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Oncohistone mutations enhance chromatin remodeling and alter cell fates

Abstract

Whole-genome sequencing data mining efforts have revealed numerous histone mutations in a wide range of cancer types. These occur in all four core histones in both the tail and globular domains and remain largely uncharacterized. Here we used two high-throughput approaches, a DNA-barcoded mononucleosome library and a humanized yeast library, to profile the biochemical and cellular effects of these mutations. We identified cancer-associated mutations in the histone globular domains that enhance fundamental chromatin remodeling processes, histone exchange and nucleosome sliding, and are lethal in yeast. In mammalian cells, these mutations upregulate cancer-associated gene pathways and inhibit cellular differentiation by altering expression of lineage-specific transcription factors. This work represents a comprehensive functional analysis of the histone mutational landscape in human cancers and leads to a model in which histone mutations that perturb nucleosome remodeling may contribute to disease development and/or progression.

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Fig. 1: A library of cancer-associated histone mutations.
Fig. 2: An H2A/H2B dimer exchange assay identifies histone mutations that destabilize nucleosomes.
Fig. 3: Asymmetric mutant H2B nucleosomes are destabilized.
Fig. 4: A chromatin remodeling assay uncovers histone mutations that affect nucleosome sliding.
Fig. 5: Globular domain mutations alter gene expression and impair cell differentiation.
Fig. 6: Proposed model of how histone core mutations contribute to cancer.

Data availability

RNA-seq raw data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE146959. Source data are provided with this paper. All other data are included within this paper, Extended Data and Supplementary Information files.

Code availability

A custom R script was used to count individual DNA barcodes from sequencing data in MN library experiments and was previously reported24. Custom Python (v.2.7.10) scripts were used to fit and determine Thalf values in MN thermal stability experiments and to calculate kobs values in ACF remodeling experiments are available at https://github.com/jdbagert/oncohistonelibrary.

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Acknowledgements

We thank T. Carroll from the Bioinformatics Resource Center at The Rockefeller University and J. Majewski and E. Bareke from McGill University for help and guidance with the RNA-seq analyses. We thank W. Wang and the Princeton Genomics Core Facility for designing the Illumina adaptor sequences and performing NGS on DNA-barcoded MN library experimental samples. We thank A.J. Burton for providing the dead CfaC peptide and H. Liu for providing Cy3-tagged histone H3. Funding support includes grant nos. P01CA196539 (T.W.M. and C.D.A.) and R37-GM086868 (T.W.M.), and we thank members of the Allis and Muir laboratories, especially those members of our collective P01 team. J.D.B. was funded by a postdoctoral fellowship from the US National Institutes of Health (grant no. GM123659). M.M.M. was funded by a postdoctoral fellowship from the US National Institutes of Health (grant no. GM131632). A.L.P. was funded by an Anderson Graduate Fellowship in Cancer Research from the Anderson Cancer Center. F.W. was supported by a postdoctoral fellowship from the German Research Foundation (WO 2039/1-1). B.A.N. was funded by the National Cancer Institute (grant no. 1K08CA245212) and we acknowledge the Memorial Sloan Kettering Cancer Center Support Grant/Core grant (no. P30 CA008748). L.F. was supported by the C. H. Li Memorial Scholar Fund Award.

Author information

Affiliations

Authors

Contributions

A.L.P. and B.E.D. contributed equal to this study. M.M.M. prepared the DNA-barcoded MN library, purified ACF, performed all remodeling experiments and prepared all samples for NGS. J.D.B. prepared Nap1, performed all dimer exchange experiments, prepared heterotypic nucleosomes and performed statistical analyses. B.E.D. prepared the humanized yeast library. A.L.P. performed all mammalian cell experiments. F.W. expressed ACF. M.M.M., J.D.B., B.E.D., A.L.P., C.D.A. and T.W.M. analyzed the data. M.M.M., J.D.B., B.E.D., A.L.P., F.W., C.D.A. and T.W.M. conceived the project with input from B.A.N. and L.F. C.D.A. and T.W.M. supervised the study. M.M.M., J.D.B. and T.W.M. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to C. David Allis or Tom W. Muir.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Chemical Biology thanks Andrew Andrews, Suzanne Baker and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Selection criteria for histone mutations to include in high-throughput libraries.

a, Distributions of histone mutations (tumor mutational burden ≤ 10) across the four canonical histones were fit to a Poisson distribution (red line) to identify sites of outlier mutational frequency (p < 0.05, red bars). b, The set of amino acid mutations observed at each histone site was assessed for overrepresentation of specific histone mutations, adjusting for codon bias. Mutants with a Benjamini FDR-adjusted p-value < 0.03 are shown in red.

