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A competitive regulatory mechanism of the Chd1 remodeler is integral to distorting nucleosomal DNA

Nature Structural & Molecular Biology volume 32, pages 1445–1455 (2025)Cite this article

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Abstract

The Chd1 chromatin remodeler repositions nucleosomes into evenly spaced arrays, a characteristic of most eukaryotic genes. Here we show that the yeast Chd1 remodeler requires two activating segments to distort nucleosomal DNA into an A-form-like conformation, a critical first step in nucleosome sliding. As shown by cryo-electron microscopy, these two activating segments together pack against the ATPase motor, where they are poised to stabilize the central ATPase cleft. These activating elements contact the ATPase at locations that are incompatible with binding of NegC, an autoinhibitory segment located between the two activators. NegC inhibits sliding by antagonizing the activators through steric competition and constraining activator placement, giving rise to directional nucleosome sliding. Given that activator reinforcement of the ATPase cleft is needed for DNA distortion, this first step in remodeling appears to provide a natural checkpoint for regulation of chromatin remodeler activity.

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Fig. 1: NegC of Chd1 is flanked on both sides by activators.
Fig. 2: The Chd1-catalyzed DNA distortion is assisted by both activators and antagonized by NegC.
Fig. 3: Chd1 activators bind to the ATPase motor in a manner that is incompatible with NegC binding.
Fig. 4: Chd1 activators and contacts lining the central cleft antagonize the closed state of the ATPase.
Fig. 5: A model for conformational regulation of the ATPase motor.

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Data availability

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB): EMD-49060, EMD-49061, EMD-49062, EMD-49063 and EMD-49064. The coordinates have been deposited in the PDB: 9N6H, 9N6I and 9N6K. The raw uncorrected micrographs and final stacks have been deposited into EMPIAR with an ID EMPIAR-12601. Analysis used PDB codes 3MWY, 5JX5 and 7TN2. Source data are provided with this paper.

Code availability

Data from biochemical experiments and the Python scripts used to plot them are available via GitHub at https://doi.org/10.5281/zenodo.15022462 (ref. 61).

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Acknowledgements

We thank A. Martinez for introducing some of the amino acid substitutions of Chd1 described in this study. We thank the Johns Hopkins Integrated Imaging Center and I. Dobbie for use of the Typhoon scanner. We thank S. H. Cho at the Penn State Cryo-Electron Microscopy Facility for assistance with EM screening and data collection and A. Wier for his support and data collection at the Frederick National Laboratory. The results generated in this study were computed on hardware generously provided to us through the NVIDIA Academic Grant by NVIDIA Corporation. This work was supported by the NIH (grant no. R01-GM084192 to G.D.B.). 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 HSSN261200800001E.

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Author notes

  1. Wesley S. Glime

    Present address: Medical College of Wisconsin, Milwaukee, WI, USA

  2. These authors contributed equally: Ilana M. Nodelman, Heather J. Folkwein.

Authors and Affiliations

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

    Ilana M. Nodelman, Wesley S. Glime & 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

    Heather J. Folkwein & Jean-Paul Armache

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  1. Ilana M. Nodelman
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Contributions

I.M.N. and G.D.B. conceived of project and designed research. I.M.N. purified all proteins and nucleosomes and generated all biochemical data. H.J.F. and J.-P.A. obtained, processed and analyzed the cryo-EM data. W.S.G. optimized the nucleosome binding assay and performed preliminary decentering experiments. I.M.N. and G.D.B. analyzed data and wrote the paper. J.-P.A. and G.D.B. supervised research. All authors edited and approved the manuscript.

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

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Extended data

Extended Data Fig. 1 Conservation of regulatory elements across remodeler families.

a. Sequence alignment highlighting the Guide-Strand Displaced (GSD)/brace II helix and NegC elements. b. Structural alignment of the isolated crystal structures of yeast S. cerevisiae Chd1 (3MWY) and M. thermophila ISWI (5JXR), superimposed on ATPase lobe 2.

