ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

Abstract

ATP-dependent chromatin remodellers regulate access to genetic information by controlling nucleosome positions in vivo1. However, the mechanism by which remodellers discriminate between different nucleosome substrates is poorly understood. Many chromatin remodelling proteins possess conserved protein domains that interact with nucleosomal features2. Here we used a quantitative high-throughput approach, based on the use of a DNA-barcoded mononucleosome library, to profile the biochemical activity of human ISWI family remodellers in response to a diverse set of nucleosome modifications. We show that accessory (non-ATPase) subunits of ISWI remodellers can distinguish between differentially modified nucleosomes, directing remodelling activity towards specific nucleosome substrates according to their modification state. Unexpectedly, we show that the nucleosome acidic patch3 is necessary for maximum activity of all ISWI remodellers evaluated. This dependence also extends to CHD and SWI/SNF family remodellers, suggesting that the acidic patch may be generally required for chromatin remodelling. Critically, remodelling activity can be regulated by modifications neighbouring the acidic patch, signifying that it may act as a tunable interaction hotspot for ATP-dependent chromatin remodellers and, by extension, many other chromatin effectors that engage this region of the nucleosome surface4,5,6,7,8,9.

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Figure 1: A diverse library of modified nucleosomes.
Figure 2: A high-throughput nucleosome remodelling assay for ISWI family chromatin remodellers.
Figure 3: Specialization of ISWI remodellers for diverse nucleosome modifications.
Figure 4: The nucleosome acidic patch is crucial for remodelling and regulatable by histone PTMs.

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Acknowledgements

We thank current and former members of the Muir laboratory for discussions and comments; W. Wang and J. Wiggins from the Princeton Sequencing Core Facility; C. Hannon and S. Blythe for writing a custom R script for managing sequencing data; and L. Guerra and L. Beh for advice with data analysis. The SNF2h, ACF1, WSTF, CHRAC-15, and CHRAC-17 coding sequences were a gift from R. Kingston; the RSF1 coding sequence was a gift from D. Reinberg; the BPTF coding sequence was a gift from C. D. Allis; F.W. was funded by a postdoctoral fellowship from the German Research Foundation (DFG); T.P. was funded by a postdoctoral fellowship from the US National Institutes of Health (GM112365); and this research was supported by the US National Institutes of Health grants P01 CA196539, R37 GM086868 and R01 GM107047.

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Contributions

G.P.D. purified all remodellers, performed all remodelling and nucleosome library experiments, and processed all sequencing data; G.P.D prepared the nucleosome library with contributions from G.P.L., M.M.M., U.T.T.N., T.P., Z.Z.B., F.W., J.B., R.P. and S.B.P.; G.P.D. and G.P.L. purified RCC1 and Sir3 constructs for nucleosome library binding experiments; G.P.L. performed the qPCR analysis; G.P.D. and K.L.D. synthesized and purified the LANA peptides; J.D.B. performed the PCA analysis; C.D.A. oversaw the preparation of crotonylated histones; G.P.D, J.D.B. and T.W.M. analysed the data; G.P.D., M.M.M. and T.W.M. conceived the project; T.W.M. supervised the study; G.P.D. and T.W.M. wrote the manuscript.

Corresponding author

Correspondence to Tom W. Muir.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks B. Cairns, F. Pugh and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Characterization of barcoded 601 (BC-601) DNA.

a, BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, 0.5× TBE, 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b, BC-601 DNA for nucleosome 99 (Supplementary Table 1) was CpG methylated by the M.SssI methyltransferase (NEB) according to the manufacturer’s instructions and characterized by digestion with the RsaI restriction enzyme, which is sensitive to CpG methylation, and PstI, which is not. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Analysis of the quality and integrity of the nucleosome library.

a, b, Analysis of individual nucleosome preparations (a) and the final library after pooling of nucleosomes (b) by native gel electrophoresis and staining with ethidium bromide. c, Antibody pull-down of library members using an anti-H3K4me3 antibody. Every nucleosome member containing an H3K4me3 mark (red) was efficiently isolated relative to other library members (black). Notably, the antibody was also able to pull down a nucleosome possessing solely the H3K4me2 mark (blue), indicating a lack of antibody specificity in this case. This experiment was performed once. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Characterization of recombinant ISWI chromatin remodellers.