Extended Data Fig. 2 Generation of a barcoded MN library for high-throughput profiling of histone mutations.

a, Schematic of barcoded MN library assembly and use in in vitro ChIP-seq assays. b, Native gel analysis of individually assembled barcoded MNs that were combined to create the nucleosome library (see panel c). c, Native gel analysis of the DNA-barcoded mutant MN library. The upper band shows the combined signal from all assembled MNs included in the library. The lower band shows free DNA from nonnucleosomal DNA controls. d, Assessment of quality of MN library members by deep sequencing, as determined by the amount of free barcoded DNA associated with each member. The plot shows normalized reads from library aliquots that were either treated with PstI restriction enzyme or untreated. The cuttable DNA control, as well as DNA exposed as a result of poor nucleosome assembly, are digested in the PstI-treated sample and thus exhibit lower read counts. The black line represents an idealized scenario in which all MNs are 100% assembled (y = x). This function yields an R2 value of 0.98, indicating that MN library members are well-assembled in the library. Data are presented as the mean ± s.d. (n = 3). e, Unstable nucleosomes (H2BE76K20,21) do not exhibit spontaneous exchange of DNA with fluorescently labeled nucleosomes, even under conditions of biased stoichiometry and high temperature. Samples containing individual or mixtures of unlabeled H2BE76K MNs and fluorescently tagged (H2B-Fluor) MNs were incubated at the indicated temperatures for 1 h and then assessed by native gel electrophoresis to determine whether exchange of fluorescent histones occurs. Numbers indicate ratios of MNs in a given sample. H2BE76K MNs were assembled on DNA with a 15 bp 3’ overhang (15 bp 3’OH), whereas H2B-Fluor MNs were assembled on DNA with no 3’ overhang (0 bp 3’OH) so the two species could be distinguished based on differential gel migration. Additionally, H2B-Fluor MNs were assembled on DNA with a 15 bp 3’ overhang as a positive control for DNA exchange. Source data

Extended Data Fig. 3 Structurally perturbative histone mutations cause growth defects in humanized yeast.

a, A humanized yeast library facilitates high-throughput analysis of the effects of histone mutations on yeast viability. A plasmid shuffle assay is used to select for yeast that can subsist solely on mutant human histones. b, Serial dilutions of humanized yeast with mutant histones after 5FOA selection. Control cells are grown without 5FOA to show strain growth prior to selection for human histones. c, Yeast carrying wild-type and select histone mutants show similar growth rates prior to 5FOA selection. This indicates that growth differences observed on 5FOA plates are not attributable to differential growth rates prior to selection for cells able to subsist solely on mutant human histones.

Extended Data Fig. 4 A histone dimer exchange assay reveals mutations that destabilize nucleosomes.

