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Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state

Nature Structural & Molecular Biology volume 29, pages 121–129 (2022)Cite this article

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Abstract

Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. Here we report a complex of the Chd1 remodeler bound to a nucleosome in a nucleotide-free state, determined by cryo-EM to 2.3 Å resolution. The remodeler stimulates the nucleosome to absorb an additional nucleotide on each strand at two different locations: on the tracking strand within the ATPase binding site and on the guide strand one helical turn from the ATPase motor. Remarkably, the additional nucleotide on the tracking strand is associated with a local transformation toward an A-form geometry, explaining how sequential ratcheting of each DNA strand occurs. The structure also reveals a histone-binding motif, ChEx, which can block opposing remodelers on the nucleosome and may allow Chd1 to participate in histone reorganization during transcription.

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Fig. 1: Structure of the Chd1 remodeler bound to a nucleosome in the nucleotide-free state.
Fig. 2: DNA distortions induced by the nucleotide-free state of the Chd1 ATPase motor.
Fig. 3: Interactions between the Chd1 ATPase motor and DNA at SHL2.
Fig. 4: The Chd1 ChEx segment binds the histone core.
Fig. 5: The ChEx segment competes with H2A/H2B acidic patch binding elements.
Fig. 6: ChEx can block other remodelers from sliding nucleosomes.
Fig. 7: Model for DNA translocation based on changes in duplex geometry.

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Data availability

The raw cryo-EM data have been deposited in EMPIAR (EMPIAR-10876). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank as EMD-25479 (nucleosome-bound Chd1), EMD-25480 (nucleosome-bound Chd1 with well-defined DBD), EMD-25483 (nucleosome-ChEx) and EMD-25481 (nucleosome-only). Atomic models built using cryo-EM data have been deposited in the RCSB Protein Data Bank with PDB codes 7TN2 (nucleosome-bound Chd1) and 7SWY (nucleosome-only). The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD025287. This study included analysis of previously determined nucleosome–remodeler complexes (PDB codes 5O9G, 6IRO, 6PWF, 6IY2, 6IY3, 6FML), nucleosome–LANA complex (1ZLA) and nucleosome-only models (1KX3, 1KX5, 3UT9, 5F99, 5Y0D, 6IPU, 6WZ5, 6ZHX, 7OHC). Source data are provided with this paper.

Code availability

Scripts to analyze and visualize the structures have been deposited at GitHub (https://github.com/gdbowman/).

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Acknowledgements

We thank R. Levendosky for Snf2h protein, K. Tripp and the Center for Molecular Biophysics at Johns Hopkins for fluorometer use, and C. Bator at the Huck Institutes of the Life Sciences Cryo-Electron Microscopy Facility for the initial cryo-EM data collection. We thank S. Abini-Agbomson for his help in setting up and troubleshooting the GraFix procedure, and U. Baxa and A. Wier for their support and data collection at the Frederick National Laboratory. This work was supported by NIH grants R01-GM084192 (G.D.B.) and DP2-GM140926 (S.D.F.). This research was also supported, in part, by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract no. HSSN261200800001E.

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Author notes

  1. These authors contributed equally: Ilana M. Nodelman, Sayan Das.

Authors and Affiliations

  1. Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    Ilana M. Nodelman, Stephen D. Fried & Gregory D. Bowman

  2. Department of Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA

    Sayan Das & Jean-Paul Armache

  3. Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Anneliese M. Faustino & Stephen D. Fried

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Contributions

I.M.N., G.D.B. and J.-P.A. conceived the project. I.M.N. produced all nucleosomes and Chd1 variants. S.D. performed GraFix for cryo-EM. J.-P.A. processed and analyzed cryo-EM data. Atomic models were built by J.-P.A., with contributions from G.D.B. J.-P.A., G.D.B. and I.M.N. analyzed the structures. S.D.F. and A.M.F. performed and analyzed MS experiments. I.M.N. and G.D.B. performed and analyzed biochemical experiments and wrote the paper. All authors contributed figures and edited and approved the manuscript.

