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Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome

Nature volume 579, pages 448–451 (2020)Cite this article

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Abstract

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and −1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase–nucleosome interactions to regulate RSC activity5. The RSC–nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.

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Fig. 1: RSC–nucleosome complex structure.
Fig. 2: RSC ATPase and ARP modules.
Fig. 3: RSC sandwiches the nucleosome.

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Data availability

The coordinate file for the RSC–nucleosome complex structure was deposited with the Protein Data Bank with accession code 6TDA. The cryo-EM maps used for model building were all deposited with the Electron Microscopy Data Base with accession code EMD-10465.

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Acknowledgements

We thank current and former members of the Cramer Laboratory, including S. Osman, G. Kokic, P. Seweryn, S. Schilbach, S. Neyer and H. Hillen. F.R.W. was supported by a Boehringer Ingelheim Fonds PhD fellowship. H.U. was supported by the Deutsche Forschungsgemeinschaft (SFB860). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935, EXC 2067/1-390729940), the European Research Council Advanced Investigator Grant TRANSREGULON (grant agreement no. 693023), and the Volkswagen Foundation.

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Authors and Affiliations

  1. Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany

    Felix R. Wagner, Christian Dienemann, Haibo Wang, Dimitry Tegunov & Patrick Cramer

  2. Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry, Göttingen, Germany

    Alexandra Stützer & Henning Urlaub

  3. University Medical Center Göttingen, Institute of Clinical Chemistry, Bioanalytics Group, Göttingen, Germany

    Henning Urlaub

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Contributions

F.R.W. carried out all experiments and data analysis unless stated otherwise. C.D. assisted with data collection and model building. A.S. and H.U. carried out cross-linking and mass spectrometry analysis. H.W. helped with nucleosome biochemistry. D.T. helped with cryo-EM data processing. P.C. designed and supervised the project. F.W. and P.C. wrote the manuscript with input from all authors.

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Correspondence to Patrick Cramer.

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Peer review information Nature thanks Blaine Bartholomew and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Preparation and characterization of RSC–nucleosome complex.

a, Preparation of endogenous Rsc2-containing isoform of the RSC complex from S. cerevisiae. Analysis of purified RSC by size-exclusion chromatography and SDS–PAGE showed high purity and homogeneity with stoichiometric subunits as assessable by Coomassie stain. Subunit identity was confirmed by mass spectrometry. The table shows the expected molecular weights of the RSC subunits. For gel source data, see Supplementary Fig. 1. b, Assembly of the RSC–nucleosome complex. SDS–PAGE analysis of fractions 7–20 of a 10–25% sucrose-gradient ultracentrifugation. Complex formation was successful as demonstrated by the comigration of histones with the RSC complex. The unbound over-stoichiometric nucleosomes only migrated to fractions 7 and 8 (black arrow). Fraction 16 in the presence of cross-linker was used for cryo-EM grid preparation (dashed box). c, Location of cross-linking sites mapped onto the structure. BS3 cross-links that appeared at least in triplicates were mapped onto the RSC-nucleosome structure. Lysine residues involved in the cross-linking network are shown as blue spheres and cross-linked residues are connected with lines indicating permitted (blue) and non-permitted (red) cross-linking distances. Of the mapped cross-links, 87.5% are within the permitted cross-linking distance, which was set to 30 Å. The remaining 12.5% of non-permitted cross-links probably reflect ambiguity caused by the presence of two identical Rsc8 subunits in the structure as well as flexibility of the complex in buffer or arise from technical errors. d, The cross-linking network between subunits of the RSC–nucleosome complex. Subunits are coloured as in Fig. 1. Cross-links with a score above 2.5 are shown. A comprehensive list of cross-links is presented in Supplementary Data 1. Cross-linking mass-spectrometry experiments were performed in duplicates with similar results.

Extended Data Fig. 2 Cryo-EM analysis of the RSC–nucleosome complex.

a, Representative cryo-EM micrograph of the RSC–nucleosome complex shows homogeneously distributed individual particles. b–d, Two-dimensional class averages of the RSC–nucleosome complex (b), the ATPase–nucleosome subcomplex (c) and the nucleosome subcomplex (d). e, FSC plots reveal the overall resolutions of the cryo-EM reconstructions. f, Cryo-EM processing workflow for the reconstructions of the RSC–nucleosome complex, the ATPase–nucleosome subcomplex and the nucleosome subcomplex. Particle distribution after 3D classifications is indicated below the corresponding map. The final maps are shown in colour. The masks used for focused classifications and refinements are colour coded corresponding to the final maps they were used for. Views are generally rotated by 180° with respect to Fig. 1c, left. g, Local resolution estimation of the combined ATPase–nucleosome map as implemented in RELION42. We note that the resolution of the peripheral area with the ATPase module is overestimated. h–j, Angular distribution plot for all particles contributing to the final reconstructions of the RSC–nucleosome complex (h), the ATPase–nucleosome subcomplex (i) and the nucleosome subcomplex (j).