Extended Data Fig. 2 Biochemical analyses of Chd1 variants.

a. Nucleosome decentering assay, similar to Fig. 1c. The fraction decentered corresponds to intensity of bottom two bands over total band intensity, with data representing mean +/− SD and the number of independent replicates (n) given below each bar. Nucleosome alone and wildtype data are the same as Fig. 1c, shown here for reference. P-values (two-tailed Welch’s t-Test) are indicated with ***, p < 0.0001. Individual data points and p-values are given in Source Data File 5. b. Nucleosome sliding assay with end-positioned 80N0 601 nucleosomes, similar to Figs. 1d and 3e. Data are presented as mean values +/− SD from n independent replicates as indicated beside each label. Wildtype data are the same as in Fig. 1d, shown here for reference. Nucleosome sliding rates given in Supplementary Table 1. c. DNA distortion assay, monitoring entry DNA movement with H2B(S53C) cross-linking, similar to Figs. 2b and 3d. Nucleosome alone and wildtype data are the same as Fig. 2, shown here for reference. d. Brace I residue I838 is largely buried by GSD/brace II helix and C-terminal activator. e. GSD/brace II residues Leu865 and Leu869 pack in the cleft formed by protrusion 1 (lobe 1) and brace I (lobe 2). f. DNA dyad shift, monitored by H3(M120C) cross-linking, similar to Fig. 4. For all bar graphs, individual data points are shown, with data presented as mean values +/− SD and the number of independent replicates (n) given below each bar. Wildtype data are the same as Fig. 4, shown here for reference. P-values (two-tailed Welch’s t-Test) are indicated with ***, p < 0.0001; **, p < 0.001; *, p < 0.01; n.s, not significant. Data values and p-values for bar graphs are given in Source Data File 5.

Source data

Extended Data Fig. 3 The GSD/brace II helix and NegC core of Chd1 antagonize nucleosome binding.

a. On native acrylamide gels, titration of Chd1 in the presence of competitor DNA produces two supershifted bands that represent 1:1 and 2:1 Chd1:nucleosome complexes. For these experiments, FAM-labeled 40N40 601 nucleosomes (2 nM) were incubated with increasing amounts of Chd1 (2.44–5000 nM) in the presence of DNA competitor (1 mg/ml salmon sperm DNA) and 1 mM AMP-PNP, and separated on 4.25% (60:1) native polyacrylamide gels. b. Data and fits from native gel nucleosome binding experiments to two Kd values, representing affinities for the two sides of the nucleosome. For clarity, error bars are not shown. This plot shows data and fits in AMP-PNP conditions. Number of independent replicates for wild type and each variant in AMPPNP conditions are: Chd1[wildtype], n = 5; Chd1[GSD/brace II]864-902-flex, n = 3; Chd1[GSD/brace II]864-871-flex, n = 3; Chd1[GSD/brace II]L865N/L869N, n = 4; Chd1[activator]876-881, n = 3; Chd1[NegC]884-889-flex, n = 8; Chd1[NegC]890-895-flex, n = 3; Chd1[NegC]884-902-flex, n = 3; Chd1[NegC]L886N/L889N/L891N, n = 3; Chd1[NegC]896-901-flex, n = 3; Chd1[NegC]902-907-flex, n = 4; Chd1[activator]F917N/L918N/F921N, n = 3; Chd1[activator]W932A, n = 3; Chd1[brace I]I843N, n = 3; Chd1[protrusion 2]M652Q, n = 3. c. Comparison of apparent nucleosome binding affinities for Chd1 variants in different nucleotide conditions. These 2D plots show the higher affinity apparent Kd value (Kd1,app) along y, and the lower affinity apparent value (Kd2,app) along x. The vertical dashed lines (at 7 µM) show the confidence limit for Kd2,app. Number of independent replicates for wild type and each variant in ADP and nucleotide-free conditions, respectively, are as follows: Chd1[wildtype], n = 7,9; Chd1[GSD/brace II]864-902-flex, n = 3,4; Chd1[GSD/brace II]864-871-flex, n = 7,7; Chd1[GSD/brace II]L865N/L869N, n = 4,4; Chd1[activator]876-881, n = 4,3; Chd1[NegC]884-889-flex, n = 3,7; Chd1[NegC]890-895-flex, n = 4,4; Chd1[NegC]884-902-flex, n = 3,3; Chd1[NegC]L886N/L889N/L891N, n = 3,4; Chd1[NegC]896-901-flex, n = 4,5; Chd1[NegC]902-907-flex, n = 3,3; Chd1[NegC]901-902-flexinsert, n = 3,4; Chd1[activator]F917N/L918N/F921N, n = 3,3; Chd1[activator]W932A, n = 3,3; Chd1[brace I]I843N, n = 4,3; Chd1[protrusion 2]M652Q, n = 5,3. Number of replicates for wild type and each variant in AMPPNP conditions is listed in Extended Data Fig. 3b. Affinities and standard deviations are reported in Supplementary Table 3. Data are given in Source Data File 6.