Corresponding authors

Correspondence to Gregory D. Bowman or Jean-Paul Armache.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Tom Owen-Hughes, Sebastian Eustermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann, Carolina Perdigoto and Sara Osman were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM raw data and analysis of the 2.3 Å Chd1-nucleosome complex.

a, Representative cryo-EM micrographs of Chd1-nucleosome complex. b, Selected 2D class averages generated from the particles used to reconstruct the Chd1-nucleosome complex. c, Four orthogonal views of the 2.3 Å structure. Coloring of nucleosome and Chd1 domains is according to Fig. 1. d, Euler angle distribution of all the particles used in the 2.3 Å 3D reconstruction. The distribution of particles in a specific orientation is proportional to the length of each cylinder. e, The final 2.3 Å reconstruction of the Chd1-nucleosome complex colored according to local resolution with blue and red representing the highest and lowest resolution, respectively. f, Four orthogonal views of the Chd1 ATPase and chromodomains from the 2.3 Å reconstruction, without the nucleosome density, colored according to local resolution. g, FSC curve calculated between two independent half-maps from refinements in CryoSPARC (2.3 Å, blue) and Relion (2.4 Å, purple) reported at 0.143 FSC cutoff. The Relion and CryoSPARC refinements were independent from each other. Arrows indicate the reported resolutions in Ångstroms.

Extended Data Fig. 2 Views of 2.3 Å resolution density maps of the Chd1-nucleosome complex.

The coloring of the figure follows that from Fig. 1 (guide DNA strand, yellow; tracking DNA strand, orange; ChEx, magenta; double chromodomains, light blue; ATPase lobe 1, purple; ATPase lobe 2, blue). a-f, Selected views of Chd1-ATPase interactions with DNA. g,h, Views of the DNA duplex at SHL2 and SHL3.

Extended Data Fig. 3 Overview of 3D classification and reconstruction of the Chd1-nucleosome complex dataset.

Flowchart of data processing, refinement, and classification towards the final reconstructions of Chd1-nucleosome dataset (left). Single asterisk marks the bifurcation where particles were classified according to presence of the DNA-binding domain (bottom right). Double asterisk marks the classification of the nucleosome alone or nucleosome with only ChEx (top right). Triple asterisk marks the classification for the ‘bridge’ region connecting the brace helix and the DBD. Darker squares report significant reconstructions.

Extended Data Fig. 4 Cryo-EM analysis of nucleosome-bound Chd1 with defined DBD, nucleosome-only and nucleosome-ChEx subsets.

a, Selected 2D class averages generated from the particles used to reconstruct the Chd1-nucleosome complex at 2.7 Å with the well-defined DNA-binding domain. b, Four orthogonal views of the 2.7 Å structure containing the well-defined DNA-binding domain. Coloring of nucleosome and Chd1 domains is done according to Fig. 1. c, Euler angle distribution of all the particles used in the 2.7 Å 3D reconstruction. d, The final 2.7 Å map of the Chd1-nucleosome complex colored according to local resolution; blue represents the highest resolution and red the lowest. e, Selected 2D class averages generated from the particles used to obtain the nucleosome-only reconstruction at 2.6 Å. f, Four orthogonal views of the 2.6 Å structure of the nucleosome. g, Euler angle distribution of all the particles used in the 2.6 Å 3D reconstruction. h, The final 2.6 Å nucleosome reconstruction colored according to local resolution; blue represents the highest resolution and red the lowest. i, Selected 2D class averages generated from the particles used to reconstruct the nucleosome-bound ChEx at 2.9 Å. j, Four orthogonal views of the 2.9 Å structure of ChEx bound to the nucleosome. Coloring of the nucleosome and ChEx is done according to Fig. 1. k, Euler angle distribution of all the particles used in the 2.9 Å 3D reconstruction. l, The final 2.9 Å reconstruction of the nucleosome-bound ChEx colored according to local resolution; blue represents the highest resolution and red the lowest. m, n, o, FSC curves were calculated between two independently refined half-maps, before (red) and after (blue) masking, and reported at 0.143 FSC cutoff. Shown are the curves for the nucleosome-Chd1 complex with well-defined DBD (2.7 Å, m), the nucleosome alone (2.6 Å, n), and nucleosome with ChEx only (2.9 Å, o). Arrows indicate the reported resolutions in Ångstroms.

