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Structure and regulation of the human INO80–nucleosome complex

Naturevolume 556pages391395 (2018) | Download Citation

Abstract

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines1. Biochemical studies2,3,4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2—a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)—in complex with nucleosomes5,6,7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding8, histone exchange9 and nucleosome spacing10,11,12 remain poorly understood. Although some remodellers work as monomers13, others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5–IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain—unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

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Acknowledgements

We thank A. Siebert and Y. Chaban at eBIC for assistance with data collection, and C. Aylett for help in preparing the nucleosome model. Electron microscopy access and support were provided by the UK national electron Bio-Imaging Centre (eBIC) (proposal EM14769), funded by the Wellcome Trust, MRC and BBSRC. The work was funded by the Wellcome Trust (D.B.W. and X.Z.), Cancer Research UK (D.B.W.) and an Imperial College President’s PhD Scholarship (R.A.).

Reviewer information

Nature thanks B. Bartholomew, O. Llorca and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. These authors contributed equally: Rafael Ayala, Oliver Willhoft.

Affiliations

  1. Section of Structural Biology, Dept. Medicine, Imperial College London, London, UK

    • Rafael Ayala
    • , Oliver Willhoft
    • , Ricardo J. Aramayo
    • , Martin Wilkinson
    • , Elizabeth A. McCormack
    • , Lorraine Ocloo
    • , Dale B. Wigley
    •  & Xiaodong Zhang

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Contributions

D.B.W. and X.Z. designed the studies. R.A., R.J.A., M.W. and O.W. performed the cryo-electron microscopy analysis, and built and refined the structural models. O.W., E.A.M. and L.O. prepared the samples. O.W. conducted the biochemical experiments. D.B.W. and X.Z. wrote the manuscript with input from all the authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Dale B. Wigley or Xiaodong Zhang.

Extended data figures and tables

  1. Extended Data Fig. 1 Analysis of INO80–nucleosome complex sample.

    a, MST experiment of INO80 with nucleosome flanked by 60 and 12 base pair overhangs (60N12) (±3 mM ADP•BeF3). Raw data (top) were processed to analyse binding and cooperativity (bottom). Data points represent mean values with s.d.; n = 3 experimentally independent replicates. b, Gel of electron microscopy sample (INO80 + nucleosome). Two loadings are shown to enable assessment of INO80 stoichiometry (left) or histones (right). n = 3 independent experimental measurements. c, DNA sequence of the 50N25 nucleosome used for the structure determination. The Widom sequence (yellow) is flanked by 50 base pairs on one side and 25 base pairs on the other. A three-base single strand overhang that remained from the restriction cleavage site is depicted in lowercase. For gel source data, see Supplementary Fig. 1. Source data

  2. Extended Data Fig. 2 Cryo-electron microscopy data processing of INO80–nucleosome complex.

    a, A typical micrograph out of the 5,479 micrographs generated. b, Representative 2D classes (from 100 generated) obtained with RELION from 775,804 particles. c, Image processing scheme. Data were processed by two parallel pathways to obtain maps for model building.

  3. Extended Data Fig. 3 Quality of the structures.

    a, Local resolution map of the INO80–nucleosome complex (4.8 Å) (left) and cut away (right). b, Angular distribution of these particles. c, Local resolution map of the INO80 complex (3.8 Å) (left) and cut away (right). d, Angular distribution of these particles. e, Corrected FSC curves of the reconstructions.

  4. Extended Data Fig. 4 Assessment of various structural features in the INO80–nucleosome complex.

    a, Overall fold of the INO80-I and motor domains. b, Locations of the INO80-I, motor domains and IES2 regions relative to the RUVBL1–RUVBL2 hexamer. c, Sequence alignment of the C-terminal regions of human and yeast IES2. The built part of the human IES2 structure is indicated by a yellow bar. Asterisks indicate lysine residues in yeast Ies2 that crosslink to Ino80-HN (red) or Ino80-HC (blue). d, Representative density from two regions of the INO80. Insert: top, Density in the deposited 4.8 Å INO80-nucleosome map; bottom, improvement in density in the 3.8 Å map, which facilitated model building. e, Coordinates of IES2 showing formation of β-sheet secondary structure with RUVBL1 (chain E) and RUVBL2 (chain D) within the 3.8 Å map. f, Left, fit of ARP5 into 4.8 Å map (left). Centre, DNA and motor domains fit into the 4.8 Å map (centre). Right, perpendicular view of centre panel that shows the DNA crossing the motor domains.

