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The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling

Nature Structural & Molecular Biology volume 25, pages 823–832 (2018)Cite this article

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Abstract

Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180-kDa Arp8 module of Saccharomyces cerevisiae INO80 and establish its role in recognition of extranucleosomal linker DNA. Arp8 engages N-actin in a manner distinct from that of other actin-fold proteins and thereby specifies recruitment of the Arp4–N-actin heterodimer to a segmented scaffold of the helicase-SANT-associated (HSA) domain of Ino80. The helical HSA domain spans over 120 Å and provides an extended binding platform for extranucleosomal entry DNA that is required for nucleosome sliding and genome-wide nucleosome positioning. Together with the recent cryo-electron microscopy structure of INO80Core–nucleosome complex, our findings suggest an allosteric mechanism by which INO80 senses 40-bp linker DNA to conduct highly processive chromatin remodeling.

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Fig. 1: Crystal structure of the 180-kDa Ino80HSA-Arp4–N-Actin–Arp8 complex.
Fig. 2: The Arp4–N-actin heterodimer is a conserved structural module of chromatin complexes.
Fig. 3: Arp8 recruits Arp4–N-actin to a segmented ‘two-plug’ scaffold of Ino80HSA.
Fig. 4: Extranucleosomal DNA binding by the Arp8 module is critical for INO80 nucleosome sliding and genome-wide nucleosome positioning.
Fig. 5: Structural model of the INO80Core+Arp–nucleosome complex.
Fig. 6: Conserved architecture of N-actin–Arp modules in INO80/SWR1 and SWI/SNF family chromatin remodelers.

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References

  1. Jiang, C. & Pugh, B. F. Nucleosome positioning and gene regulation: advances through genomics. Nat. Rev. Genet. 10, 161–172 (2009).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. Krietenstein, N. et al. Genomic nucleosome organization reconstituted with pure proteins. Cell 167, 709–721.e12 (2016).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Hopfner, K. P., Gerhold, C. B., Lakomek, K. & Wollmann, P. Swi2/Snf2 remodelers: hybrid views on hybrid molecular machines. Curr. Opin. Struct. Biol. 22, 225–233 (2012).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Clapier, C. R., Iwasa, J., Cairns, B. R. & Peterson, C. L. Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nat. Rev. Mol. Cell Biol. 18, 407–422 (2017).

    Article  PubMed  CAS  Google Scholar 

  5. Dion, V., Shimada, K. & Gasser, S. M. Actin-related proteins in the nucleus: life beyond chromatin remodelers. Curr. Opin. Cell Biol. 22, 383–391 (2010).

    Article  PubMed  CAS  Google Scholar 

  6. Shen, X., Mizuguchi, G., Hamiche, A. & Wu, C. A chromatin remodelling complex involved in transcription and DNA processing. Nature 406, 541–544 (2000).

    Article  PubMed  CAS  Google Scholar 

  7. Peterson, C. L., Zhao, Y. & Chait, B. T. Subunits of the yeast SWI/SNF complex are members of the actin-related protein (ARP) family. J. Biol. Chem. 273, 23641–23644 (1998).

    Article  PubMed  CAS  Google Scholar 

  8. Cairns, B. R., Erdjument-Bromage, H., Tempst, P., Winston, F. & Kornberg, R. D. Two actin-related proteins are shared functional components of the chromatin-remodeling complexes RSC and SWI/SNF. Mol. Cell 2, 639–651 (1998).

    Article  PubMed  CAS  Google Scholar 

  9. Cao, T. et al. Crystal structure of a nuclear actin ternary complex. Proc. Natl Acad. Sci. USA 113, 8985–8990 (2016).

    Article  PubMed  CAS  Google Scholar 

  10. Schubert, H. L. et al. Structure of an actin-related subcomplex of the SWI/SNF chromatin remodeler. Proc. Natl Acad. Sci. USA 110, 3345–3350 (2013).

    Article  PubMed  Google Scholar 

  11. Szerlong, H. et al. The HSA domain binds nuclear actin-related proteins to regulate chromatin-remodeling ATPases. Nat. Struct. Mol. Biol. 15, 469–476 (2008).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Meagher, R. B., Kandasamy, M. K., Smith, A. P. & McKinney, E. C. Nuclear actin-related proteins at the core of epigenetic control. Plant Signal. Behav. 5, 518–522 (2010).

