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The structure of the NuA4–Tip60 complex reveals the mechanism and importance of long-range chromatin modification

Nature Structural & Molecular Biology volume 30, pages 1337–1345 (2023)Cite this article

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Abstract

Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site. Neither the molecular mechanism of this activity nor its physiological importance are known. Here we report the structure of the Pichia pastoris NuA4 complex, with its core resolved at 3.4-Å resolution. Three subunits, Epl1, Eaf1 and Swc4, intertwine to form a stable platform that coordinates all other modules. The HAT module is firmly anchored into the core while retaining the ability to stretch out over a long distance. We provide structural, biochemical and genetic evidence that an unfolded linker region of the Epl1 subunit is critical for this long-range activity. Specifically, shortening the Epl1 linker causes severe growth defects and reduced H4 acetylation levels over broad chromatin regions in fission yeast. Our work lays the foundations for a mechanistic understanding of NuA4’s regulatory role and elucidates how its essential long-range activity is attained.

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Fig. 1: Structure of the yeast NuA4 core complex.
Fig. 2: The unique environment of the Act1–Arp4–Eaf1HSA module.
Fig. 3: Ensuring selective use of Tra1.
Fig. 4: Shortening the Epl1 linker uncovers the HAT module.
Fig. 5: A long Epl1 linker is essential for S. pombe viability.
Fig. 6: The long Epl1 linker is important for NuA4 long-range activity.

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Data availability

The cryo-EM maps have been deposited in the electron microscopy database (EMBD) under accession codes EMD-15869 (NuA4-WT-overall), EMD-15881 (NuA4-WT-core), EMD-15896 (NuA4-SL1-overall), EMD-15897 (NuA4-SL1-core), EMD-14980 (NuA4-SL3-overall), EMD-15066 (NuA4-SL3-core), EMD-15070 (NuA4-SL3-tra1-tail) and EMD-15067 (NuA4-SL3-tra1-ring). A composite map was also deposited for NuA4-SL3 (EMD-14989). The model coordinates for core NuA4 derived from the SL3 composite map were deposited in the PDB database under the accession code 7ZVW. Original cryo-EM data are available on request. Source data are provided with this paper.

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Acknowledgements

We are grateful to S. Piatti and L. Pillus for kindly sharing S. cerevisiae strains. We thank the IGBMC mass spectrometry service for helpful advise and discussions. We acknowledge support from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National pour la Recherche Scientifique (CNRS), the Nanotumor Consortium, with financial support from the ITMO Cancer of AVIESAN (National Alliance for Life Sciences & Health) within the framework of the Cancer Plan (P.S.), the Ligue contre le Cancer (P.S.), the University of Strasbourg Institute for Advanced Study (USIAS) for a fellowship within the French national program ‘investment for the future’ (IdEx-Unistra) (P.S., E.S.), the Agence Nationale de la Recherche (ANR) grants ANR-17-CE12-0022 (P.S.), ANR-15-CE11–0022–03 (D.H.), and ANR-10-LABX-0030-INRT to IGBMC. We acknowledge the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC.

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Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Integrated Structural Biology Department, Illkirch, France

    Alexander Fréchard, Rozalie Hexnerova, Corinne Crucifix, Gabor Papai, Ekaterina Smirnova, Patrick Schultz & Adam Ben-Shem

  2. Centre National de la Recherche Scientifique, UMR7104, Illkirch, France

    Alexander Fréchard, Rozalie Hexnerova, Corinne Crucifix, Gabor Papai, Ekaterina Smirnova, Patrick Schultz & Adam Ben-Shem

  3. Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France

    Alexander Fréchard, Rozalie Hexnerova, Corinne Crucifix, Gabor Papai, Ekaterina Smirnova, Patrick Schultz & Adam Ben-Shem

  4. Université de Strasbourg, Illkirch, France

    Alexander Fréchard, Rozalie Hexnerova, Corinne Crucifix, Gabor Papai, Ekaterina Smirnova, Patrick Schultz & Adam Ben-Shem

  5. Equipe labellisée Ligue Contre le Cancer, Illkirch, France

    Alexander Fréchard, Rozalie Hexnerova, Corinne Crucifix, Gabor Papai, Ekaterina Smirnova, Patrick Schultz & Adam Ben-Shem

  6. Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), Université de Montpellier, CNRS, Montpellier, France

    Céline Faux, Conor McKeon, Florie Lo Ying Ping & Dominique Helmlinger

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Contributions

A.B.-S., D.H. and P.S. designed the study; A.B.-S. devised the NuA4 purification method; A.B.-S. and C.C. defined conditions for grid preparation and freezing; C.C., A.F. and G.P. prepared cryo-EM specimens; G.P. collected and A.F. analyzed cryo-EM data; A.F. and A.B.-S. interpreted the maps by fitting crystal coordinates and model building; E.S. and R.H. performed HAT activity assays, F.L.Y.P., C.M. and C.F. performed all S. cerevisiae and S. pombe experiments, D.H., P.S. and A.B.-S. supervised the work; D.H., A.F. and P.S. prepared figures; A.B.-S., D.H., A.F. and P.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Dominique Helmlinger, Patrick Schultz or Adam Ben-Shem.

