Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo, and hir2Δ strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4. Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2Δ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1, and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2Δ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4, thereby promoting genome stability.
Bibliographical noteFunding Information:
We are grateful to the members of the Basrai laboratory for helpful discussions and comments on the manuscript. We gratefully acknowledge Sue Biggins for reagents and advice and Kathy McKinnon of the National Cancer Institute Vaccine Branch fluorescence-activated cell sorting (FACS) Core for assistance with FACS analysis. M.A.B. and P.S.M. are supported by the National Institutes of Health (NIH) Intramural Research Program at the National Cancer Institute and D.L. by the NIH Intramural Research Program at the National Library of Medicine. This research was also supported by grants from the NIH to C.L.M. (R01HG005084), to C.B. and C.L.M. (R01HG005853) and to C.B. and M.C. (R01HG005853), from the Canadian Institute of Health Research to C.B. and M.C. (FDN-143264) and the Lewis-Sigler Fellowship to A.B. C.L.M. and C.B. are fellows in the Canadian Institute for Advanced Research (CIFAR, https:// www.cifar.ca/) Genetic Networks Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
© 2018 by the Genetics Society of America.
- Chromosome segregation
- Gene regulation
- Histone chaperone