Dataset Information

Evidence for the nuclear import of monomeric histones H3.1 & H4

ABSTRACT: We present here evidence that histones H3.1 and H4 can be imported into the nucleus as monomers in human cells. Using a tether-and-release system to study the cytosolic phase and import dynamics of newly synthesised histones, we find that H3.1 and H4 can be maintained as stable monomers in the cytosol in a tethered state. Cytosolically tethered histones are bound tightly to Importin- proteins (predominantly IPO4), but not to the 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, dissociation with IPO4 and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones under steady-state conditions reveals an excess of H3 specifically associated with sNASP, suggesting that NASP can maintain 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, with sNASP acting as a potential nuclear receptor for monomeric histone H3.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Juan Ramon Hernandez-Fernaud

LAB HEAD: Andrew Bowman

PROVIDER: PXD009915 | Pride | 2018-09-10

REPOSITORIES: Pride

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Publications

Evidence for the nuclear import of histones H3.1 and H4 as monomers.

Apta-Smith Michael James MJ Hernandez-Fernaud Juan Ramon JR Bowman Andrew James AJ

The EMBO journal 20180903 19

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 his ...[more]

PMID: 30177573

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Project description:The N-terminal regions of histone proteins (histone tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly due to their inherently disordered nature. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably due to the poor N-terminal coverage provided by pepsin, the dominant protease in HX-MS studies. Inspired from histone-clipping events, we profiled the activity of Cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent and showed optimum activity in a slightly reducing and non-denaturing environment. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3 and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. When in a mononucleosome, we find evidence for inter- or intramolecular interactions within the H4 tail but not for H3.1. Further, EX1 kinetics observed in the respective monomers are absent when in the mononucleosome, indicating stabilization of the histone-folds. The data obtained here may serve as a baseline towards the comparison of nucleosome dynamics harboring different histone variants or post-translational modifications. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones and nucleosomes that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

Dataset Information

Evidence for the nuclear import of monomeric histones H3.1 & H4

Publications

Evidence for the nuclear import of histones H3.1 and H4 as monomers.

Similar Datasets

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