Extended Data Fig. 3 Cryo-EM analysis of the free RSC complex.

a, Representative cryo-EM micrograph of the free RSC complex shows homogeneously distributed individual particles. b, Two-dimensional class averages of the free RSC complex. c, Cryo-EM processing workflow for the reconstruction of the free RSC complex. Particle distribution after 3D classifications is indicated below the corresponding map. The final maps after focused 3D refinement and masks are depicted in colour. Views are generally rotated by 180° with respect to Fig. 1c, right. d, Angular distribution plot for all particles contributing to the final reconstruction of the free RSC complex. e, Two views of the combined RSC core map coloured according to the local resolution as implemented in RELION42. f, FSC plots of the maps used for model building of the RSC core complex.

Extended Data Fig. 4 Cryo-EM densities for selected RSC regions.

a–c, Examples of map quality. a, Close-up of the Rsc4 β-sheet shows clear separation of individual strands. b, The high quality of the map for the ZZ zinc-finger of Rsc8 allowed backbone tracing and placement of side chains as well as for the zinc ion. c, Coiled-coil helices of the two Rsc8 subunits with density for one helix. d, View along the exit DNA in the direction of the nucleosome showing the lowpass-filtered maps for the modules ATPase, ARP, DIM, arm and body, and the nucleosome. At the site where the H2A C-terminal tail protrudes from the nucleosome near Sfh1, there is low-resolution density connecting the arm module and the nucleosome. Density bridging form the ARP module to the exit DNA close to the H3 histone tail can be observed. e, Density representing the finger helix (green) at the acidic patch of the nucleosome (indicated by H2A in yellow). Side-chain density is visible for conserved arginine residues. f, Interaction of RSC with the nucleosome is sterically impaired by the flexibly bound ubiquitin moiety at H2B lysine 123 (K120 in human). The Sfh1 finger helix and the ubiquitin moiety (ubiquitylated nucleosome PDB code 6NOG)81 overlap after superposition of nucleosomes.

Extended Data Fig. 5 Structure of RSC body and arm modules, cancer mutations and remodeller families.

a, Cartoon representation of RSC core viewed as in Fig. 1. Important structural elements are labelled. b, Conservation between SWI/SNF complexes RSC (yeast) and PBAF (human). Residues that are identical (blue) or conserved (light blue) in human PBAF highlighted on the RSC structure (grey). Purple spheres depict identical residues that show missense mutations in various cancers. c, Comparison of overall structure of RSC with complexes of INO80 (yeast INO80)82 and CHD (yeast CHD1)83 families. ATPase motor domains are shown in orange, DNA is shown in blue. With regard to the INO80 family, the ATPase of the SWR1 complex also binds SHL +2 (ref. 84), whereas the ATPase of the INO80 complex binds SHL −6 (refs. 82,85). The ARP module of INO80 contacts exit DNA, which is not the case in RSC. The INO80 complex also contacts both faces of the histone octamer82, resembling the sandwiching interactions made by RSC on a topological level. With respect to the CHD family, the ATPase motor of yeast Chd1 also binds SHL +2, but its DNA-binding region engages with exit DNA near the nucleosome, leading to a different DNA trajectory83,86. With respect to the ISWI family, the ATPase motor binds SHL +2 (ref. 87), but other interactions have not been structurally resolved (not shown).

Extended Data Fig. 6 Course of polypeptide chains of architectural subunits Sth1, Rsc8 and Rsc58 and ATPase–nucleosome interactions.

