A Genome-Wide Screen Reveals a Role for the HIR Histone Chaperone Complex in Preventing Mislocalization of Budding Yeast CENP-A

Abstract

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 (, ,, ) and a Cse4-specific E3 ubiquitin ligase,, 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 , and 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 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 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.

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast

“…We investigated the physiological consequences of defects in Cse4 proteolysis in met30-6 and cdc4-1 strains. Increased stability of overexpressed Cse4 in psh1Δ, slx5Δ and hir2Δ strains correlates with its mislocalization to non-centromeric regions [24][25][26]33]. We reasoned that the strong defects in proteolysis of endogenous Cse4 may contribute to its mislocalization and CIN in met30-6 and cdc4-1 strains.…”

“…The rationale for this is based on the essential role of the N-terminus for its interactions with kinetochore proteins, Ub-mediated proteolysis and post-translational modifications (PTMs) of Cse4 [19,26,27,[54][55][56][57][58][59][60][61][62]. Furthermore, we recently showed that hir mutants, which are defective in proteolysis of overexpressed Cse4, are sensitive to GAL-CSE4 but not GAL-cse4Δ129 (Cse4 lacking the N-terminal domain) [33]. Growth assays showed that GAL-cse4Δ129 did not result in lethality in WT, met30-6 or cdc4-1 strains (Fig 2A), suggesting that the N-terminus of Cse4 is required for the SDL of GAL-CSE4.…”

“…Defects in Cse4 proteolysis contribute to GAL-CSE4-mediated SDL in psh1Δ, doa1Δ, slx5Δ and hir2Δ strains [19,[24][25][26]33]. Hence, we examined the stability of overexpressed HA-Cse4 in WT, met30-6 and cdc4-1 strains using whole cell extracts from strains grown at the GO analysis of negative genetic interactor genes with GAL-CSE4 for biological processes.…”

“…Chromatin immunoprecipitation was performed with two biological replicates as described previously [33,94]. Wild type, met30-6, and cdc4-1 strains expressing HA-Cse4 were grown logarithmically in YPD at 25˚C.…”

“…Psh1-mediated proteolysis of Cse4 is also regulated by the FACT (Facilitate Chromatin Transcription/transactions) complex and Casein kinase 2 (CK2) [29,30]. In addition to ubiquitin ligases, chromatin associated complexes, such as the SWI/ SNF, HIR and kinetochore protein Spt4, prevent mislocalization of Cse4 to non-centromeric regions [31][32][33][34]. Recently, it was shown that the cell cycle regulated expression of Cid and Cnp1 contribute to preventing their mislocalization to non-centromeric regions in flies and fission yeast, respectively [35,36].…”