A specific role for importin-5 and NASP in the import and nuclear hand-off of monomeric H3
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Evaluation Summary:
This is a very fundamental study that challenges the paradigm that histones H3 and H4 are imported to the nucleus primarily as heterodimers. Instead this study provides compelling evidence that H3 and H4 are imported by importin 5 as monomers and dimerize on chaperones in the nucleus. The work is of relevance to colleagues studying nuclear import and epigenetic regulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Core histones package chromosomal DNA and regulate genomic transactions, with their nuclear import and deposition involving importin-β proteins and a dedicated repertoire of histone chaperones. Previously, a histone H3-H4 dimer has been isolated bound to importin-4 (Imp4) and the chaperone ASF1, suggesting that H3 and H4 fold together in the cytoplasm before nuclear import. However, other studies have shown the existence of monomeric H3 in the nucleus, indicating a post-import folding pathway. Here, we report that the predominant importin associated with cytoplasmic H3 is importin-5 (Imp5), which hands off its monomeric cargo to nuclear sNASP. Imp5, in contrast to Imp4, binds to both H3 and H4 containing constitutively monomeric mutations and binds to newly synthesised, monomeric H3 tethered in the cytoplasm. Constitutively monomeric H3 retains its interaction with NASP, whereas monomeric H4 retains interactions specifically with HAT1 and RBBP7. High-resolution separation of NASP interactors shows the ‘s’ isoform but not the ‘t’ isoform associates with monomeric H3, whilst both isoforms associate with H3-H4 dimers in at least three discrete multi-chaperoning complexes. In vitro binding experiments show mutual exclusivity between sNASP and Imp5 in binding H3, suggesting direct competition for interaction sites, with the GTP-bound form of Ran required for histone transfer. Finally, using pulse-chase analysis, we show that cytoplasm-tethered histones do not interact with endogenous NASP until they reach the nucleus, whereupon they bind rapidly. We propose an Imp5-specific import pathway for monomeric H3 that hands off to sNASP in the nucleus, with a parallel H4 pathway involving Imp5 and the HAT1-RBBP7 complex, followed by nuclear folding and hand-off to deposition factors.
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Evaluation Summary:
This is a very fundamental study that challenges the paradigm that histones H3 and H4 are imported to the nucleus primarily as heterodimers. Instead this study provides compelling evidence that H3 and H4 are imported by importin 5 as monomers and dimerize on chaperones in the nucleus. The work is of relevance to colleagues studying nuclear import and epigenetic regulation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study, the authors use a powerful mix of histone mutations, biochemistry, and proteomics to show that histones H3 and H4 are imported into the nucleus as monomers by Imp5 where they are transferred to NASP and HAT1-RBBP7 respectively. This challenges the standing paradigm that H3 and H4 are imported into the nucleus as heterodimers.
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Reviewer #2 (Public Review):
Bowman et al. provided solid data to support the existence of a soluble pool of monomeric H3 in cells. This soluble pool of monomeric H3 associates with Importin-5 and sNASP in cytosol and nucleus, respectively. They also characterized H3 handover between Importin-5 and sNASP. They went further to demonstrate that monomeric H4 may similarly transfer between Importin-5 and HAT1-RBBP7 complex. The existence of a soluble pool of monomeric H4 is not fully confirmed. I suggest the authors discuss some of the limitations of the current manuscript in the Discussion section. Overall, the key conclusions of the manuscript are well supported.
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Reviewer #3 (Public Review):
The goal of this paper is to describe how newly synthesized histones are imported into the nucleus.
Prior biochemical purifications suggest that H3-H4 dimers fold in the cytoplasm, are regulated by the sNASP histone chaperone, and translocate to the nucleus in association with the ASF1 histone chaperone and the importin-4 (Imp4) karyopherin. However, using an imaging-based approach, the authors previously showed that histones H3 and H4 can be imported into the nucleus as monomers.
Here, the authors show that new, cytoplasmic H3.1 and H4 monomers are bound by HSPA8 and importin-5 (Imp5). Imp5 then translocates monomeric histones into the nucleus and transfers H3.1 to sNASP. They further propose that the previously observed cytosolic H3-H4 dimers are not new histones but rather old nucleosomal histones that …
Reviewer #3 (Public Review):
The goal of this paper is to describe how newly synthesized histones are imported into the nucleus.
Prior biochemical purifications suggest that H3-H4 dimers fold in the cytoplasm, are regulated by the sNASP histone chaperone, and translocate to the nucleus in association with the ASF1 histone chaperone and the importin-4 (Imp4) karyopherin. However, using an imaging-based approach, the authors previously showed that histones H3 and H4 can be imported into the nucleus as monomers.
Here, the authors show that new, cytoplasmic H3.1 and H4 monomers are bound by HSPA8 and importin-5 (Imp5). Imp5 then translocates monomeric histones into the nucleus and transfers H3.1 to sNASP. They further propose that the previously observed cytosolic H3-H4 dimers are not new histones but rather old nucleosomal histones that diffuse into the cytoplasm, which are then re-imported via Imp4. Therefore, folding of H3-H4 dimers exclusively occurs in the nucleus.
The authors certainly provide compelling evidence that monomeric histones are imported into the nucleus via Imp5. Constitutively monomeric histone mutants co-purified with Imp5 and the association was recapitulated in vitro. A wide range of exciting techniques is used to address how monomeric histones are handled in cells (i.e., biochemical, FRAP, imaging of cytoplasmic tethered and released histones, proximity-dependent protein labeling, etc). The aim of finding how monomeric histones are imported into the nucleus is certainly attained. More data could however support some of the conclusions regarding the association of histones to ASF1 and Imp4 and whether they truly exclusively represent evicted nucleosomal histones that diffused out of the nucleus.
Otherwise, the data shown here is certainly important for the field, as it provides an explanation of how monomeric histones are handled in the cytoplasm.
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