    Article  PubMed  CAS  Google Scholar 

  13. Son, E. Y. & Crabtree, G. R. The role of BAF (mSWI/SNF) complexes in mammalian neural development. Am. J. Med. Genet. C 166C, 333–349 (2014).

    Article  CAS  Google Scholar 

  14. Hodges, C., Kirkland, J.G. & Crabtree, G.R. The many roles of BAF (mSWI/SNF) and PBAF complexes in cancer. Cold Spring Harb. Perspect. Med. 6, (2016).

  15. Shen, X., Ranallo, R., Choi, E. & Wu, C. Involvement of actin-related proteins in ATP-dependent chromatin remodeling. Mol. Cell 12, 147–155 (2003).

    Article  PubMed  CAS  Google Scholar 

  16. Tosi, A. et al. Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex. Cell 154, 1207–1219 (2013).

    Article  PubMed  CAS  Google Scholar 

  17. Gerhold, C. B. & Gasser, S. M. INO80 and SWR complexes: relating structure to function in chromatin remodeling. Trends Cell Biol. 24, 619–631 (2014).

    Article  PubMed  CAS  Google Scholar 

  18. Papamichos-Chronakis, M., Watanabe, S., Rando, O. J. & Peterson, C. L. Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity. Cell 144, 200–213 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Udugama, M., Sabri, A. & Bartholomew, B. The INO80 ATP-dependent chromatin remodeling complex is a nucleosome spacing factor. Mol. Cell. Biol. 31, 662–673 (2011).

    Article  PubMed  CAS  Google Scholar 

  20. Chen, L. et al. Subunit organization of the human INO80 chromatin remodeling complex: an evolutionarily conserved core complex catalyzes ATP-dependent nucleosome remodeling. J. Biol. Chem. 286, 11283–11289 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Jonsson, Z. O., Jha, S., Wohlschlegel, J. A. & Dutta, A. Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex. Mol. Cell 16, 465–477 (2004).

    Article  PubMed  CAS  Google Scholar 

  22. Zhou, C. Y. et al. The yeast INO80 complex operates as a tunable DNA length-sensitive switch to regulate nucleosome sliding. Mol. Cell 69, 677–688.e9 (2018).

    Article  PubMed  CAS  Google Scholar 

  23. Kapoor, P., Chen, M., Winkler, D. D., Luger, K. & Shen, X. Evidence for monomeric actin function in INO80 chromatin remodeling. Nat. Struct. Mol. Biol. 20, 426–432 (2013).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  24. Eustermann, S. et al. Structural basis for ATP-dependent chromatin remodelling by the INO80 complex. Nature 556, 386–390 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Ayala, R. et al. Structure and regulation of the human INO80–nucleosome complex. Nature 556, 391–395 (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Gerhold, C. B. et al. Structure of Actin-related protein 8 and its contribution to nucleosome binding. Nucleic Acids Res. 40, 11036–11046 (2012).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Saravanan, M. et al. Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex. Proc. Natl Acad. Sci. USA 109, 20883–20888 (2012).

    Article  PubMed  Google Scholar 

  28. Zhao, K. et al. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 95, 625–636 (1998).

    Article  PubMed  CAS  Google Scholar 

  29. Dominguez, R. & Holmes, K. C. Actin structure and function. Annu. Rev. Biophys. 40, 169–186 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. von der Ecken, J. et al. Structure of the F-actin-tropomyosin complex. Nature 519, 114–117 (2015).

    Article  PubMed  CAS  Google Scholar 

  31. Huang, W. et al. Structural insights into micro-opioid receptor activation. Nature 524, 315–321 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  32. Rasmussen, S. G. et al. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  33. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    Article  PubMed  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  35. Bakshi, R., Prakash, T., Dash, D. & Brahmachari, V. In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins. Biochem. Biophys. Res. Commun. 320, 197–204 (2004).

    Article  PubMed  CAS  Google Scholar 

  36. Yen, K., Vinayachandran, V., Batta, K., Koerber, R. T. & Pugh, B. F. Genome-wide nucleosome specificity and directionality of chromatin remodelers. Cell 149, 1461–1473 (2012).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  37. Mueller-Planitz, F., Klinker, H., Ludwigsen, J. & Becker, P. B. The ATPase domain of ISWI is an autonomous nucleosome remodeling machine. Nat. Struct. Mol. Biol. 20, 82–89 (2013).

