Structural insights into assembly and function of the RSC chromatin remodeling complex

Abstract

SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.

Extended Data Fig. 1. Cryo-EM structure determination of the Sth1-Arp7-Arp9-Rtt102 RSC subcomplex (RSC^SAR)

a, Workflow for cryo-EM data acquisition and structure determination. Our processing only revealed density for the ARP module. b, Schematic for model building using Coot and Rosetta. Chains from PDB 5TGC and PDB 4I6M were used as a starting model. c, The top 10 Rosetta models were deposited as PDB 6VZG and the top model is shown with its ribbon thickness and color indicating the RMSD among all 10 models. d, The ARP module form SWI/SNF (left) has a single, unbroken α-helix for the HSA domain while that of RSC (right) comprises two α-helices separated by a loop. e, Our cryo-EM has density for a known ATP binding site in Arp7. This site is catalytically dead and the ATP is likely from endogenous ATP pools.

Extended Data Fig. 2. Cryo-EM structure determination of RSC^SAR bound to the nucleosome.

a, Workflow for cryo-EM data acquisition and structure determination. b, 3D classification with alignment was used to find ~300,000 particles with better ARP module density. This dataset reached a final global FSC resolution of ~3.9 Å with a local resolution range of 3.4–15 Å. c, Partial signal subtraction followed by 3D classification without alignment was used to identify a sub-set of particles with an alternate DNA conformation at the nucleosome DNA exit site.

Extended Data Fig. 3. Multi-body refinement of RSC^SAR bound to the nucleosome in the ADP-BeF₃ state.

a, The consensus refinement of RSC^SAR-nucleosome from Extended Data Fig. 2b was used to generate 3 masks and as a starting model for multi-body refinement. b, PCA was performed in Relion-3 and used to generate maps that show the conformational heterogeneity present in our data set. Several of these maps are shown overlaid. c, Eigenvalue plots for the top three eigenvectors. d, The maps generated in panel (b) were used to build PDB models. 10 models for each eigenvector were generated and are shown superimposed. The main sources of heterogeneity in our data set are a rocking of the ATPase domain (eigenvector 1) and rocking of the ARP module along two different axes (eigenvector 2 and 3).

Extended Data Fig. 4. Model building of RSC^SAR-nucleosome (ADP-BeF₃) and comparison with Snf2-nucleosome (ADP-BeF₃).

a, Model building schematic for RSC^SAR-nucleosome (ADP-BeF₃). Snf2-nucleosome (PDB 5Z3U) and the T. thermophilus Snf2 crystal structure (PDB 5HZR) were used as a starting point for model building and refinement using Coot, Rosetta, and phenix.real_space_refine. The ARP module built in Extended Data Fig.1 was rigid-body docked into the map, yielding a near-complete model for all components in our sample. b, PDB 5Z3U is shown docked into our cryo-EM map. c, The same view as in panel (b) but showing Sth1-nucleosome (ADP-BeF₃). d, The top Rosetta model is shown with its ribbon thickness and color indicating the RMSD among the top 10 models. e, The RSC^SAR-nucleosome structure is shown with the HSA and postHSA domains of Sth1 colored in a rainbow from N to C terminus. Residues 385–391 are shown as a cylinder to signal the linkage between the two domains and indicate that the density is compatible with an alpha helix, although this region is not included in the deposited.

Extended Data Fig. 5. The ARP module of RSC only interacts with the HSA domain of Sth1.

a, Our model of RSC^SAR-nucleosome (ADP-BeF₃). b, Model of RSC-nucleosome (PDB 6KW3) shown in the same orientation as panel (a) (left) and rotated 90° (right). The ARP module only interacts with the HSA domain in both RSC and RSC^SAR. c, Multiple RSC-nucleosome (ADP-BeF₃) cryo-EM structures were aligned. Left, all components shown and colored using the same convention. Right, Arp7–Arp9–Rtt102 and all components of the SRC are omitted to highlight the conformational flexibility of the Sth1 HSA domain (orange) among the three structures. The HSA domain from RSC^SAR is included in this panel to indicate that, despite their conformational flexibility, the HSA domains from the three RSC-nucleosome structures have a similar general orientation relative to the HSA from RSC^SAR.

Extended Data Fig. 6. Comparison of the nucleosome-bound ATPase domain of Snf2, RSC, and RSC^SAR in the ADP-BeF₃ nucleotide state.

a, The PDB models for Snf2, RSC^SAR, and RSC are shown with the same orientation and domain coloring. All three structures are from S.cerevisae, bound to the nucleosome and in the ADP-BeF₃ nucleotide state. b-d, The ATPase domain of each structure from (a) was docked into the cryo-EM map of RSC^SAR. The postHSA domain (b), Protrusion 1 domain (c), and Brace Helices (d) were colored according to their respective PDB model in each panel. Cyan: RSC^SAR, Magenta: RSC (PDB 6KW3), Orange: Snf2 (PDB 5Z3U).

Extended Data Fig. 7. Comparison of cryo-EM maps of Snf2-nucleosome in the apo, ADP, and ADP-BeF₃ states and RSC^SAR-nucleosome (ADP-BeF₃).