a, Purified chromatin remodellers were run a 4–20% Mini-PROTEAN TGX gel (Bio-Rad) and for 35 min at 180 V. Proteins were stained with Coomassie. The composition of each remodeller–remodelling complex is depicted above each respective lane on the gel. Expected molecular weights: SNF2h: 122 kDa, ACF1: 179 kDa, CHRAC-15: 14.7 kDa, CHRAC-17: 16.9 kDa, WSTF: 171 kDa, TIP5: 208 kDa, RSF1: 164 kDa; migrates at higher apparent molecular weight, SNF2L: 121 kDa, BPTF: 338 kDa, RbAp46: 47.8 kDa. b, All remodellers display ATP-dependent nucleosome remodelling activity as detected by a restriction enzyme accessibility assay. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Nucleosome remodelling activity is negligible in the absence of ATP.

Bar graphs show individual DNA cleavage rates (kMN, remodelling rates in the case of nucleosomes; see Supplementary Table 3) from library remodelling experiments for each member of the library in the presence of the indicated chromatin remodeller with and without ATP. Rate values were rank ordered and are displayed from low to high. The dashed red line represents the rate of remodelling of unmodified nucleosomes. The related graphs for the ACF complex can be found in Fig. 2d. Data are represented as the mean of experimental replicates ± s.e.m. (n = 3).

Extended Data Figure 5 Principal component (PC) analysis of library remodelling data.

Percentages show the fractions of the variance accounted for by each PC. Individual nucleosomes are shown in light blue, and PC weight values for each remodeller are shown in either orange or black. Weights are scaled by a factor of 2 for visibility. a, PC1 vs. PC2 and PC1 vs. PC3 are plotted. b, PC2 vs. PC3 are plotted as in Fig. 3a. Nucleosomes driving differences in remodeller activity were numbered as in Supplementary Table 1 and grouped by their location in PC space (Supplementary Table 4).

Extended Data Figure 6 Alteration of histone–DNA contacts affects remodelling activity.

a, Modified histone residues in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. PTMs are numbered and labelled on the nucleosome structure. Values were capped at −2 and 2 for display purposes. b, Histone mutants present in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. The heatmap is displayed as in a. Locations of each mutation are individually labelled on the nucleosome structure. Values were capped at −3 and 3 for display purposes. All histones are unmodified unless otherwise specified.

Extended Data Figure 7 Remodelling of nucleosomes containing modifications preferred by histone recognition domains.

Library remodelling data generated by the NoRC (a), WICH (b), and NURF (c) complexes for nucleosomes containing residues known to interact with histone binding modules in accessory subunits of each complex (NoRC: TIP5; WICH: WSTF; NURF: BPTF). Literature binding specificities are displayed in corresponding tables on the right. Bar graphs display log2 values of the rate of remodelling of individual nucleosome library members (kMN) relative to unmodified nucleosomes (kunmod.). Data are represented as the ratio of the mean of experimental replicates ± s.e.m. (n = 3). Note that H3KpolyAc includes the H3K14ac modification (Supplementary Table 1). All histones are unmodified unless otherwise specified. BRD, bromodomain; PHD, PHD-finger; PHD–BRD, tandem PHD-finger–bromodomain module; ND, not determined.

Extended Data Figure 8 Remodelling assays carried out on individual nucleosomes measured via standard gel-based read-out to validate library data.

a, Activity of the NURF complex towards H3K4me3+H4K16ac relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). b, Activity of the ACF complex on unmodified and acidic patch mutant nucleosomes. c, Remodelling of unmodified nucleosomes is inhibited by the presence of the LANA peptide when compared to a LANA peptide with key binding residues mutated (LRS to AAA). d, Activity of the ACF complex towards nucleosomes modified near the acidic patch (H2BK108ac and H2BS112GlcNac) relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). Gel images of example replicates used to generate densitometry measurements in each subpanel are shown above respective graphs. a, c, and d use a restriction enzyme accessibility assay. b uses a nucleosome repositioning electrophoretic mobility shift assay. All histones are unmodified unless otherwise specified. All data are represented as the mean of experimental replicates ± s.e.m. (n = 3). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 High-throughput chromatin remodelling and binding data.

a, Heat-map displaying ISWI remodelling data (as in Fig. 2b) against the nucleosome library with CHD4 data for comparison. Rows were sorted on the basis of values for SNF2h (low to high). b, Heat map displaying binding of chromatin factors RCC1 and Sir3 against the nucleosome library relative to unmodified nucleosomes. Values were capped at −4 and 4 for display purposes. All data are represented as the mean of experimental replicates (n = 3).