Extended Data Fig. 5 Nucleosome-Chd1 complexes in the absence of nucleotide are more resistant to competitor DNA when the 601[TA-rich + 1] sequence is used.

a, 40-601-40 nucleosomes (30 nM) were preincubated with 120 nM Chd1 in the presence or absence of ATP𝛄S for 15 min at room temperature, with or without salmon sperm DNA. Reactions were separated on 4.25% native acrylamide gels. Shown are representative gels. b, Quantification of nucleosome binding in the presence of competitor. Fits to averaged data points gave apparent Kd values of 0.43 ± 0.15 mg ml −1 and 0.11 ± 0.05 mg ml −1 for nucleotide-free Chd1[wt] for 601[TArich +1] and 601[canonical], respectively, and 0.11 ± 0.05 mg ml −1 and 0.07 ± 0.04 mg ml −1 for ATP𝛄S-bound Chd1[wt] for 601[TArich +1] and 601[canonical], respectively. Data shown are averages of six replicates. Error bars represent standard deviations.

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Extended Data Fig. 6 DNA parameters.

Shown are DNA parameters calculated with (a) CURVES + 35 and (b) 3DNA64, with the X-axes indicating the distance from the dyad. For the Chd1-bound structure in the nucleotide-free state, yellow bars represent the Chd1-bound (TA-rich) side and gray bars represent the unbound (TA-poor) side, and brown indicates overlap of the bars. For the 601 sequences, solid lines show parameter values for the TA-rich sides and dotted lines show those of the TA-poor sides.

Extended Data Fig. 7 Absorption of single nucleotides on the nucleosome.

Shown are crystal and cryo-EM structures of the nucleosome, aligned based on the histone core. Each view shows the DNA minor groove facing away from the histone core. Note that in each case, the bulging strand, which remains base-paired, contains an additional nucleotide.

Extended Data Fig. 8 Variability in density for the Chd1 DNA-binding domain, exit DNA, and the bridge.

a, Overview of a filtered Chd1-nucleosome reconstruction where the DNA-binding domain is well-defined. b, Zoomed-in views showing different sub-classes obtained from the dataset, exhibiting variability in the exit DNA and the DNA-binding domain. c-f, Visualization of variability of the connection (‘bridge’) between the brace helix and the DNA-binding domain. c, Model of the nucleosome-bound Chd1. d, Model of the nucleosome-bound Chd1 model converted into a map, filtered to 10 Å. e, Separated unmodeled density (purple) from the 2.3 Å Coulomb potential density reconstruction as shown against the map from d. f, Unmodeled densities (various colors) from subsets obtained using focused classification, shown against the map from d.

Extended Data Fig. 9 Nucleosome sliding assays of Chd1 variants.

