Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin.

Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.

Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae.

Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to noncentromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A have been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) lead to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, and SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to noncentromeric regions, leading to CIN when Cse4 is overexpressed.