Abstract
Nucleosomes are basic repeating units of chromatin and form regularly spaced arrays in cells. Chromatin remodelers alter the positions of nucleosomes and are vital in regulating chromatin organization and gene expression. Here we report the cryo-EM structure of chromatin remodeler ISW1a complex from Saccharomyces cerevisiae bound to the dinucleosome. Each subunit of the complex recognizes a different nucleosome. The motor subunit binds to the mobile nucleosome and recognizes the acidic patch through two arginine residues, while the DNA-binding module interacts with the entry DNA at the nucleosome edge. This nucleosome-binding mode provides the structural basis for linker DNA sensing of the motor. Notably, the Ioc3 subunit recognizes the disk face of the adjacent nucleosome through interacting with the H4 tail, the acidic patch and the nucleosomal DNA, which plays a role in the spacing activity in vitro and in nucleosome organization and cell fitness in vivo. Together, these findings support the nucleosome spacing activity of ISW1a and add a new mode of nucleosome remodeling in the context of a chromatin environment.
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Data availability
Coordinates and EM maps have been deposited in the EMDataResource and PDB under accession codes EMD-32992 (PDB 7X3T, ISW1a-diNCP); EMD-32995 (PDB 7X3W, N1-motor); EMD-32996 (PDB 7X3X, N1-RA) and EMD-32994 (PDB 7X3V, N2-Ioc3). MNase-seq datasets are available in the National Center for Biotechnology Information Gene Expression Omnibus repository (GSE240192). Source data are provided with this paper.
Code availability
Scripts to analyze MNase-seq data have been deposited at GitHub (https://github.com/siayouyang/MNase-seq_analysis_workflow).
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Acknowledgements
We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for the cryo-EM facility (crosslinked dataset) and the computational facility support on the cluster of Bio-Computing Platform. This work was supported by the National Key Research and Development Program (grant nos. 2022YFA1302700 and 2019YFA0508902 to Z.C.), the National Natural Science Foundation of China (grant nos. 32130016 and 31825016 to Z.C.), Beijing Frontier Research Center for Biological Structure and Tsinghua–Peking Joint Center for Life Sciences.
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L.L. prepared the sample and performed the biochemical analysis. K.C. performed the EM analysis. Y.S. and P.H. prepared MNase-seq samples and performed MNase-seq analysis. Y.Y. conducted the yeast genetics assays. Z.C. wrote the manuscript with help from all authors. Z.C. directed and supervised all the research.
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Extended data
Extended Data Fig. 1 CryoEM analysis of the Isw1a-dinucleosome complex.
(a) Representative negative stain images, (b) cryoEM images, and (c) 2D classification of the ISW1a complex bound to the dinucloesome. (d) Flowchart of the cryo-EM data processing. (e) Angular distributions of cryo-EM particles in the final round of refinement of the masked dataset. (f) Gold standard Fourier shell correlation (FSC) curves, showing the resolutions of 5.4 Å, 3.2 Å, 2.9 Å, 3.1 Å and 3.1 Å for the ISW1a-dinucleosome complex, N1 (bound with arginine anchors, RAs), N2 (bound with the Finger helix, FH), N1-motor, and N2-Ioc3, respectively.
Extended Data Fig. 2 Local density maps of the Isw1a-dinucleosome complex.
(a) nucleosomal DNA; (b) H2A-H2B of the N1 nucleosome; (c) H2A-H2B of the N2 nucleosome; (d) motor domain of Isw1; (e) HSS domain of Isw1 (f) the CLB domain of Ioc3 and (g) HLB domain of Ioc3.
Extended Data Fig. 4 Additional biochemical analyses of the Isw1a complex.
(a) The ISW1a complex slides the closely packed 50N15N50 dinucleosome to more evenly spaced positions. This experiment was repeated independently for three times.The dinucleosomes 38N38N39 (evenly spaced nucleosomes, lane 1) and 32N33N50 (one nucleosome moved, lane 2) showed similar migration patterns as those of the ISW1a-remodeling products B2 and B3, respectively. (b–d) the chromatin remodeling activities of the WT and indicated mutant ISW1a complex towards the nucleosome substrates 50N15N50 (b), 0N80 (c), and 10N10N60 (d). Representative gels are shown. Quantification of the initial substrates remodeled are shown on the right in (b) and (c); quantification of the initial substrate (B1) and final product (B4) are shown in the middle and right panels of (d), respectively. Error bars indicate SD of the mean(n = 3).
Extended Data Fig. 5 Tension estimation imposed on NegC of Isw1.
(a) The distance between the RA and the motor domain of Isw1 through a disordered NegC domain as reported in current study. The NegC connects the C-terminus of the motor domain with the RA motif, spanning a distance ∼74 Å through a disordered sequence of 108 aa (residues 657–764). (b) The distance between the RA and the motor domain of Isw1 through the fully folded NegC domain as predicted by AlphaFold (ID: AF-P38144-F1). The distance is ∼47 Å through a disordered sequence of 24 aa (residues 741–764). (c) Tension estimated by the worm-like-chain model imposed on the disordered sequence of the melted (back line) and folded (red line) NegC. Assuming the persistent length of 1 nm for the polypeptide, the tensions are estimated to be ∼1 pN and ∼6 pN for the melted and folded NegC, respectively.
Extended Data Fig. 6 Interaction between the HLB domain of Ioc3 and the DNA.
(a) Structural comparison of the HSS-Ioc3 DNA-binding module bound to the dinucleosome (color coded) and the free DNA (colored grey, PDB code 2Y9Z)3. The structures of Ioc3 are aligned. (b) Interaction between the HLB domain and the DNA. The electrostatic potential of the HLB domain is calculated by Pymol.
Extended Data Fig. 8 Additional analysis of the nucleosome organization of the WT and Ioc3 mutant yeast cells.
To ensure the data quality, three independent datasets are measured, and each dataset includes three biological replicates of the WT samples, and one set of the Ioc3 mutants. (a, b) Nucleosome shift analysis of dataset1 derived from the 1108 overlapping genes in the Venn diagram of Fig. 5b. (a) Histograms of the number of genes having a given nucleosome shift (1-bp bins). (b) List of median shifts of the +1 to +4 promoter nucleosomes of the 1108 overlapping genes. The shifts of one of the WT cells are used as control for Wilcoxon-Mann-Whitney test shown as heat-map in Fig. 5d. (c–e) Nucleosome shift analysis of dataset2. All genes with at least one significantly shifted promoter nucleosomes are included. (c) Histograms of the number of genes having a given nucleosome shift (1-bp bins). (d) List of median shifts of the +1 to +4 promoter nucleosomes. The shifts of one of the WT cells are used as control for two-sided Wilcoxon-Mann-Whitney test shown as heat-map in (e). (f–h) Equivalent plots for dataset3.
Extended Data Fig. 9 Four specific loci with shifted nucleosomes.
(a) Smoothed dyad density showing downstream shifting of the promoter nucleosomes detected in three independent datasets. (b) Raw dyad density of the RAD53 promoter nucleosomes without gaussian smoothing (black bars).
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Li, L., Chen, K., Sia, Y. et al. Structure of the ISW1a complex bound to the dinucleosome. Nat Struct Mol Biol 31, 266–274 (2024). https://doi.org/10.1038/s41594-023-01174-6
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DOI: https://doi.org/10.1038/s41594-023-01174-6
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