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ANP32E is a histone chaperone that removes H2A.Z from chromatin

Nature volume 505, pages 648–653 (2014)Cite this article

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Abstract

H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 Å resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E−/− cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators.

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Figure 1: Immunopurification of e-H2A, e-H2A.Z and e-ANP32E predeposition complexes from soluble nuclear fractions.
Figure 2: The ANP32E ZID domain interacts with H2A.Z αC-helix.
Figure 3: Specific recognition of the H2A.Z/H2B pair by ANP32E.
Figure 4: Specific removal of H2A.Z from the nucleosome by ANP32E.
Figure 5: Genomic localization and chromatin enrichment of H2A.Z is dependent on ANP32E.

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Gene Expression Omnibus

Protein Data Bank

Data deposits

ChIP-Seq datasets have been deposited in GEO under accession number GSE51579. X-ray crystallographic coordinates and structure factor files have been deposited in Protein Data Bank under accession number 4CAY.

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Acknowledgements

We thank I. Davidson, P. Antony, J. Cavarelli and D. Moras for reading the manuscript, and V. Cura and A. McEwen for help during data collection. This work was supported by CNRS, INSERM, Université de Strasbourg, and by grants from INCa (INCa_4496 and INCa_4454), ANR (VariZome, ANR-12-BSV8-0018-01; Nucleoplat, NT09_476241), the Association pour la Recherche sur le Cancer, La Fondation pour la Recherche Médicale, La Ligue Nationale contre le Cancer (Equipe labellisée, to A.H. and S.D.), the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-01) and by Instruct (ESFRI). A.O. acknowledges the Association pour la Recherche sur le Cancer for Financial support. K.P. was supported by la Ligue Nationale contre le Cancer.

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  1. Arnaud Obri, Khalid Ouararhni and Christophe Papin: These authors contributed equally to this work.

Authors and Affiliations

  1. Département de Génomique Fonctionnelle et Cancer, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg, CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France,

    Arnaud Obri, Khalid Ouararhni, Christophe Papin, Isabelle Stoll, Ludovic Roy & Ali Hamiche

  2. Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg, CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France,

    Marie-Laure Diebold, Martin Marek & Christophe Romier

  3. Equipe labélisée Ligue contre le Cancer, INSERM/Université Joseph Fourier , Institut Albert Bonniot, U823, Site Santé-BP 170, 38042 Grenoble Cedex 9, France,

    Kiran Padmanabhan & Stefan Dimitrov

  4. Division of Cellular and Molecular Research, Laboratory of Inflammation Biology, National Cancer Centre, Singapore, Singapore

    Patrick T. Reilly & Tak W. Mak

  5. The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, Ontario, Canada,

    Tak W. Mak

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Contributions

A.H. conceived and supervised the project. A.O. and K.O. built constructs and performed complex purifications and chromatin assembly/eviction assays. C.R., M.L.D. and M.M. solved the ANP32E-ZID–H2A.Z/H2B structure. C.P. and K.P. performed ChIP-seq experiments. P.T.R. and T.W.M. provided the ANP32E−/− mice. I.S. and K.O. generated the ANP32E−/− MEF cells. C.P. and L.R. conducted all bioinformatics analyses. A.H., C.R. and S.D. designed experiments, analysed data and wrote the paper.

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Correspondence to Stefan Dimitrov, Christophe Romier or Ali Hamiche.

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Extended data figures and tables

Extended Data Figure 1 ANP32E and p400.com are stably associated in an H2A.Z subcomplex.

a, Control (Ctrl) and e-H2A- and e-H2A.Z-expressing cells were stained with anti-HA (green) and DAPI (blue) (upper panel). Extracts from control and e-H2A.Z-expressing HeLa cells were blotted with a monoclonal anti-H2A.Z antibody (lower panel). b, Silver staining of e-H2A.Z predeposition complex fractionated on a glycerol gradient (upper panel). Fractions were pooled as indicated at the top of the gel (low molecular mass complexes, medium molecular mass complexes and high molecular mass complexes) and analysed by mass spectrometry. Lower panel, immunoblotting of fractions containing e-H2A.Z nuclear subcomplexes with the indicated antibodies. c, Mass spectrometry analyses of the different H2A.Z subcomplexes. d, GST pull-down assays show that ANP32E directly interacts through its N-terminal domain with MRGBP.

Extended Data Figure 2 Mapping of the ANP32E and H2A.Z interaction domains.

a, b, GST pull-down assays using ANP32E deletion mutants. Upper panels, the acidic very C-terminal conserved domain of ANP32E is required for the interaction with H2A.Z/H2B. Lower panels, immunoblotting using anti-Flag antibody of Flag-tagged H2A.Z. c, Deletion analysis of the ANP32E very C-terminal conserved domain. The 215–240 amino-acid region is the minimal domain required for interaction with H2A.Z/H2B. d, Same as in a and b, using H2A.Z C-terminal deletion mutants. Deletion of a region encompassing the H2A.Z M6 cassette impairs binding to ANP32E.

