A specific role for Importin-5 and NASP in the import and nuclear hand-off of monomeric H3

Abstract

Core histones package chromosomal DNA and regulate genomic transactions, with their import and deposition involving a dedicated repertoire of molecular chaperones. Histones H3 and H4 have been predominantly characterised as obligate heterodimers, however, recent findings have alluded to the existence of a significant pool of monomeric histone H3 in the nucleoplasm. Using a combination of in vitro and in vivo experiments, here we show that monomeric H3 and H4 use an Importin 5 (Imp5) dependent pathway for their nuclear import, distinct from Importin 4 (Imp4) previously described for H3-H4 dimers. Using mutants that disrupt the histone fold, we show monomeric H3 loses its interaction with Imp4, but retains interactions with Imp5 and the chaperone NASP. H4 monomeric mutants similarly bind Imp5 and not Imp4, however, they lose interaction with NASP, retaining their interaction with the HAT1-RBBP7 complex instead. In vitro experiments revealed that Imp5 and NASP are mutually exclusive in their binding, suggesting a facilitated hand-off mechanism. Furthermore, new H3 accumulates rapidly in a NASP-bound complex after nuclear translocation. NASP can assemble into three distinct co-chaperoning complexes, including a novel complex containing NASP, H3 and the putative ubiquitin ligase UBR7, a NASP-H3-H4-RBBP7 subcomplex and the previously characterised NASP-H3-H4-ASF1-HAT1-RBBP7 multi-chaperoning complex. Here we propose an alternative import pathway and folding mechanism for monomeric H3 and H4 that involves Imp5, rather than Imp4, and hands off to nuclear chaperones NASP, RBBP7 and HAT1.