Source data

Extended Data Fig. 4 M652Q enhances nucleosome binding and does not by itself substantially contribute to a NegC phenotype.

a. The location of the GSD/brace II helix in autoinhibited Chd1 clashes with the guide strand. Shown is a superpositioning of the isolated and inhibited crystal structure of yeast Chd1 (3MWY) aligned with a nucleosome-bound cryo-EM structure (7TN2). The DNA guide strand (yellow) from the bound nucleosome penetrates the GSD/brace II helix, suggesting that this location would conflict with DNA binding. b. Two views highlighting the hydrophobic packing of the GSD/brace II helix against ATPase lobe 2 in the autoinhibited Chd1 structure (3MWY). c. Nucleosome binding plots comparing the Chd1[protrusion 2]M652Q variant to wildtype, a GSD/brace II variant, and a NegC variant. The nucleotide conditions for binding are indicated above each plot. Data are presented as mean values +/− SD from n independent replicates. Number of independent replicates for wild type and each variant in AMPPNP, ADP and nucleotide-free conditions, respectively, are as follows: Chd1[wildtype], n = 5,7,9; Chd1[GSD/brace II]864-871-flex, n = 3,7,7; Chd1[NegC]884-889-flex, n = 8,3,7; Chd1[protrusion 2]M652Q, n = 3,5,3. Also see Extended Data Fig. 3. d. A native gel and quantitation of decentering experiments for Chd1[protrusion 2]M652Q, with and without a chromodomain disruption. Data are presented as mean values +/− SD from n independent replicates as indicated below each bar. Nucleosome alone and wildtype data are the same as Fig. 1c, shown here for comparison. P-values (two-tailed Welch’s t-Test) are indicated with ***, p < 0.0001; **, p < 0.001; *, p < 0.01; n.s, not significant. Individual data points and p-values are given in Source Data File 7. e. Progress curves for nucleosome sliding of 80N0 Widom 601 nucleosomes. The Chd1[protrusion 2]M652Q variant is ~6-fold slower than wildtype. Data are presented as mean values +/− SD from n independent replicates as indicated beside each label. Sliding rates are given in Supplementary Table 1.

Source data

Extended Data Fig. 5 Cross-linking of the Chd1 ATPase to nucleosomal DNA shows that disruption of the GSD/brace II helix allows for more robust interactions at superhelix location 2 (SHL2).

After labeling with the photo-crosslinker azidophenacyl bromide (APB), V721C variants of Chd1[wildtype] and Chd1[GSD/braceII]864-871-flex were incubated with dual labeled (Cy5)40-601-19(FAM) nucleosomes in different nucleotide conditions, UV irradiated, processed, and then the cleaved DNA fragments were resolved on urea denaturing gels. The cross-linking site indicated corresponds to the ATPase lobe 2 binding to SHL2 DNA, as previously reported19. These two scans show cross-linking to the two strands (and thus two sides) of the Widom 601 sequence. This gel is a representative of 3 independent experiments.