a, Back view of RSC. The Sth1 subunit of RSC starts with its N terminus in the body module and tracks through it, turning around with a contact helix and loop. Forming the central helix I, the hook and the central helix II it folds back and forth tightly interweaving the body module before it exits with its HSA region through the ARP module to build the ATPase module. b, RSC with the domains of the two Rsc8 subunits highlighted in blue. Both Rsc8 start N-terminal with their SWIRM domains in the arm module where they support the two repeat domains of Sfh1 in a similar manner. They then follow distinct paths through the arm towards the body module where they contribute with both their SANT and ZZ zinc-finger domains. Here the two domains of each subunit form different contacts with various interactions partners and whereas one ZZ zinc-finger domain is tightly packed at the body and DNA-interaction module interface, the other seems to extend from the body, presumably as additional interaction surface. Both Rsc8 subunits unite again with their C-terminal long helices in a coiled-coil fold on the opposite side of the body module. c, Rsc58 N-terminal bromodomain attaches to the top of the body module. Then, Rsc58 follows an interwound path through the body module via the central and connector loop. It turns back, docking to the body with a three-helix bundle and stabilizing the module with its C-terminal end. d, Contacts of Sth1 ATPase motor (orange) with the nucleosome. View as in Fig. 1c, left, but rotated by 45° around a horizontal axis. Arrows indicate directionality of DNA translocation.

Extended Data Fig. 7 DNA recognition and NDR formation.

a, Space-filling RSC–nucleosome structure with DIM (green) and SnAC (orange) densities. View on the top as in Fig. 1c, left, but rotated by 90° around the vertical and horizontal axis. Arrows indicate directionality of DNA translocation. Number of upstream DNA base pairs relative to SHL −7 is provided. b, Schematic of a promoter before (top) and after (bottom) RSC remodelling shows NDR formation by sliding the flanking –1 and +1 nucleosomes away from the NDR centre. Arrows indicate the transcription start site.

Extended Data Fig. 8 Sequence alignments for the Sth1 ATPase domain and HSA region.

a, Sequence alignment of the S. cerevisiae Sth1 ATPase domain to the homologous Snf2 ATPase domain of the same organism. Secondary structure elements are represented in orange according to the cryo-EM structure of the Snf2 ATPase (PDB entry 5Z3U)17. Residues modelled in the Snf2 structure are topped by a black line with helical regions shown as cylinders and sheet regions as arrows. The Sth1 residues modelled in this work are indicated with a black dashed line below. ATPase motifs are underlined. Invariant residues are coloured in dark blue and conserved residues in light blue. The alignment was generated with MSAProbs71 within the MPI Bioinformatics Toolkit60 and visualized using ESPript88. b, Sequence alignment of the HSA regions from S. cerevisiae homologues Sth1 and Snf2. Illustration and generation of the alignment as in a.

Extended Data Table 1 Subunit composition of RSC and related chromatin remodelling complexes
Full size table

Supplementary information

Supplementary Figure

Supplementary Figure 1 | Uncropped gel images with size marker. Uncropped gel images for Extended Data Figs. 1a and 1b. * Sample from another unrelated experiment, not from the size exclusion chromatography run.

Reporting Summary

Supplementary Table

Supplementary Table 1 | RSC subunit modelling. Modelling details for the RSC complex. Density that could not be assigned to a subunit was modelled with a poly alanine backbone in chain X. The domains of the two Rsc8 subunits could not be connected. Therefore, they are clustered by proximity and combined in one chain (L) spaced by a 443 amino acid number offset.

Supplementary Table

Supplementary Table 2 | Cryo-EM data collection, refinement and validation statistics. * Dataset from two collections were combined. Datasets from three collections were combined, † not tilted, ‡ 25° tilt. # Reported value is based on visual inspection, because the software-generated values were overestimated. § Map7: chains A, B, C, D, E, F, G, H, I (2-124), J (24-146), K (390-408) and S (393-1006); Map8: chains K (151-389), L, M, N, O, P, Q, R, S (48-297) and X.

Supplementary Data

Supplementary Data 1 | RSC-nucleosome BS3 crosslinks. List of intra- and inter-subunit lysine-lysine crosslinks as identified by LC-MS analyses and subsequent database search using pLink 2. The respective scores of cross-link identification are listed as well as the number of CSMs (cross-linked spectra matches). The numbering for the histone H3 is shifted because the N-terminal PEP sequence was omitted for purification (instead of MPEPAKSAP... it is MAKSAP...). The numbering for the Rsc2 protein includes the remains of C-terminal TAP-tag after TEV cleavage.

Video 1

Overview of RSC structure. The video shows the structure of RSC rotating around a vertical axis. It first depicts the low pass-filtered cryo-EM  map, showing the five lobes of RSC and the nucleosome with exit DNA extending from it. It then shows the high-resolution cryo-EM maps for RSC modules, and finally the structural model as a ribbon representation with subunits in different colours (colour code as in Fig. 1).

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Wagner, F.R., Dienemann, C., Wang, H. et al. Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome. Nature 579, 448–451 (2020). https://doi.org/10.1038/s41586-020-2088-0

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