The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity.

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.

Ypt4 and lvs1 regulate vacuolar size and function in Schizosaccharomyces pombe.

The yeast vacuole plays key roles in cellular stress responses. Here, we show that deletion of lvs1, the fission yeast homolog of the Chediak-Higashi Syndrome CHS1/LYST gene, increases vacuolar size, similar to deletion of the Rab4 homolog ypt4. Overexpression of lvs1-YFP rescued vacuolar size in ypt4Δ cells, but ypt4-YFP did not rescue lvs1Δ, suggesting that lvs1 may act downstream of ypt4. Vacuoles were capable of hypotonic shock-induced fusion and recovery in both ypt4Δ and lvs1Δ cells, although recovery may be slightly delayed in ypt4Δ. Endocytic and secretory trafficking were not affected, but ypt4Δ and lvs1Δ strains were sensitive to neutral pH and CaCl2, consistent with vacuolar dysfunction. In addition to changes in vacuolar size, deletion of ypt4 also dramatically increased cell size, similar to tor1 mutants. These results implicate ypt4 and lvs1 in maintenance of vacuolar size and suggest that ypt4 may link vacuolar homeostasis to cell cycle progression.