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

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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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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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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Correspondence to Karl-Peter Hopfner.

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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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Supplementary Dataset 1

Uncropped gel images

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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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