Each structure is shown with cryo-EM map and corresponding PDB model. All structures are bound to the nucleosome but this region is omitted for clarity. The same map at the same contour level is shown opaque (top) and transparent (bottom). Note that density for the Brace Helix I is absent in the ADP-BeF₃ state but present in the apo and ADP-bound states.

Extended Data Fig. 8. Structural comparison of the ATPase domain of SWI/SNF remodelers in the apo, ADP, and ADP-BeF₃ nucleotide states.

a, Domain schematic of Sth1 from RSC^SAR. b, Structural alignment of the ATPase domain of Snf2 apo with Snf2 - ADP (left), Snf2-ADP-BeF₃ (middle), and RSC^SAR- BeF₃ (right). All structures are colored using the domain convention shown in (a). c, All structures from (b) are aligned and colored grey, except for the Brace Helices which are colored by model: Snf2 apo, pink; Snf2 ADP, purple; Snf2 ADP-BeF₃, tan; RSC^SAR BeF₃, yellow. PDB codes for structures shown: Snf2 apo (5X0Y), ADP (5Z3O), ADP-BeF3 (5Z3U).

Extended Data Fig. 9. Conformation of the nucleosomal DNA in the cryo-EM structure of RSC^SAR-nucleosome (ADP-BeF₃).

a, The models of Snf2 (PDB 5Z3U) and RSC^SAR were aligned. A ribbon representation of the nucleosomal DNA is shown for Snf2 (grey) and Sth1 (colored by SHL). Super helical Locations for the aligned models are shown inside the cryo-EM density of RSC^SAR. b, RSC^SAR with a canonical conformation of the nucleosome. Top panel shows map and model from the initial 3.9 Å consensus refinement (Extended Data Fig. 2b). The nucleosomal DNA is colored purple and basic residues in the N-lobe are colored blue. c, RSC^SAR with a peeled conformation of the nucleosomal DNA exit site. Top panel shows map and model from the 4.3 Å refinement of a subset of particles with a peeled DNA conformation (Extended Data. Fig. 2c). The nucleosomal DNA is colored orange and basic residues in the N-lobe are colored blue. c, Alignment of canonical and peeled DNA structures. Note that basic residues in the N-lobe appear to make contact with the exit site DNA in the peeled conformation. d, SHL6 shifts between the canonical and peeled DNA conformations.

Extended Data Fig. 10. A basic patch in RSC^SAR binds to the nucleosome and is important for remodeling.

a, Different maps from the RSC^SAR consensus refinement are shown in the same orientation. A region of unassigned density is circled in yellow b, The same maps shown in panel (a) with red indicating regions of the maps that are not accounted for by the PDB model of the nucleosome. c, Schematic of the RSC^SAR construct used in nucleosome remodeling assays. Sequences of Sth1 mutants used for remodeling assays are shown below the wild type sequence. Residues mutated in the RSC^SAR-L and RSC^SAR-R variants are noted (salmon, L; green, R). d, Remodeling assay of wildtype, RSC^SAR-All Alanine, RSC^SAR-L, and RSC^SAR-R mutants. WT data are the same as in Fig. 6b. e, Remodeling assay of wildtype and RSC^SAR-R mutant. f, Remodeling assay of wildtype and RSC^SAR-L mutant. Error is reported as standard deviation where n=3 replicates.

Fig.1 |. Cryo-EM structure of the Sth1-Arp7-Arp9-Rtt102 RSC subcomplex (RSC^SAR) bound to a nucleosome.

a, Schematic representation of RSC^SAR, with Sth1 domains and their boundaries indicated. The same color scheme is used in all figures. b, 3.9 Å Cryo-EM map of RSC^SAR bound to a nucleosome. c, Molecular model of the RSC^SAR:nucleosome complex. d, e, Large rotations relate the position of the ARP module in RSC^SAR and RSC. d, Structure of a RSC:nucleosome complex (PDB 6KW3) with the portion corresponding to RSC^SAR shown with the same colors introduced above. The N-terminal portion of Sth1 preceding the HSA, which is absent in the Sth1 construct used in RSC^SAR, is shown in dim purple. The Substrate Recognition Complex (SRC) of RSC is shown in grey. e, RSC^SAR is shown with the nucleosome and Sth1 in the same orientation as that in (d). The position occupied by the ARP module in RSC (d) is shown in dim colors. The movements required to convert the ARP module from its position in RSC^SAR to that in RSC—a 120° in-plane rotation and a 90° rotation about the HSA helix—are indicated. f, Close up of the regulatory hub located at the base of the HSA helix, consisting of the C-terminal end of the HSA helix, the postHSA and P1.

