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. Amongst the top five lead candidates of the screen that showed increased nuclear YFP-CENP-A fluorescence were depletions of histone chaperones (CHAF1B/p60 and CHAF1A/p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibit CENP-A mislocalization, CIN phenotypes, and increased enrichment of CENP-A in the chromatin fraction. 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 have identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.