a, Overview of the Chd1-nucleosome complex, highlighting the opposite-gyre DNA interacting loop (residues 475-481), a loop on the chromodomains that contacts the DNA-binding domain (residues 295-302), and conserved residues on the DNA-binding domain that contact the chromodomains (residues 1199-1202). b, Quantification of nucleosome sliding reactions. Sliding reactions were carried out at room temperature with 200 nM Chd1 and 40 nM FAM-80-601-0 nucleosomes. In the top graphs, data are presented as mean values ± SD, with lines showing the fit to the averaged data at each time point. Each variant was measured in multiple independent experiments: Chd1[wt] (n = 11); Chd1[K478D/G479A/K480D/K481A] (n = 6); Chd1[∆475-481] (n = 8); Chd1[∆296-302] (n = 7); Chd1[D1033A/E1034A/D1038A] (n = 6); Chd1[R1199A/D1200A] (n = 6); Chd1[D1201A/P1202A] (n = 5). The bar graph shows mean sliding rates ± SD, calculated from individual fits. For comparison of rates to Chd1[wt], a two-tailed t-test yielded the following p values: Chd1[K478D/G479A/K480D/K481A] (p = 2.5×10−6); Chd1[∆475-481] (p = 7.6×10−12); Chd1[∆296-302] (p = 0.29); Chd1[D1033A/E1034A/D1038A] (p = 9.0×10−4); Chd1[R1199A/D1200A] (p = 8.1×10−3); Chd1[D1201A/P1202A] (p = 0.022).

Extended Data Fig. 10 The Chd1 ChEx segment, devoid of secondary structure, lays over the histone surface similarly to extended peptide segments of histone chaperones.

a, Interactions of ChEx region with histone core. The main chain amide of T125 hydrogen bonds to the C-terminal end of alpha helix 1 of H2B. Neighboring this region are interactions with the acidic patch: R126 ChEx hydrogen bonds with E56 of H2A, and the arginine anchor, R130, hydrogen bonds with residues in the canonical acidic patch binding pocket (E61, D90, E92). Adjacent to the acidic pocket, the conserved Y137 of ChEx packs against a hydrophobic surface of H2A, consisting of L65, A69, L85, and A86. The aromatic ring of Y137 is protected from solvent by V135 of ChEx. Several side chain/main chain hydrogen bonds are formed between H2A and ChEx around Y137: the backbone of Y137 hydrogen bonds with side chains of H2A D72 and N73; the backbone amide of I139 hydrogen bonds with the H2A N73 side chain; and the side chain of ChEx N138 hydrogen bonds with the backbone carbonyl of H2A D72. A second tyrosine (Y141) of ChEx packs against the aliphatic regions of side chains of R52 and K56 of the αN helix of H3. At the C-terminal end of ChEx, a group of acidic residues interacts with a cluster of basic residues on H3 (R42, R52, and K56). ChEx residues are labeled in magenta, and hydrogen bonds 3.2 Å or less and 3.3–3.5 Å are shown as green dashes or yellow dots, respectively. b, Comparison of nucleosome-binding footprints of the Chd1 ChEx segment with the C-terminal domain (CTD) of the FACT subunit Spt16. The FACT structure (6UPK) was aligned with the Chd1-nucleosome structure by superimposing the bound H2A-H2B dimers of each structure. With this alignment, the Spt16 CTD clashes with ChEx at the H3 binding interface and with the DNA at SHL4.5.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Table 1.

Reporting Summary

Supplementary Data 1

Crosslinking MS data for the Chd1–nucleosome complex.

Supplementary Video 1

Overview of two cryo-EM structures of the Chd1–nucleosome complex in the nucleotide-free state. The maps resolved at 2.3 Å resolution had poor density for exit DNA and the DNA-binding domain. The maps at 2.7 Å resolution resulted from focused subclassification for the DNA-binding domain, which also shows stronger density for exit DNA.

Supplementary Video 2

A tour of the interface between the Chd1 ATPase motor and nucleosomal DNA at SHL2. The electron density, shown as a mesh, is from a 2.3 Å cryo-EM map.

Supplementary Video 3

Comparison of the nucleotide-free Chd1–nucleosome structure to other remodeler–nucleosome complexes. All structures shown here were nucleotide-free or ADP-bound, with a similar ‘open’ ATPase conformation.

Supplementary Video 4

Morphing video illustrating the relative changes in the nucleosomal DNA upon binding of Chd1 in the nucleotide-free state. See also Fig. 2.

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Nodelman, I.M., Das, S., Faustino, A.M. et al. Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state. Nat Struct Mol Biol 29, 121–129 (2022). https://doi.org/10.1038/s41594-021-00719-x

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