Fig.2 |. The structure of the HSA–postHSA–P1 regulatory hub changes in the presence of the ARPs

a, Cryo-EM structure of Snf2 bound to the nucleosome in the presence of ADP-BeF₃ (PDB 5Z3U). Tan: nucleosome; Blue: ATPase domain; Yellow: Brace Helices; Dark pink: P1 domain; Green: postHSA domain. b, Two views showing the interaction of postHSA–P1 domains and the Brace Helices. c, Structure of RSC^SAR bound to the nucleosome in the presence of ADP-BeF₃ (this work). Coloring is the same as in panel (a) plus Orange: HSA domain; Dark Green: Arp7; Light Green: Arp9; Grey: Rtt102. d, Equivalent views as those in (b), showing the interaction of HSA–postHSA–P1 domains and the Brace Helices. The cryo-EM density for RSC^SAR is included in this panel. A pseudo four-helix bundle is formed by the HSA–postHSA–P1 domains. The green arrow highlights the longer postHSA helix present in RSC^SAR vs. Snf2 (compare (b) and (d)).

Fig.3 |. Binding of the ARP module induces folding of the HSA helix

a, Molar ellipticity plot for the HSA peptide in the presence (orange) and absence (blue) of the ARP module. Raw data are shown in grey, with the colored line representing a smoothed trace. The HSA+ARPs data was calculated by subtracting the plots shown in (b) and calculating molar ellipticity based on the HSA peptide (see Methods). b, Circular dichroism plot for the ARP module (green) and the ARP module plus HSA peptide (orange). Raw, unsmoothed data are shown. All spectra were collected with identical protein concentrations and with complexes at equimolar ratios.

Fig.4 |. RSC^SAR peels off nucleosomal DNA at the exit site.

a, 3D classification of RSC^SAR particles showed that ~25% have a peeled DNA conformation at the DNA exit site. Two cryo-EM maps are shown representing these populations. Blue: nucleosomal DNA with unpeeled exit site, Pink: nucleosomal DNA with peeled exit site. b, The exit site DNA in the published RSC-nucleosome cryo-EM structure showed a peeled conformation that was stabilized by accessory subunits in RSC not present in RSC^SAR. This panel shows a density generated from a molecular model (PDB 6KW3); only the ATPase domain of Sth1 and the nucleosome were included to simplify the comparison with the structures in (a). Yellow: DNA from RSC-nucleosome model. c, An overlay of the three structures (RSC^SAR peeled; RSC^SAR unpeeled, and RSC) shows that RSC^SAR is able to induce a DNA conformation at the exit site comparable to that of full RSC in the absence of the accessory subunits present in the latter. Colors equivalent to panels a and b.

Fig.5 |. A basic stretch in Sth1 binds to the nucleosome acidic patch

a, The RSC^SAR map was manually segmented and colored grey for the nucleosome and blue for Sth1. Inset: the nucleosome is shown colored by electrostatic potential, red (acidic) and blue (basic). The region of Sth1 that sits on top of the nucleosome’s acidic patch is highlighted with a red dashed ellipsoid and a separate interaction with helix 1 of H2B is highlighted with a yellow ellipsoid. b, Same as panel (a) but with several nucleosome-binding proteins shown in ribbon representation: Sir3 (PDB 3TU4); RCC1 (PDB 3MVD); LANA peptide (PDB 1ZLA); CENP-C peptide (PDB 4INM); and PRC1 (PDB 4R8P). c, Close up of the acidic patch and the “arginine anchor” motif. Cryo-EM density from a sharpened map of RSC^SAR is shown, with density for the arginine anchor colored blue.

Fig.6 |. Mutations in a basic region of Sth1 disrupt nucleosome remodeling

a, Schematic of the RSC^SAR construct used in nucleosome remodeling assays. Sth1_301–1097 was used for cryoEM analysis whereas Sth1_301–1395 was used for activity assays. A “basic patch” that follows the ATPase and SnAc domains is highlighted, with a sequence alignment shown below. Sequences of Sth1 mutants used for remodeling assays are shown below the wild type sequence. b, Restriction enzyme accessibility assay of wildtype and mutant RSC^SAR. The LANA peptide binds the acidic patch and has been shown to reduce remodeling rates in other systems. Error is reported as standard deviation of the mean of n=3 technical replicates and curves were fit to a one-phase association non-linear regression curve.

Fig. 7 |. The ARP module: assembly and regulatory role in the RSC complex.

This figure places the findings of this work in the context of the full RSC complex (highlighted by rounded white rectangles). a, Arp7–Arp9–Rtt102 (“ARPs”) mediate the folding of Sth1’s HSA helix and formation of the ARP module (Arp7–Arp9–Rtt102 + Sth1_HSA). In the presence of a folded HSA helix, a regulatory hub containing the postHSA and P1 segments is assembled where the HSA connects to the ATPase domain of Sth1. All subunits and motifs are colored following the schematic shown in Fig. 1a. A dimmed nucleosome is included in this panel as an orientation reference. b, The structure of RSC^SAR bound to a nucleosome. c, The structure of RSC bound to a nucleosome (PDB 6KW3) shown in the same orientation as RSC^SAR in (b). The ARP module from RSC^SAR is shown in dim colors to highlight the different positions it adopts in RSC^SAR and RSC. The rotations relating its conformation in RSC^SAR to that in RSC, which have their pivot point in the regulatory hub, are indicated in grey. d, Same structure as in (c), rotated to highlight that RSC can interact with the acidic patches on both sites of the nucleosome using its Sth1 and Sfh1 subunits.