Extended Data Fig. 6 Cryo-EM raw data and summary of the combined (2:1 and 1:1) and 1:1 Chd1[L886G/L889G/L891G]-nucleosome complexes.

a. Three representative cryo-EM micrographs of the Chd1[L886G/L889G/L891G]-nucleosome complex. Chd1-nucleosome complexes are highlighted with green circles; GroEL complexes are highlighted with orange circles. Note that GroEL particles showed no systematic adjacency with Chd1-nucleosome particles. b. Representative 2D class averages generated from the final particles used for the reconstruction of the combined (2:1 and 1:1) Chd1-nucleosome 2.37 Å complex. c. Four orthogonal views of the combined (2:1 and 1:1) Chd1-nucleosome 2.37 Å structure. Coloring of Chd1 domains is given in 1D schematic. d. Representation of the Euler angle distribution of final particles in the combined Chd1-nucleosome 2.37 Å complex. The length of each cylinder representing a specific orientation is proportional to the number of particles. e. Local resolution of the combined (2:1 and 1:1) Chd1-nucleosome 2.37 Å complex, colored in accordance with the resolution values (Å), from highest (blue) to lowest (red). f. Fourier Shell Correlation (FSC) curve of the combined (2:1 and 1:1) Chd1-nucleosome 2.37 Å structure calculated between two independent half maps from the refinement in CryoSPARC (2.37 Å) at the FSC cutoff 0.143. g. Conical FSC (cFSC) curve of the combined (2:1 and 1:1) Chd1-nucleosome 2.37 Å structure calculated between two independent half maps with a conical mask of a specified half angle and axis in Fourier space, calculated in CryoSPARC. The cFSC shows correlations at each spacial frequency and the spread of resolution values over direction. The mean of the correlations at each spacial frequency are shown by the blue line, the minimum and maximum by the light blue shading, and the standard deviation by the dark blue shading. The crossings of 0.143 are given by the green histogram. Lines and arrows indicate the axis of rotation between subsequent views. h. Representative 2D class averages generated from the final particles used for the reconstruction of the (1:1) Chd1-nucleosome 2.54 Å complex. i. Four orthogonal views of the (1:1) Chd1-nucleosome 2.54 Å structure. j. Representation of the Euler angle distribution of the final particles in the (1:1) Chd1-nucleosome 2.54 Å structure. The length of each cylinder representing a specific orientation is proportional to the number of particles. k. Local resolution of the (1:1) Chd1-nucleosome 2.54 Å complex, colored in accordance with the resolution values (Å), from highest (blue) to lowest (red). l. Fourier Shell Correlation (FSC) curve of the (1:1) Chd1-nucleosome 2.54 Å structure calculated between two independent half maps from the refinement in CryoSPARC at the FSC cutoff 0.143. m. Conical FSC (cFSC) curve of the (1:1) Chd1-nucleosome 2.54 Å structure calculated between two independent half maps with a conical mask of a specified half angle and axis in Fourier space, calculated in CryoSPARC. The mean of the correlations at each spacial frequency are shown by the blue line, the minimum and maximum by the light blue shading, and the standard deviation by the dark blue shading. The crossings of 0.143 are given by the green histogram. Lines and arrows indicate the axis of rotation between subsequent views. Cryo-EM statistics are given in Table 1.

Source data

Extended Data Fig. 7 Cryo-EM raw data and summary of the 2:1 Chd1[L886G/L889G/L891G]-nucleosome complexes.

