Molecular Determinants of Chromosome Transmission and Cell Cycle Regulation

Division Of Basic Sciences - Nci

Investigators

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Abstract

We use multi-organismal (yeast, xenograft mouse and human cells) and multi-disciplinary (genetic, cell biology, biochemical and genome-wide) approaches to study chromosomal stability. We have pioneered studies in these areas and determined that a balanced stoichiometry of kinetochore proteins, topology of centromeric chromatin and post-translational modifications of centromere associated proteins restrict the localization of Cse4/CENP-A to centromeric chromatin and prevent CIN. Provided below is a summary of our studies with budding yeast and human cells during the last year (2024-2025). For studies with budding yeast, we showed that Set1-mediated cell cycle dependent methylation of evolutionarily conserved Dam1/DASH complex (Ska1 in metazoans) plays a crucial role in the maintenance of kinetochore assembly for faithful chromosome segregation (Mishra et al., PloS Genetics 2025). In the second study we showed that evolutionarily conserved Mck1 kinase interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability . Our results showed that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability (Zhang et al., Genetics 2024). In the third study we developed a novel in vivo assay for examining the interaction of histone H4 with Cse4. Our results showed that the Cse4-H4 interaction facilitates conformational changes in Cse4 for its sumoylation and mislocalization (Ohkuni et al., Nucleic Acids Res 2024). In the fourth study we showed that altered stoichiometry of histone H3 and expression of oncohistone mutation H3 E97K contributes to mislocalization of Cse4 and CIN. Oncohistone mutations in the globular domain of histone H3 such as H3 E97K occur in several cancers. These results highlight the importance of histone H3 stoichiometry in preventing mislocalization of Cse4 for chromosomal stability and suggest that oncohistone H3 mutations may contribute to CIN in human cancers (Ohkuni et al., Nucleic Acids Res. 2025). For the second project with human cells we focused on identification of pathways that prevent mislocalization of Cse4 and CIN. In the first study we defined a novel role for Beta-TrCP-mediated proteolysis of Mis18Beta in preventing mislocalization of CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in Beta-TrCP depleted cells is dependent on high levels of Mis18Beta as depletion of Mis18Beta suppresses mislocalization of CENP-A in these cells. We propose that deregulated expression of Mis18Beta (Sethi et al, Mol. Cell. Biol. 2024). In the second study we pursued in depth studies with DNAJC9 a histone co-chaperone identified amongst the top candidates in a genome-wide siRNA screen for gene depletions that lead to increased nuclear CENP-A levels. Our results show that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN (Balachandra et al, EMBO J 2024). In the third study we pursued studies with Protein Phosphatase 1 Regulatory Subunit (PNUTS) another hit in the genome-wide siRNA screen for gene depletions that lead to increased nuclear CENP-A levels. Our results showed that depletion of PNUTS resulted in high nuclear CENP-A levels throughout the cell cycle in a PP1-dependent manner, mislocalization of CENP-A on mitotic chromosomes, defects in kinetochore integrity and CIN phenotypes. These studies highlight the importance of phospho-regulation mediated by PNUTS in preventing CENP-A mislocalization and CIN (Balachandra et al., Mol. Cell. Biol. 2025). In summary, our studies using multi-organismal and multi-disciplinary approaches have provided 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.