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Nature Structural & Molecular Biology thanks Alan Cheung, TaeSoo Kim, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Subunit characterization of the purified P. pastoris NuA4 complex.

a, Colloidal coomassie blue stained SDS-PAGE analysis of the P. pastoris NuA4 complex purified from a SBP-tagged Eaf1 subunit. Bdc1 is the bromodomain protein homologous to human BRD8 and S. pombe Bdc1. The SDS-PAGE analysis was performed three times. Left lane shows molecular weight markers in KDa. b, Proteomic analysis of the purified NuA4 complexes. For each identified protein subunit, the table shows the peptide spectrum matches (PSM) counts, the number of residues, the PSM values normalized by subunit length (NSAFs) and the NSAF value normalized to Tra1. For each protein values were averaged over 3 experiments. c, Subunit names and composition of the NuA4 complexes purified from the P. pastoris, S. cerevisiae and S. pombe yeast species.

Source data

Extended Data Fig. 2 Subunit and domain organization of the yeast NuA4 complex.

a, Multiple sequence alignment between the bromodomain-containing subunit associated with purified P. pastoris NuA4, the S. pombe Bcd1 subunit and BDR8 subunits from human, drosophila, mouse, rat and chicken. The conserved C-terminal bromodomain is boxed. b, NuA4 subunits are organized into an enzymatic HAT (or Piccolo) module, an activator binding Tra1 module, an actin module and a TINTIN module interconnected by a Scaffolding module. Protein domains are abbreviated as follows: YEATS (Yaf9, ENL, AF9, Taf14, Sas5 domain), SANT (Swi3, Ada2, N-Cor, TFIIIB domain), DIS (disordered region), YIR (Yaf9 interacting region), TIR (Tra1 Interacting region), HSA (Helicase SANT associated), MYST HAT (MOZ, Ybf2/Sas3, Sas2, Tip60 family of Histone Acetyl Transferase), ING (domain found in the inhibitor of Growth family of proteins), PHD (plant homeodomain), HEAT (α-helical repeats found in Huntingtin, Elongation factor 3, Protein phosphatase 2A and Tor kinase), FAT (four helix bundle found in FRAP, ATM and TRRAP), FRB (N-terminal FKBP12-Rapamycin Binding kinase domain), FATC (kinase domain important for assembly).

Extended Data Fig. 3 Cryo-EM data analysis strategy and resolution assessment of the cryo-EM structure of WT P. pastoris NuA4.

a, Image processing strategy used to obtain a map of the full P. pastoris NuA4 complex. b, Refinement steps of the volume masked as shown by the dotted contour. The map colored in rainbow spectrum represent the local resolution of the reconstruction of the masked volume. c, Fourier Shell Correlation (FSC) curves are depicted as a function of resolution in Å for the entire NuA4 complex (black) and the masked volume (red).

Extended Data Fig. 4 Cryo-EM data analysis and resolution assessment of the cryo-EM structures of Epl1-SL1 and Epl1-SL3 NuA4 mutants.

a, Image processing used to obtain the maps of the NuA4 complex containing the Epl1-SL1 mutation and of the masked lobe. b, Image processing used to obtain the maps of the NuA4 complex containing the Epl1-SL3 mutation and of three masked domains depicted by dotted lines. c, Fourier Shell Correlation (FSC) curves are depicted as a function of resolution in Å for the entire NuA4 Epl1-SL1 complex (black) and the masked areas (red). d, Fourier Shell Correlation (FSC) curves are depicted as a function of resolution in Å for the entire NuA4 Epl1-SL3 complex (black) and the three masked areas (red, green and blue).

Extended Data Fig. 5 High resolution structure of the yeast NuA4 complex.

Representative regions illustrating the quality of the cryo-EM map and the high-resolution structural features. Cryo-EM map and atomic model showing that side chains are clearly identified. a, Part of the Eaf1 subunit b, ATP molecule bound in the Arp4 active site. c, Part of the Tra1 subunit. d, Interaction network between the Swc4, Epl1 and Eaf1 subunits in the neck region.