a. Representative 2D class averages of (2:1) Chd1-nucleosome 2.61 Å structure generated from the final particles used for the reconstruction of the complex. b. Four orthogonal views of the (2:1) Chd1-nucleosome 2.61 Å structure. Coloring of Chd1 domains is given in 1D schematic. c. Representation of the Euler angle distribution of the final particles in the (2:1) Chd1-nucleosome 2.61 Å structure. The length of each cylinder representing a specific orientation is proportional to the number of particles. d. Local resolution of the (2:1) Chd1-nucleosome 2.61 Å structure, colored in accordance with the resolution values (Å), from highest (blue) to lowest (red). e. Fourier Shell Correlation (FSC) curve of the (2:1) Chd1-nucleosome 2.61 Å structure, calculated between two independent half maps from the refinement in CryoSPARC at the FSC cutoff 0.143. f. Conical FSC (cFSC) curve of the (2:1) Chd1-nucleosome 2.61 Å structure, calculated between two independent half maps with a conical mask of a specified half angle and axis in Fourier space, calculated in CryoSPARC. The cFSC shows correlations at each spacial frequency and the spread of resolution values over direction. The mean of the correlations at each spacial frequency are shown by the blue line, the minimum and maximum by the light blue shading, and the standard deviation by the dark blue shading. The crossings of 0.143 are given by the green histogram. g. Representative 2D class averages of (2:1) Chd1-nucleosome 2.88 Å structure, including a well-resolved DNA-binding domain, generated from the final particles used for the reconstruction of the complex. h. Four orthogonal views of the 2.88 Å structure. i. Representation of the Euler angle distribution of final particles in the (2:1) Chd1-nucleosome 2.88 Å complex with a well-resolved DNA-binding domain. The length of each cylinder representing a specific orientation is proportional to the number of particles. j. Local resolution of the (2:1) Chd1-nucleosome 2.88 Å complex with a well-resolved DNA-binding domain, colored in accordance with the resolution values (Å), from highest (blue) to lowest (red). k. Fourier Shell Correlation (FSC) curve of the (2:1) Chd1-nucleosome 2.88 Å complex with a well-resolved DNA-binding domain, calculated between two independent half maps from the refinement in CryoSPARC at the FSC cutoff 0.143. l. Conical FSC (cFSC) curve of the (2:1) Chd1 nucleosome 2.88 Å structure with a well-resolved DNA-binding domain, calculated between two independent half maps with a conical mask of a specified half angle and axis in Fourier space, calculated in CryoSPARC. The mean of the correlations at each spacial frequency are shown by the blue line, the minimum and maximum by the light blue shading, and the standard deviation by the dark blue shading. The crossings of 0.143 are given by the green histogram. Lines and arrows indicate the axis of rotation between subsequent views. Cryo-EM statistics are given in Table 1.

Source data

Extended Data Fig. 8 Flowchart of the cryo-EM data processing.

Data processing, classification and refinement flowchart of the Chd1[L886G/L889G/L891G]-nucleosome dataset.

Extended Data Fig. 9 Select regions of electron density maps.

a. The DNA duplex under the ATPase at SHL2. b. The GSD/brace II helix. c. The brace stabilizer, bound to brace I. d. Part of the NegC competitor (Y926) beside a bound portion of NegC. e. A lower contoured view of density for the NegC competitor and C-terminal helix. Density shown in a-d were from the combined high-resolution map; density in e was from 2.90 Å map.

Extended Data Fig. 10 Nucleosome binding titrations indicate that Chd1[GSD/brace II]864-871-flex binds more tightly to the TA-rich side of the nucleosome.

This graph shows fits to a two-Kd model, where the Chd1[GSD/brace II]864-871-flex variant was titrated against 40N40 nucleosomes made with either canonical Widom 601 or the 601[TArich+1] sequence. For these experiments, the higher affinity site becomes stronger with 601[TArich+1] nucleosomes, suggesting that the higher affinity site for Chd1[GSD/brace II]864-871-flex is the TA-rich side. Data are presented as mean values +/− SD from n independent replicates. Data are given in Source Data File 10.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Data

Uncropped gels of purified samples.

Source data

Source Data Fig. 1

Source data of quantified decentering and sliding data.

Source Data Fig. 2

Source data of quantified DNA distortion entry data.

Source Data Fig. 3

Source data for quantified DNA distortion entry and sliding rate data.

Source Data Fig. 4

Source data for quantified DNA distortion dyad data.

Source Data Extended Data Fig. 2 and Table 2

Source data for quantified decentering, sliding, DNA distortion entry and DNA distortion dyad data.

Source Data Extended Data Fig. 3 and Table 3

Source data for fraction bound data and calculated Kd values for nucleosome binding.

Source Data Extended Data Fig. 4 and Table 4

Source data for fraction bound, fraction centered and fraction shifted.

Source Data Extended Data Fig. 6 and Table 6

Source data for FCS plots.

Source Data Extended Data Fig. 7 and Table 7

Source data for FCS plots.

Source Data Extended Data Fig. 10 and Table 10

Source data for binding plot.

Source Data All Figures

Uncropped gels.

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Nodelman, I.M., Folkwein, H.J., Glime, W.S. et al. A competitive regulatory mechanism of the Chd1 remodeler is integral to distorting nucleosomal DNA. Nat Struct Mol Biol 32, 1445–1455 (2025). https://doi.org/10.1038/s41594-025-01556-y

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