Structures of the ISWI–nucleosome complex reveal a conserved mechanism of chromatin remodeling

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Abstract

Chromatin remodelers are diverse enzymes, and different models have been proposed to explain how these proteins work. Here we report the 3.3 Å-resolution cryogenic electron microscopy (cryo-EM) structures of Saccharomyces cerevisiae ISWI (ISW1) in complex with the nucleosome in adenosine diphosphate (ADP)-bound and ADP-BeFx-bound states. The data show that after nucleosome binding, ISW1 is activated by substantial rearrangement of the catalytic domains, with the regulatory AutoN domain packing the first RecA-like core and the NegC domain being disordered. The high-resolution structure reveals local DNA distortion and translocation induced by ISW1 in the ADP-bound state, which is essentially identical to that induced by the Snf2 chromatin remodeler, suggesting a common mechanism of DNA translocation. The histone core remains largely unperturbed, and prevention of histone distortion by crosslinking did not inhibit the activity of yeast ISW1 or its human homolog. Together, our findings suggest a general mechanism of chromatin remodeling involving local DNA distortion without notable histone deformation.

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Fig. 1: Overall structures of ISW1–nucleosome complex in the ADP-BeFx-bound and ADP-bound states.
Fig. 2: Conformational change of ISW1 following activation.
Fig. 3: Comparison of the ISW1–nucleosome and Snf2–nucleosome complexes in the ADP-BeFx-bound state.
Fig. 4: Comparison of the ISW1– and Snf2–nucleosome complexes in the ADP-bound state.
Fig. 5: Unperturbed structure of the histone core bound by ISW1.
Fig. 6: Biochemical analysis of chromatin remodeling activity of ISWI.

Data availability

Coordinates and EM maps have been deposited in the EMDataResource and Protein Data Bank (PDB) under accession codes EMD-9718 (PBD ID 6IRM, native complex, ADP-BeFx); EMD-9719 (PBD ID 6IRN, crosslinked complex, ADP-BeFx); and EMD-9720 (PBD ID 6IRO, crosslinked complex, ADP). Source data for Figs. 2c,d, 6a–d and Supplementary Fig. 1a are available with the paper online. All other data that support the findings of this study are available from the corresponding author upon request.

Change history

  • 23 April 2019

    In the version of this article initially published, the date of publication (13 March 2019) was incorrect. The correct date is 14 March 2019. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for the cryo-EM facility (crosslinked dataset) and the computational facility support on the cluster of Bio-Computing Platform. We thank the Electron Microscopy Laboratory of Peking University for collecting the non-crosslinked dataset, X. Wang and P. Yang for providing the EV71 virus and H. Deng and C. Zhang for mass-spectrometry analysis. K. Chen, R. Su and J. Wang performed Rosetta analysis. J. Lei wrote the software. This work was supported by the National Key Research and Development Program (nos. 2017YFA0102900 and 2014CB910100 to Z.C., 2016YFA0500700 to N.G.), the National Natural Science Foundation of China (nos. 31570731, 31270762 and 31630046 to Z.C., 31725007 and 31630087 to N.G.) and the Advanced Innovation Center for Structural Biology, Tsinghua–Peking Joint Center for Life Sciences.

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L. Yan prepared the samples and performed the biochemical analysis. H. Wu, X. Li and N. Gao performed the EM analysis. Z. Chen wrote the manuscript with help from all authors. Z. Chen directed and supervised all of the research.

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Correspondence to Ning Gao or Zhucheng Chen.

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Integrated supplementary information

Supplementary Figure 1 Cryo-EM structure analysis of the native ISW1–nucleosome complex in the ADP-BeFx-bound state without fixation.

(a) Chromatin remodeling activity of the mutant ISW1 (ISW1ΔL) with the H4-like L3 loop replaced with a flexible sequence. Control: reaction in the absence of ATP. Error bars indicate s.d. (n=3). Domain architecture of ISW1 is shown and the relative position of L3 is highlighted with a magenta line. (b) Representative Cryo-EM micrograph of the non-cross-linked sample. (c) FFTs of image in (b) with the Thon rings extending to ~3.5 Å. (d) 2D class averages of the cryo-EM particles. (e) Flowchart of cryo-EM data processing for the non-crosslinked dataset. (f) Angular distributions of cryo-EM particles in the final round of refinements of the ISW1-NCP and the (ISW1)2-NCP-complexes. (g) Resolution estimation of the EM maps. Gold standard Fourier shell correlation (FSC) curves, showing the overall nominal resolutions of 3.56 Å, 3.87 Å, 4.11 Å, 4.20 Å, 4.42 Å for the NCP masked region of the ISW1-NCP-complex, ISW1-NCP-complex, ISW1-NCP-SHL-2-complex and ISW1-NCP-SHL2-complex, and (ISW1)2-NCP-complex, respectively. Source data

Supplementary Figure 2 Essentially identical structures of the ADP-BeFx-bound ISW1 complex at different sides of the nucleosome and with two methods of sample preparation.

(a-b) Fitting the final structure of the complex into the SHL-2 (a) and SHL2 (b) maps, respectively. The maps were reconstructed with the samples prepared without glutaraldehyde fixation. (c) Structural alignment of H3-H4 of the SHL+2 (color-coded) and SHL-2 (grey) complexes. (d) Structural alignment of ISW1 of the SHL+2 (color-coded) and SHL-2 (grey) complexes. (e) Two different views of the structures determined with different methods of sample preparation. The structure determined with glutaraldehyde fixation is color coded, with AutoN in magenta. The structure without fixation is in grey. Dashed line, disordered region of AutoN.

