Abstract
Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.
Highlights
Each cell division requires the doubling of both DNA and histone content, with half of the histones being of parental origin and half being newly synthesised
During analyses of histone chaperone localisation, we observed a striking discrepancy between biochemical fractionation and immunofluorescence in the sub-cellular localisation of a number of histone chaperones
Whilst s/tNASP, HAT1, RbAp46 and ASF1A appear overwhelmingly cytosolic using a standard NP-40 lysis protocol (Suzuki et al, 2010), with CAF1p60 almost split, they appear entirely nuclear when probed by immunofluorescence
Summary
Each cell division requires the doubling of both DNA and histone content, with half of the histones being of parental origin and half being newly synthesised. Whilst much effort has gone into studying the dynamics of recycled parental histones (Prior et al, 1980; Jackson, 1987, 1990; Katan-Khaykovich & Struhl, 2011; RadmanLivaja et al, 2011; Alabert et al, 2015), less is known about the program for newly synthesised histone incorporation. As they form the stable core of the nucleosome and are the substrates for the majority of post-translational marks, histones H3 and H4 are often at the forefront of these investigations. A number of importin-b proteins have been suggested to provide chaperoning roles for basic nuclear cargo including ribosomal proteins and linker histones (Jakel et al, 2002), but not, as yet, the core histones
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