    Article  PubMed  CAS  Google Scholar 

  38. Yen, K., Vinayachandran, V. & Pugh, B. F. SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes. Cell 154, 1246–1256 (2013).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Brahma, S., Ngubo, M., Paul, S., Udugama, M. & Bartholomew, B. The Arp8 and Arp4 module acts as a DNA sensor controlling INO80 chromatin remodeling. Nat. Commun. https://doi.org/10.1038/s41467-018-05710-7 (2018).

  40. Schwarz, M. et al. Single‐molecule nucleosome remodeling by INO80 and effects of histone tails. FEBS Lett. 592, 318–331 (2018).

    Article  PubMed  CAS  Google Scholar 

  41. Brahma, S. et al. INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers. Nat. Commun. 8, 15616 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Clapier, C. R. et al. Regulation of DNA translocation efficiency within the chromatin remodeler RSC/Sth1 potentiates nucleosome sliding and ejection. Mol. Cell 62, 453–461 (2016).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  43. Liu, X., Li, M., Xia, X., Li, X. & Chen, Z. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure. Nature 544, 440 (2017).

    Article  PubMed  CAS  Google Scholar 

  44. Turegun, B., Baker, R. W., Leschziner, A. E. & Dominguez, R. Actin-related proteins regulate the RSC chromatin remodeler by weakening intramolecular interactions of the Sth1 ATPase. Commun. Biol. 1, 1 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Turegun, B., Kast, D.J. & Dominguez, R. Subunit Rtt102 controls the conformation of the Arp7/9 heterodimer and its interactions with nucleotide and the catalytic subunit of SWI/SNF remodelers. J. Biol. Chem. (2013).

  46. Aramayo, R. J. et al. Cryo-EM structures of the human INO80 chromatin-remodeling complex. Nat. Struct. Mol. Biol. 25, 37–44 (2018).

    Article  PubMed  CAS  Google Scholar 

  47. Watanabe, S. et al. Structural analyses of the chromatin remodelling enzymes INO80-C and SWR-C. Nat. Commun. 6, 7108 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  48. Yamada, K. et al. Structure and mechanism of the chromatin remodelling factor ISW1a. Nature 472, 448–453 (2011).

    Article  PubMed  CAS  Google Scholar 

  49. Lin, C.-L. et al. Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex. Nucleic Acids Res. 45, 7249–7260 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  50. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

    Article  PubMed  CAS  Google Scholar 

  51. Conrath, K. E. et al. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob. Agents Chemother. 45, 2807–2812 (2001).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  52. Trowitzsch, S., Bieniossek, C., Nie, Y., Garzoni, F. & Berger, I. New baculovirus expression tools for recombinant protein complex production. J. Struct. Biol. 172, 45–54 (2010).

    Article  PubMed  CAS  Google Scholar 

  53. Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2004).

    Article  PubMed  CAS  Google Scholar 

  54. Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998).

    Article  PubMed  CAS  Google Scholar 

  55. Levendosky, R.F., Sabantsev, A., Deindl, S. & Bowman, G.D. The Chd1 chromatin remodeler shifts hexasomes unidirectionally. eL ife 5 (2016).

  56. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  57. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  58. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  59. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  60. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  61. Afonine, P. V. et al. FEM: feature-enhanced map. Acta Crystallogr. D Biol. Crystallogr. 71, 646–666 (2015).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  62. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  PubMed  CAS  Google Scholar 

  63. The PyMOL Molecular Graphics System, Version 1.8. (Schrodinger, LLC, 2015).

  64. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).

    Article  PubMed  CAS  Google Scholar 

  65. Kirchhofer, A. et al. Modulation of protein properties in living cells using nanobodies. Nat. Struct. Mol. Biol. 17, 133–138 (2010).

    Article  PubMed  CAS  Google Scholar 

  66. Keilhauer, E. C., Hein, M. Y. & Mann, M. Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS). Mol. Cell. Proteomics 14, 120–135 (2015).

    Article  PubMed  CAS  Google Scholar 

  67. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  PubMed  CAS  Google Scholar 

  68. Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  69. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    Article  PubMed  CAS  Google Scholar 

  70. Favicchio, R., Dragan, A. I., Kneale, G. G. & Read, C. M. Fluorescence spectroscopy and anisotropy in the analysis of DNA-protein interactions. Methods Mol. Biol. 543, 589–611 (2009).