Extended Data Figure 3 ANP32E-ZID is highly conserved.

a, Multiple alignment of ANP32E from various organisms, indicating that ANP32E is a vertebrate-specific H2A.Z histone chaperone. The ANP32E-ZID defines the minimal H2A.Z/H2B interacting domain and corresponds to the primary conserved C-terminal region of ANP32E. b, Multiple alignment of the human ANP32 protein family showing the presence of a specific insertion in ANP32E embedded in its ZID domain. The numbering above the sequences corresponds to human ANP32E. Sequence conservation is shown by shading, from red (high) to green/blue (low).

Extended Data Figure 4 H2A.Z αC-helix undergoes a large conformational change upon ANP32E-ZID binding.

a–c, The conformation of the α3–αC region of H2A.Z is shown in the H2A.Z nucleosome (a), the ANP32E-ZID–H2A.Z/H2B complex (b) and as a superposition of views from panels a and b (c), with the ANP32E-ZID αN-helix hidden (upper panels) or shown (lower panels). These views highlight the extension of the H2A.Z αC-helix occurring upon ANP32E-ZID binding and show that the ANP32E-ZID αN-helix occupies a position previously filled by the H2A.Z docking domain.

Extended Data Figure 5 Stereo views of the interactions made by ANP32E-ZID and the H2A.Z/H2B pair.

a, Close-up view of the interaction between ANP32E-ZID (blue) αN, H2A.Z (red) α3–αC and H2B (light grey) α2 helices. b, Close-up view of the interaction between ANP32E-ZID and the H2A.Z/H2B region involved in DNA interaction close to the entry/exit points of the nucleosome. c, Alignment of the ANP32E-ZID showing the conservation of the residues (yellow triangles) that interact with the H2A.Z/H2B pair.

Extended Data Figure 6 Mutational analysis of ANP32E-H2A.Z interaction.

a, GST pull-down experiments and anti-Flag immunoblotting showing that a single glycine insertion at position 101 of H2A.Z (H2A.Z-G101) prevents its interaction with ANP32E. b, Multiple alignment of human canonical and variant H2A histones showing the presence of an extra residue (generally a glycine) in their M6 cassette compared with H2A.Z (yellow diamond). c, Co-expression in bacteria of full-length ANP32E, either wild type or mutant, with the H2A.Z/H2B pair. *Proteolytic fragments of ANP32E. d, Purification (left panel) and western blotting (right panel) of the in vivo complexes incorporating ANP32E either wild type or mutated on the residues highlighted in panel e. e, Schematic view of human ANP32E-ZID showing the residues (yellow triangles) that interact with the H2A.Z/H2B pair and that are mutated in the m1m2 mutant.

Extended Data Figure 7 Specific removal of H2A.Z from the nucleosome by ANP32E.

a, Effects of increasing amounts of competitor supercoiled plasmid DNA on ANP32E-mediated H2A.Z eviction from nucleosomes reconstituted on the negatively supercoiled human α-satellite 360 bp DNA minicircle corresponding to topoisomer −1. The reaction products were analysed on native PAGE. b, c, Effects of increasing amounts of ANP32E (b) or NAP1 (c) on H2A.Z/H2B or H2A/H2B dimer deposition on (H3/H4)2 tetrasome particles reconstituted on topoisomer −1. d, Mononucleosome assembly to generate H2A or H2A.Z mononucleosomes by salt-jump dialysis. e, Effects of increasing amounts of e-ANP32E complex (e-ANP32E.com) on H2A.Z incorporation on bead-immobilized H2A nucleosomes. e-ANP32E.com is not able to catalyse replacement of H2A with H2A.Z in vitro.

Extended Data Figure 8 Genome-wide distribution of H2A.Z in ANP32E WT and knockout MEF cells.

a, Heat-map view of genome-wide H2A.Z binding sites in wild-type and knockout cells at promoter-proximal sites (left panel) and distal sites (right panel). b, Heat-maps of H2A.Z-occupied and H2A.Z-free TSS in MEF cells from wild-type and ANP32E knockout MEFs (left-most panels). Sub-clusters in H2A.Z-occupied TSS (middle panel) were generated to highlight different distribution patterns of H2A.Z (percentage fractions are indicated) in ANP32E wild-type (blue) and knockout (red) MEFs (right panels). c, H2A.Z preferentially binds CpG-containing promoters.

Extended Data Figure 9 Steric hindrances that cause H2A.Z/H2B eviction from the nucleosome upon ANP32E binding.

a, Superposition (middle panel) of the ANP32E-ZID–H2A.Z/H2B complex (left panel) onto the H2A.Z/H2B pair in a nucleosomal context (right panel). Several steric hindrances (indicated by arrows) are observed at the DNA level as well as at the interface with the nucleosomal H3/H4 pair (coloured green and pink, respectively) that are incompatible with stable H2A.Z/H2B binding to the nucleosome.

Extended Data Table 1 Mass spectrometry analysis of the e-H2A, e-H2A.Z, e-ANP32E and e-H2A.ZNKLLG complexes
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Obri, A., Ouararhni, K., Papin, C. et al. ANP32E is a histone chaperone that removes H2A.Z from chromatin. Nature 505, 648–653 (2014). https://doi.org/10.1038/nature12922

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