Structure and regulation of the human INO80-nucleosome complex

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 machines ¹ . Biochemical studies^2-4 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 nucleosomes^5-7 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 sliding ⁸ , histone exchange ⁹ and nucleosome spacing^10-12 remain poorly understood. Although some remodellers work as monomers ¹³ , others work as highly cooperative dimers^{11, 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.

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

a, MST experiment of hINO80 with 60N12 nucleosome (+/- 3mM ADP•BeF₃). Raw data (above) were processed to analyse binding and cooperativity (below). Data points represent mean values with SD, where n = 3 experimentally independent replicates. b, Gel of EM sample (hINO80 + nucleosome). Two loadings are shown to allow assessment of INO80 stoichiometry (left) or histones (right). n=3 independent experimental measurements. c, DNA sequence of the nucleosome (50N25) 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. An additional 3 base single strand overhang left over from restriction cleavage site is depicted in lower case. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 2. Cryo-EM data processing of INO80-nucleosome complex.

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

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.

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 beta-sheet secondary structure with RUVBL1 (chain E) and RUVBL2 (chain D) within the 3.8 Å map. f, (i) Fit of Arp5 into 4.8 Å map. (ii) DNA and motor domains fit into the 4.8 Å map. (iii) Perpendicular view of (ii), showing the DNA crossing the motor domains.

Extended Data Fig. 5

Comparisons of INO80-nucleosome interactions with those of Chd1 and Snf2. Viewed from the top of nucleosome, showing that all the motor domains are located on one side while Arp5-Ies6 (green) contacts the other side of the DNA wrap. Chd1 induces an unwrapping of the DNA at the SHL -7 position due 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 so likely presents an incomplete picture of interactions or DNA distortions within the nucleosome in the complex.

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 visualised by SDS-PAGE and Coomassie staining. n=1. b, A comparison of Arp5-Ies6 and Arp5 nucleosome binding activity assayed by EMSA, demonstrating a lack of nucleosome binding activity by Arp5 at in vivo relevant concentrations in the absence of Ies6. Nucleosomes were labelled with AlexaFluor 488 (Thermo Fisher Scientific). Reaction species were visualised 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 Figure 1. n=2 biologically independent experiments in all the graphs. Error bars represent SD from the mean values.

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

a, Individual particles (selected out of 775,804) with RUVBL1-RUVBL2 oriented similarly showing different orientations of SC1 (dashed lines). b, 2D class averages (~30 particles each) showing different orientations of SC1 relative to the RUVBL1-RUVBL2. c, Projections of the 3D reconstruction along the same angles of those in b, confirming the extra density as SC1. The white bar represents 100 Å.

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

a, Schematic diagram illustrating the histone tail truncations used in this study. b, Initial nucleosome sliding rates of human nucleosomes lacking 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 summarised in Figure 6a of the main text. n=2 biologically independent experiments in all the graphs. Error bars represent SD from the mean values.

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

a, ATPase data and Hill coefficients for data shown in Figure 6c of the main text. b, ATPase rates for mutations of the H3 tails. c, Nucleosomes carrying wildtype or mutated H3 tails show similar salt stability indicating that the mutations have not altered the stability of nucleosomes. n=2 biologically independent experiments in all the panels. Error bars represent SD from the mean values.

Fig. 1. Human INO80-nucleosome complex.

a, Ino80 subunit with functional domains labeled. b, 3D INO80-nucleosome complex reconstruction with RUVBL1-2, Ino80, Arp5, Ies2 and nucleosome structural models fitted. Scale bar, 100 Å. c, INO80 nucleosome interactions with histones and nucleosome positions labelled. INO80 contacts nucleosome at SHL-6 and SHL-3. Also shown are locations of the histone tails.

Fig. 2. Comparison of INO80 with Chd1 and a model for translocation by INO80.

a, INO80 and Chd1 nucleosome complexes viewed from the side of the nucleosome. b, Chd1 is proposed to push DNA towards the dyad axis (cyan). c, Ino80 (aligned on the nucleosome as in b) would push DNA past Arp5-Ies6 towards the dyad but from the opposite direction. d, Same as b but instead with the view aligned on the motor domains as in c.

Fig. 3. Nucleosome distortion in the INO80-nucleosome complex.

a, DNA is peeled off in Ino80 (orange) and Chd1 (yellow) compared to the free nucleosome (red). In Ino80 this is due to motor domain interaction while in Chd1 this is due to the SANT and SLIDE domain interactions. b, DNA near to the motor domains in Ino80-nucleosome (orange) is lifted compared to a canonical nucleosome (lime green), which also causes slight rotation of H2A/H2B (pink). c, Lifting of the DNA in Ino80-nucleosome complex and movement of the H3 N-terminal helix (purple) near the motor domains.

Fig. 4. INO80 is regulated by H3 tails.

a, Initial nucleosome sliding rates of human nucleosomes lacking different histone tail, using a FRET-based assay. b, Effect of increasing the extent of H3 tail truncation on nucleosome sliding. No effect is observed with 30 residues removed but a 39 residue truncation induces stimulation of sliding and, c, ATPase rates for H3 tail truncations, d, loss of cooperativity for both sliding and binding of nucleosomes, e, Lysine to glutamine mutations in the H3 tail affect both the rate and cooperativity of sliding. n=2 biologically independent experiments in all the graphs. Error bars represent SD from the mean values.