Extended Data Fig. 6 Mobility of Tra1 domains in NuA4 and SAGA.

Cryo-EM maps of NuA4 Tra1 (a) and of SAGA Tra1 (b). The Tra1 ring solenoid and the FRB/LBE domains of the Tra1 pseudo kinase are labeled HEAT and FRB/LBE, respectively. Local resolution map of the Tra1 HEAT domain in NuA4 (c) and in SAGA (d). The colored bar indicates the local resolution in Å. Cryo-EM maps of NuA4 Tra1 (e) and of SAGA Tra1 (f) enlarged and oriented to visualize the FRB/LBE domain of the pseudokinase.

Extended Data Fig. 7 Domain organization and sequence alignment of the Epl1 subunit.

a, Schematic representation of the Epl1 subunit showing in pink the N-terminal part resolved in the crystal structure of Piccolo (Xu et al., 2016a) and in violet the C-terminal part revealed in our cryo-EM map. The corresponding structures are shown and the hatched region corresponds to the unstructured linker region. Residue numbers indicate, in P. pastoris, the first unstructured residue in the crystal structure (347), the last residue in the crystal structure (358), the first residue in the cryo-EM map (419) and the last unstructured linker residue in the cryo-EM map (430). b, Multiple sequence alignment of the Epl1 subunit. c, Table showing the Epl1 linker length in different species.

Extended Data Fig. 8 Proteolytic cleavage of the Epl1 subunit to isolate the HAT module.

Colloidal Coomassie blue stained SDS-PAGE analysis of a purified P. pastoris NuA4 complex carrying a TEV cleavage site in the linker connecting the two functional parts of Epl1. NuA4 was bound to a streptavidin column via the SBP-tag on the core Eaf1 subunit prior to addition of TEV protease. Lane 1 shows the subunits retained on the streptavidin column after TEV cleavage, Lane 2 represent the composition of the sub-complex eluted from the streptavidin column after TEV cleavage and lane 3 shows the input holo-NuA4 complex. These experiments have been repeated twice with similar results and identical conclusions.

Extended Data Fig. 9 The Epl1 linker is dispensable for NuA4 activity and functions in budding yeast.

a, Schematic illustration of the S. cerevisiae Epl1 linker mutants that were constructed. The epl1-SL1 allele corresponds to a seamless deletion of residues 401–487. The epl1-SL5 allele corresponds to a longer, seamless deletion of residues 395–501 from endogenous Epl1. b, S. cerevisiae EPL1+/epl1-SL1 SDS3+/sds3Δ and EPL1+/epl1-SL5 SDS3+/sds3Δ double heterozygous diploids were sporulated, dissected, and germinated to show the growth phenotype of all four possible genotypes (top panel). Single epl1-SL1 and epl1-SL5 mutants were isolated, along with an isogenic wild-type (WT) controls, grown to exponential phase and spotted on rich media in ten-fold serial dilutions at grown at different temperatures, as indicated (bottom panel). Data are representative of four independent experiments performed with distinct mutant clones. c, Acetylated histone H4 levels in exponentially growing S. cerevisiae epl1 mutants. The control epl1-SL1 and epl1-SL5 single mutants were obtained from the tetrad analysis shown in C. The epl1-DNt sds3Δ and epl1-CNt sds3Δ double mutants were obtained from32 and corresponds to a deletion of residues 1-485 and 485–833, respectively. Western blots of total protein extracts were probed with an anti-pan-acetyl-H4 antibody. An anti-H3 antibody and Ponceau red staining were used as controls for equal loading between lanes. Data are representative of two independent experiments. Source data for c are provided as a Source Data File. d, Illustration of S. pombe Epl1 mutants. The epl1-DCt allele has a seamless deletion from residues 405 of Epl1 until its C-terminal end (residue 557). e, Tetrad analysis of a heterozygous epl1+/epl1-DCt diploid strain following sporulation and germination to show 2:2 segregation of a lethal phenotype.

Source data

Extended Data Table 1 List of yeast strains used in this study
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Source Data Extended Data Fig. 9

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Fréchard, A., Faux, C., Hexnerova, R. et al. The structure of the NuA4–Tip60 complex reveals the mechanism and importance of long-range chromatin modification. Nat Struct Mol Biol 30, 1337–1345 (2023). https://doi.org/10.1038/s41594-023-01056-x

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  • DOI: https://doi.org/10.1038/s41594-023-01056-x

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