    Article  PubMed  CAS  Google Scholar 

  71. Kiianitsa, K., Solinger, J. A. & Heyer, W.-D. NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 321, 266–271 (2003).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We are grateful to M. Moldt for technical support and J. Michaelis, Gregor Witte, Katja Lames, and Robert Byrne for discussion and technical help. We thank the Max-Planck Crystallization Facility (Martinsried, Germany), the staff of the Swiss Light Source (Villingen, Switzerland), and the European-Molecular-Biology-Laboratory/Deutsches-Elektronen-Synchotron (Hamburg, Germany) for support and measurement time. We thank S. Krebs and H. Blum at the Laboratory of Functional Genome Analysis (Gene Center, LMU Munich) for high-throughput sequencing. We thank T. Straub (Bioinformatics Core Unit, Biomedical Center, LMU Munich) for advice on bioinformatics. This work is supported by the Deutsche Forschungsgemeinschaft CRC1064 (to K.-P.H. and P.K.) and the European Research Council (ERC Advanced Grant ATMMACHINE), the Gottfried-Wilhelm-Leibniz Prize, and the Center for Integrated Protein Sciences Munich to K.-P.H. K.R.K. is supported by GRK1721. S.E. acknowledges an EMBO long-term fellowship. V.N., K.S., and M.S. acknowledge funding by Quantitative Biosciences Munich.

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Author notes

  1. Gabriele Stoehr

    Present address: OmicScouts GmbH, Freising, Germany

  2. Alessandro Tosi

    Present address: Vossius & Partner, Munich, Germany

  3. These authors contributed equally: K.R. Knoll, S. Eustermann.

Authors and Affiliations

  1. Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany

    Kilian R. Knoll, Sebastian Eustermann, Vanessa Niebauer, Gabriele Stoehr, Kevin Schall, Alessandro Tosi, Marianne Schwarz & Karl-Peter Hopfner

  2. Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

    Kilian R. Knoll, Sebastian Eustermann, Vanessa Niebauer, Gabriele Stoehr, Kevin Schall, Alessandro Tosi, Marianne Schwarz & Karl-Peter Hopfner

  3. Chair of Molecular Biology, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany

    Elisa Oberbeckmann & Philipp Korber

  4. Institute of Biophysics, Ulm University, Ulm, Germany

    Marianne Schwarz

  5. ChromoTek GmbH, Planegg, Germany

    Andrea Buchfellner

  6. Center for Integrated Protein Science, Ludwig-Maximilians-Universität München, Munich, Germany

    Karl-Peter Hopfner

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Contributions

K.R.K. and S.E. determined the structures and built atomic models. K.R.K. prepared samples for crystallization and performed biochemical analysis of the Arp8 module. S.E., A.T., M.S., and A.B identified the Arp4–N-actin binding nanobody and performed its initial characterization. K.R.K. and G.S. performed affinity enrichment mass spectrometry analysis. S.E. and K.P.H. devised with a contribution of M.S. preparation and characterization of recombinant INO80 complex. V.N. prepared mutant complexes and performed their biochemical analysis. V.N. and E.O. performed and analyzed genome-wide remodeling assays under supervision by P.K. K.S. prepared nucleosomes. S.E. and K.-P.H. designed the overall study, analyzed the results, and wrote the paper with contributions from K.R.K., V.N., E.O., and P.K.

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Supplementary Figure 1 Crystal structure of the 180 kDa Arp4–N-actin–Arp8–Ino80HSA module.

a, SDS–PAGE and Coomassie brilliant blue staining of the Arp8 module and the size-exclusion chromatography elution profile of the Arp8 module on a S200 10/300 column. Uncropped gel images are shown in Supplementary Dataset 1. b,c, Close-ups of the Arp8 module structure showing the N-actin(ATP/LAR) and Arp4(ATP) nucleotide-binding pockets with electron density (green mesh) for the bound ligands (mFo – DFc difference map with a carving radius of 20 Å around each ligand; contoured at 3σ; resulting from structure refinement with phenix.refine lacking ligands). d, Close-up of the Arp4–N-actin–Swr1HSA crystal structure N-actin(apo) nucleotide-binding pocket (PDB 5I9E). The dotted line illustrates the canonical ATP-binding site of actin. Interestingly, Asp157 would block ATP binding in the apo N-actin structure, while it is moved outward in the ATP-bound N-actin structure in b

Supplementary Figure 2 Arp4–N-actin heterodimer: a conserved structural subunit in chromatin complexes.