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–6. (PDF 11328 kb)

Reporting Summary (PDF 363 kb)

Supplementary Table 1

Histone Composition of Nucleosome Library Members. Individual histone composition of each member of the nucleosome library. Modifications include lysine acetylation (Kac), lysine mono, di, and trimethylation (Kme, Kme2, Kme3), arginine mono, asymmetric and symmetric dimethylation (Rme, Rme2a and Rme2s, respectively), lysine ubiquitylation (Kub), serine and tyrosine phosphorylation (Sph and Yph, respectively), serine modified with N-acetylglucosamine (SGlcNAc), and lysine crotonylation (Kcr). * = CpG methylation of nucleosomal DNA. (XLSX 33 kb)

Supplementary Table 2

Normalized Sequencing Reads Used to Calculate Nucleosome Remodeling Rate Constants. This table contains sequencing read counts that have been sorted by both a nucleosome identifier and multiplexing barcodes. This includes reads corresponding all 28,224 kinetic measurements to measure ISWI nucleosome remodeling as well as those for DNA Standard 2 and CHD4. The reads have already been normalized to DNA Standard 1, and are organized by enzyme, time point, with or without ATP, and by nucleosome (experimental triplicate measurements) as in Supplementary Table 1. (XLSX 371 kb)

Supplementary Table 3

Calculated Nucleosome Remodeling Rate Data. Raw rate values calculated by averaging experimental triplicate measurements from data in Supplementary Table 2 as described in the methods section of this study. Rows are organized by nucleosome number as in Supplementary Table 1. Per remodeler, the average of remodeling rate of all unmodified nucleosomes and corresponding error (considering error propagation) as well as the low threshold for measurement and corresponding error (considering error propagation) were calculated (bottom of table; see methods). Columns correspond to raw average measured rates (kslow ± ATP), standard error of the rates (kslow ± ATP SEM), final nucleosome remodeling rates used to calculate fold changes relative to unmodified nucleosomes (Final kslow +ATP, “kMN„; values are adjusting considering the calculated low threshold rate; note that these values for conditions –ATP were calculated to be negligible and thus ignored), fold change of remodeling rate relative to unmodified nucleosomes (log2k_MN/k_unmod.), and the corresponding error (considering error propagation). (XLSX 163 kb)

Supplementary Table 4

Nucleosome Library Members Contributing to Variation in Remodeler Behavior. This table contains nucleosomes grouped (by color) according to Extended Data Fig. 5b. Per remodeler, values in each column correspond to log2 values of the ratio of the rate of remodeling for a particular nucleosome to that of unmodified nucleosomes (as in Supplementary Table 3). Nucleosome modifications per histone are specified and nucleosomes are numbered as in Supplementary Table 1. (XLSX 16 kb)

Supplementary Table 5

Unique hexanucleotide nucleosome identifier barcodes. This table contains a list of unique hexanucleotide nucleosome identifier barcodes used in this study. Barcodes are numbered corresponding to the nucleosome (as in Supplementary Table 1) they are associated with. (XLSX 35 kb)

Supplementary Table 6

Unique multiplexing barcodes. This table contains a list of unique multiplexing barcodes used in this study. Barcodes are numbered arbitrarily. Typically, no more than 72 samples were sequenced in a single sequencing run. (XLSX 34 kb)

Supplementary Table 7

Raw Sequencing Reads and Calculation of Relative Binding Measurements for RCC1 and Sir3 Using the Nucleosome Library. This contains raw sequencing read counts used to calculate relative binding measurements for RCC1 and Sir3 using the nucleosome library as described in methods section ‘Processing of sequencing data for binding experiments using the nucleosome library’. (XLSX 132 kb)

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Dann, G., Liszczak, G., Bagert, J. et al. ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference. Nature 548, 607–611 (2017). https://doi.org/10.1038/nature23671

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