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The program for processing newly synthesized histones H3.1 and H4

Nature Structural & Molecular Biology volume 17, pages 1343–1351 (2010)Cite this article

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Abstract

The mechanism by which newly synthesized histones are imported into the nucleus and deposited onto replicating chromatin alongside segregating nucleosomal counterparts is poorly understood, yet this program is expected to bear on the putative epigenetic nature of histone post-translational modifications. To define the events by which naive pre-deposition histones are imported into the nucleus, we biochemically purified and characterized the full gamut of histone H3.1–containing complexes from human cytoplasmic fractions and identified their associated histone post-translational modifications. Through reconstitution assays, biophysical analyses and live cell manipulations, we describe in detail this series of events, namely the assembly of H3–H4 dimers, the acetylation of histones by the HAT1 holoenzyme and the transfer of histones between chaperones that culminates with their karyopherin-mediated nuclear import. We further demonstrate the high degree of conservation for this pathway between higher and lower eukaryotes.

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Figure 1: Subcellular distribution of H3.1-containing complexes.
Figure 2: Purification of eH3.1 from cytosolic extracts.
Figure 3: sNASP interacts with HAT1–RbAp46 and ASF1B through histone intermediates.
Figure 4: sNASP homodimerizes but binds H3–H4 heterodimers.
Figure 5: Importin-4 and ASF1B function downstream of sNASP, tNASP, and HAT1 in vivo.
Figure 6: Conservation of the H3–H4 pre-deposition pathway in Saccharomyces cerevisiae.
Figure 7: Model for nuclear import of pre-deposition replication-dependent histones.

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Acknowledgements

Funding for this project was provided by the Howard Hughes Medical Institute, by US National Institutes of Health grants GM064844 and R37GM037120 (D.R.), by the Natural Sciences and Engineering Research Council of Canada (E.I.C. and W.-H.W.K.), the Canadian Cancer Society Research Institute (J.F.G.) and by the Deutsche Akademie der Naturforscher Leopoldina (Leopoldina Fellowship Program, LPDS 2009-5, P.V.). The eH3.1 HeLa S3 cells were kindly provided by H. Tagami and G. Almouzni (Institut Curie). The HAT1–RbAp46, WT ASF1B and V94R Asf1 constructs were kind gifts from B. Stillman (Cold Spring Harbor Laboratory), H. Silljé (Max Planck Institute) and C. Mann (CEA Saclay), respectively. The baculoviruses expressing human HAT1 and RbAp46 were kindly provided by R. Kingston (Harvard Medical School). We also thank S. Mehta and S. Kim for technical support with tissue culture, G. Zhong for help with yeast TAP purification and J. Zhang for a copious amount of histones. We are grateful to L. Vales for comments on the manuscript.

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  1. Jeffrey Fillingham, Guohong Li and Haiyan Zheng: These authors contributed equally to this work and are listed in alphabetical order.

Authors and Affiliations

  1. Department of Biochemistry, Howard Hughes Medical Institute, New York University School of Medicine, New York, New York, USA

    Eric I Campos, Guohong Li, Philipp Voigt, Zhonghua Gao, Loren A Day & Danny Reinberg

  2. Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Jeffrey Fillingham, Wei-Hung W Kuo, Harshika Seepany & Jack F Greenblatt

  3. Department of Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada

    Wei-Hung W Kuo, Harshika Seepany & Jack F Greenblatt

  4. Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada

    Jeffrey Fillingham

  5. Biological Mass Spectrometry Facility at the Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey, USA

    Haiyan Zheng

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Contributions

E.I.C. performed the tissue culture–related work and biochemical purification of eH3.1, sNASP and ASF1B; protein interaction and enzymatic assays, with assistance from Z.G.; subcloning and mutagenesis; cross-linking experiments, with the assistance of P.V.; AUC runs, with the assistance of L.A.D.; and RNAi experiments. J.F. performed the work in S. cerevisiae with assistance from W.-H.W.K. and H.S. G.L. cloned sNASP and generated stable sNASP clones. H.Z. performed the mass spectrometry analyses. Experiments were performed under the supervision of D.R. and J.F.G. The manuscript was written by E.I.C. and D.R. with assistance from the other authors.

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Correspondence to Danny Reinberg.

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Campos, E., Fillingham, J., Li, G. et al. The program for processing newly synthesized histones H3.1 and H4. Nat Struct Mol Biol 17, 1343–1351 (2010). https://doi.org/10.1038/nsmb.1911

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