methylation, and phosphorylation (Boeckmann et al., 2013). We determined that evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation. Using budding yeast with a single nucleosome we provided 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. A genome-wide screen with an HDACi was used 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 allow us to understand the molecular role of PTMs of Cse4 in chromosome segregation and determine if these PTMs are conserved in human CENP-A. In the second project we have focused on the identification of pathways that prevent mislocalization of Cse4 and CIN. 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). 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 determined that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016, 2018). Our recent results show that sumoylation of the C-terminus of Cse4 promotes mislocalization to non-centromeric regions (Ohkuni et al., 2020). Genome-wide approaches have been used to identify regulators that prevent mislocalization of Cse4 to euchromatin and these studies revealed a role for histone chaperones (Ciftci-Yilmaz et al., 2018). F-box proteins Cdc4 and Met30 in Cse4 proteolysis (Au et al., 2020) and Dbf4 dependent kinase (DDK) (Eisenstatt et al., 2020). We recently reported that reduced dosage of histone H4 prevents mislocalization of Cse4 (Eisenstatt et al., 2021) 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 chromosomal stability. In the third project, we have focused on causes and consequences of mislocalization of CENP-A in human cells. Mislocalization of CENP-A has been observed in many cancers and this correlates with poor prognosis. Hence, 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 recently reported that mislocalization of overexpressed CENP-A in pseudodiploid DLD1 cell line and xenograft mouse model contribute to CIN, aneuploidy with karyotypic heterogeneity (Shrestha et al., 2021). 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. In summary, our studies using multi-organismal and multi-disciplinary approaches will provide mechanistic insights for how defects in kinetochore function contribute to aneuploidy in human cancers. We are optimistic that our studies will help translate basic science research to the clinic and aid in the diagnosis, prognosis and treatment of cancers that show overexpression of CENP-A.