  5. Extended Data Fig. 5 Comparisons of INO80–nucleosome interactions with those of Chd1 and Snf2.

    Images are viewed from the top of nucleosome, showing that all the motor domains are located on one side and that ARP5–IES6 (green) contacts the other side of the DNA wrap. Chd1 induces an unwrapping of the DNA at the SHL −7 position owing in a large part to interactions with the accessory SANT and SLIDE domains. Despite this unwrapping, the histone core remains largely unaltered. Although the Snf2–nucleosome structure does not induce unwrapping of DNA, it is only a fragment of the motor subunit and also lacks other accessory subunits of the SWI–SNF complex and so probably presents an incomplete picture of interactions or DNA distortions within the nucleosome in the complex.

  6. Extended Data Fig. 6 Interaction of human actin, ARP5 and ARP8 with human H2A–H2B dimers assessed by in vitro pulldown.

    a, Actin and actin-related proteins were all expressed with a C-terminal double-Strep tag and used as bait to capture untagged H2A–H2B dimers. The result supports the position of ARP5 in the reported structure. Assay products were visualized by SDS–PAGE and Coomassie staining. n = 1. b, A comparison of ARP5–IES6 and ARP5 nucleosome-binding activity assayed by electrophoretic mobility shift assay, which demonstrates a lack of nucleosome-binding activity by ARP5 at in vivo relevant concentrations in the absence of IES6. Nucleosomes were labelled with Alexa Fluor 488. Reaction species were visualized by fluorescent scan. n = 1. c, ARP5–IES6 and 0N100 nucleosome interaction measured by MST. d, ARP5–IES6 and H2A–H2B interaction measured by MST. For gel source data, see Supplementary Fig. 1. n = 2 biologically independent experiments in all the graphs. Error bars represent s.d. from the mean values. Source data

  7. Extended Data Fig. 7 INO80 SC1 is flexible in the INO80–nucleosome complex.

    a, Individual particles (selected out of 775,804 particles in total) with RUVBL1–RUVBL2 oriented similarly, to show different orientations of SC1 (dashed lines). b, Two-dimensional class averages (~30 particles each) showing different orientations of SC1 relative to RUVBL1–RUVBL2. c, Projections of the 3D reconstruction along the same angles of those in b, confirming the extra density as SC1. Scale bar, 100 Å.

  8. Extended Data Fig. 8 INO80 is regulated by H3 tails.

    a, Schematic of histone tail truncations used in this study. b, Initial nucleosome sliding rates of human nucleosomes that lacked different histone tails. Plots of raw data for each histone tail deletion, with Vmax obtained after fitting the data shown as a dotted line. Data are summarized in Fig. 4a. n = 2 biologically independent experiments in all the graphs. Error bars represent s.d. from the mean values. Source data

  9. Extended Data Fig. 9 INO80 is regulated by H3 tails.

    a, ATPase data and Hill coefficients for data shown in Fig. 4c. b, ATPase rates for mutations of the H3 tails. c, Nucleosomes carrying wild-type or mutated H3 tails show similar salt stability, which indicates that the mutations have not altered the stability of nucleosomes. n = 2 biologically independent experiments in all the panels. Error bars represent s.d. from the mean values. Source data

  10. Extended Data Table 1 Electron microscopy data collection, image processing and model refinement statistics

Supplementary information

  1. Supplementary Figure

    This file contains original gel images for Extended Data Fig. 1b, Extended Data Fig. 6a, 6b

  2. Reporting Summary

  3. Video 1: Rotating movie of INO80-nucleosome

    The 3D reconstruction is shown with structural models fitted in as in Fig. 1. The video was created using Chimera

Source data

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https://doi.org/10.1038/s41586-018-0021-6

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