Supplementary Figure 3 Local densities of the ISW1–nucleosome complex in the ADP-BeFx-bound state.

(a) The nucleotide binding pocket. (b) Trp600 of ISW1. (c) Arg308 of ISW1. (d) The ‘K16R17H18R19’ motif of the H4 tail. (e) SHL0 of the nucleosome (f) α2 helix of H4. (g) The minor of SHL1.5 of the nucleosome.

Supplementary Figure 4 Cryo-EM structure analysis of the ISW1–nucleosome complex in the ADP-BeFx-bound state fixed with glutaraldehyde.

(a) A representative Cryo-EM micrograph. (b) FFTs of image in (a) with the Thon rings extending to ~3.5 Å. (c) 2D class averages of characteristic projection views of Cryo-EM particles. (d) Flowchart of Cryo-EM data processing for the ISW1-152NCP-ADP-BeFx-crosslinked dataset. (e) Angular distributions of cryo-EM particles in the final round of refinements of the ISW1-NCP-complex sample. (f) Angular distributions of cryo-EM particles in the final round of refinements of the ISW1-NCP-SHL-2-complex sample. (g) Angular distributions of cryo-EM particles in the final round of refinements of the ISW1-NCP-SHL2-complex sample. (h) Resolution estimation of the EM maps. Gold standard Fourier shell correlation (FSC) curves, showing the overall nominal resolutions of 3.25 Å, 3.37 Å, 3.61 Å, 3.77 Å for the NCP masked region of the ISW1-NCP-complex, ISW1-NCP-complex, ISW1-NCP-SHL-2-complex and ISW1-NCP-SHL2-complex, respectively.

Supplementary Figure 5 Cryo-EM structure analysis of the ISW1–nucleosome complex in the ADP-bound state.

(a) A representative Cryo-EM micrograph. (b) FFTs of image in (a) with the Thon rings extending to ~3.5 Å. (c) 2D class averages of characteristic projection views of Cryo-EM particles. (d) Flowchart of Cryo-EM data processing for the ISW1-152NCP-ADP dataset. (e) Angular distributions of cryo-EM particles in the final round of refinements of the ISW1-NCP-complex sample. (f) Resolution estimation of the EM maps. Gold standard Fourier shell correlation (FSC) curves, showing the overall nominal resolutions of 3.29 Å, 3.40 Å for the NCP masked region and ISW1-NCP-complex, respectively.

Supplementary Figure 6 Local density maps of the ISW1–nucleosome complex in the ADP-bound state determined at a resolution of 3.29 Å.

(a) Local maps of the DNA at the positions 61-68. (b) Local maps of the DNA at the positions 32-49.

Supplementary Figure 7 Structural changes of ISW1 after activation.

(a) Superimposition of the structure of ISW1 (color coded) in the ADP-BeFx- nucleosome-bound state and that of MtISWI in the auto-inhibited state (grey, PDB code 5JXR) (Nature 540, 466–469, 2016). Only the region around the catalytic pocket is shown. The alignment was performed on core1 of the enzymes. (b) Superimpositions of the structure of core1 of ISWI in the activated state (color coded) and the inhibited state (grey). Dashed line, the disordered region of AutoN in the activated state. (c) Interactions with the histones H4 and H3 by ISW1 in the context of the nucleosome. (d) Comparison of the H4-binding modes in the contact of the nucleosome and free H4 peptide. MtISWI (PDB code 5JXT), grey; H4 peptide, magenta. Structural alignment was performed on core2 of the enzymes.

Supplementary Figure 8 Structure refinement of H4 within the ISW1–NCP complex in the ADP-BeFx-bound state using Rosetta.

(a) RMSDs of three regions of H4 calculated between the Rosetta-refined ten top-scoring structures and our model. (b-d) Structural comparison at the regions of H4 residues 24-32 (b), residues 45-50 (c), and residues 55-70 (d). Free NCP (PDB code 3MVD) (Nature 467, 562–566, 2010), black; our model, gold; the Rosetta-refined structures are colored differently.

Supplementary Figure 9 Little AFFECTS the remodeling activity of ISWI and Snf2 by cross-linking of H3-H4.

(a) SDS-PAGE analysis of the WT, crosslinked (X, treated with oxidized glutathione), and rescued (X+DDT) nucleosomes. H3*-H4*, crosslinked H3-H4 dimer. (b) Native PAGE analysis of the WT and crosslinked nucleosomes. (c) Mass-Spec analysis of the H3-H4 linkage by S-S bond. (d) SDS-PAGE and native PAGE analyses of crosslinked (X, treated with Cu-phenanthroline) and rescued (X+DDT) nucleosomes. (e-f) Chromatin remodeling activities of Snf2h (e) and ISW1 (f). WT nucleosome, black; crosslinked nucleosome (treated with Cu-phenanthroline), red; rescued nucleosome (treated with Cu-phenanthroline and then reverted with DTT), blue. Error bars indicate s.d. (n=3). (g) SDS-PAGE and native PAGE analyses of crosslinked (X, treated with Cu-phenanthroline) and rescued (X+DDT) H3C110A nucleosomes.

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Supplementary Figures 1–9 and Supplementary Dataset 1

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Supplementary Video 1

CryoEM map of the ‘601’ nucleosome bound by ISW1 in the ADP state

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Yan, L., Wu, H., Li, X. et al. Structures of the ISWI–nucleosome complex reveal a conserved mechanism of chromatin remodeling. Nat Struct Mol Biol 26, 258–266 (2019). https://doi.org/10.1038/s41594-019-0199-9

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