a, SDS–PAGE and Coomassie brilliant blue staining of the NactNB–Arp4–N-actin complex and the size-exclusion chromatography elution profile of the NactNB–Arp4–N-actin complex on a S200 10/300 column. Uncropped gel images are shown in Supplementary Dataset 1. b, Surface representation of the NactNB–Arp4–N-actin complex displaying the NactNB-binding epitope on the Arp4–N-actin dimer. Gray regions indicate recognition sites of the respective labeled NactNB-binding element. c, Close-up of the NactNB–Arp4–N-actin(apo) structure N-actin nucleotide-binding pocket, with the green mesh displaying electron density in the mFo – DFc difference electron density map (contoured at 3σ with a carving radius of 20 Å around ATP-binding residues Asp157, Ser14 and Gln137). The dotted line illustrate the canonical ATP-binding site of actin. d,e, Close-ups of the NactNB–Arp4–N-actin structure N-actin(ATP) and Arp4(ATP) nucleotide-binding pockets with electron density (green mesh) for the bound ligands (mFo – DFc difference map with a carving radius of 20 Å around each ligand; contoured at 3σ; resulting from structure refinement with phenix.refine lacking ligands)

Supplementary Figure 3 Arp8 recruits Arp4–N-actin to a segmented ‘two-plug’ scaffold of Ino80HSA.

a, Electron density for Ino80HSA. Shown is a feature-enhanced map (FEM) calculated by phenix.fem at a contour level of 1σ (blue mesh). Ino80HSA is shown in stick representation. b, Structures of the Arp4–N-actin–Arp8–Ino80HSA complex, the Arp4–N-actin–Swr1HSA complex (PDB 5I9E) and the Arp9–Arp7–Snf2HSA complex (PDB 4I6M) shown as cartoon representations. c, Close-ups display binding of Ino80HSA plug 1 to a hydrophobic pocket at the barbed end of Arp4 and plug2 to a hydrophobic pocket at the barbed end of Arp8

Supplementary Figure 4 Extranucleosomal DNA binding by the Arp8 module is important for nucleosome sliding and genome-wide nucleosome positioning.

a, Electrostatic surface potential of the Arp8 module (calculated with the APBS PyMol plugin; Proc. Natl. Acad. Sci. USA 98, 10037–10041, 2001) shown as surface representation. Ino80HSA is outlined by a dotted line. b, SDS–PAGE and Coomassie brilliant blue staining of the Arp8 module (WT) and the Arp8 module in complex with NactNB (WT and Ino80HSA mutant HSAα2). Size-exclusion chromatography elution profiles are shown of the different Arp8 module complexes on an S200 10/300 column. c, Arp8 module 40-bp dsDNA binding affinity measured by fluorescence anisotropy (with 20 nM dsDNA). Anisotropy is plotted against Arp8 module protein concentration and fitted to a non-linear non-cooperative 1:1 binding model (Methods). Data points and error bars represent the means ± s.d. from three independent experiments. d, Competition electrophoretic mobility shift assays with two nucleosome species (20 nM each), one with 80 bp (0N80) and one without DNA overhang (0N0), and increasing concentration of the indicated Arp8 module complexes. For Arp8 module(wt)-NactNB and Arp8 module(HSAα2)-NactNB, the same gels are shown in Fig. 4b. Experiments were performed in triplicates. e, Sequence alignment of WT Ino80HSA (residues 476–560) and the three Ino80HSA mutants. Mutated residues are highlighted in purple. f, Left, SDS–PAGE and SimplyBlue (Thermo Scientific) staining of the INO80 WT, HSAα1, HSAα2 and HSAα1/α2 complexes. Right, heat map showing color-coded quantification of the respective protein band intensity for the SDS gel in the left panel. Band intensity was determined using the profile plot implementation of ImageJ (Nat. Methods 9, 671–675, 2012). g, Electrophoretic mobility shift assays with 0N80 nucleosomes (20 nM) and increasing concentrations of the indicated INO80 complexes. Experiments were performed in triplicate. h, Graphical description of the restriction enzyme accessibility assay shown in i (adopted from Cell 167, 709–721, 2016). i, Restriction enzyme accessibility assay displaying the INO80 (18 nM) nucleosome-remodeling activity of arrays of 601-based nucleosomes under conditions identical to those in the genome-wide remodeling assays shown in Fig. 4e,f. Experiments were performed in triplicate. (Uncropped gel images are shown in Supplementary Dataset 1.)

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Knoll, K.R., Eustermann, S., Niebauer, V. et al. The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling. Nat Struct Mol Biol 25, 823–832 (2018). https://doi.org/10.1038/s41594-018-0115-8

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