a, Denaturing gel analysis of purified mouse Nap1. b, Proof-of-concept demonstration of the dimer exchange assay on wild-type and destabilized H2BE76K20,21 mutant MNs using fluorescently labeled dimers and in-gel fluorescence readout. The lower band in the ethidium bromide stain shows partially disassembled nucleosomes, which are observed only when Nap1 is added to nucleosomes without additional H2A/H2B dimers. Fluor, fluorescein. EtBr, ethidium bromide. c, Nucleosomes do not exhibit exchange of H3/H4 tetramers during the Nap1 H2A/H2B dimer exchange assay. Samples containing wild-type MNs or H2BE76K and fluorescently tagged H3 (H3-Cy3) MNs were incubated under Nap1 H2A/H2B dimer exchange conditions (see Methods) and then assessed by native gel electrophoresis to determine whether exchange of fluorescent histones occurs. Numbers indicate the relative ratios of MNs in each sample. Wild-type MNs were assembled on DNA with a 60 bp overhang (60 bp OH), whereas H2BE76K/H3-Cy3 MNs were assembled on DNA with no overhang (0 bp OH), allowing discrimination of the two nucleosome species by gel migration. Additionally, H2BE76K/H3-Cy3 MNs were assembled on DNA with a 60 bp overhang as a positive control for DNA exchange. d, Qubit quantification of MN DNA after affinity purification of nucleosomes that were subjected to the Nap1-mediated dimer exchange assay with biotinylated H2A/H2B dimers. DNA yield displays the fraction of DNA from affinity purified samples relative to input samples, indicating the extent of H2A/H2B dimer exchange. Individual WT and H2BE76K MNs were used as a proof-of-principle demonstration that destabilized MNs result in increased dimer exchange and, by extension, increased MN DNA after affinity purification. DNA yields from barcoded MN library experiments are similar to wild-type MNs, which is consistent with the resulting library data that shows that the majority of mutations have no or only moderate effects on dimer exchange (Fig. 2b). Data are presented as the mean ± s.d. (n = 2 for individual MNs, n = 4 for MN library). e, H2AE56A/K greatly enhance dimer exchange compared to other acidic patch mutations in the histone mutant library. Data are presented as the mean ± s.d. (n = 4). f, Denaturing gel analysis of purified octamers, dimers, and tetramers used to generate individual MNs for follow-up stability assays. g, Native gels of assembled mutant MNs used in follow-up dimer exchange and thermal stability assays. h, Raw data showing enhanced fluorescence for destabilizing mutants in the Nap1-mediated dimer exchange assay. i, Validation of select dimer exchange-enhancing histone mutant MNs via quantification of the in-gel fluorescence data shown in panel h. Data are presented as the mean ± s.d. (n = 3). Values from mutant MNs in e and i exhibit statistically significant differences relative to wild-type (p < 0.05) in a two-sample, unequal variances t-test, unless otherwise noted. ns, not significant. Source data

Extended Data Fig. 5 Destabilizing histone mutations perturb nucleosome thermal stability and octamer assembly.

a, Raw data for Fig. 2f showing the thermal stability of MNs with histone mutations. Data are shown in triplicate with a sigmoidal fit for the dimer melt (see Methods). The Thalf is shown for each fit as a dotted vertical line and reported as the mean ± s.d. (n = 3). b, Fast protein liquid chromatography traces of histone mutants that did not form octamers under standard assembly conditions.

Extended Data Fig. 6 Asymmetric mutant H2B nucleosomes are destabilized.

a, Denaturing gel analysis of purified wild-type, mutant, and H2A/H2B-CfaN-streptag dimers. b, Proof-of-concept assembly and purification of heterotypic nucleosomes with ubiquitinated H2B (H2Bub), which allows asymmetric nucleosomes to be distinguished by size. Numbers indicate molecular species shown in Fig. 3a. Hex., hexasomes. Het., heterotypic. c, Assembly and purification of heterotypic (het.) nucleosomes containing H2BE71K and H2BE76K. Assembly of wild-type (effectively homotypic) nucleosomes served as a control for the assembly process. 6/8 refers to hexasomes/octasomes. d, Raw data showing enhanced fluorescence for both homotypic and heterotypic destabilizing mutant MNs in the Nap1-mediated dimer exchange assay. e, Raw data showing the thermal stability of homotypic and heterotypic mutant MNs. Data are shown in triplicate with a sigmoidal fit for the dimer melt. The Thalf is shown for each fit as a dotted vertical line and reported as the mean ± s.d. (n = 3). Source data

Extended Data Fig. 7 Mutations at the histone-DNA interface and the acidic patch alter nucleosome sliding.

a, Denaturing gel showing the two subunits of the purified hACF complex. b, Time-course of ACF remodeling reactions from all MNs in the library both with ATP (red) and without ATP (black) demonstrate that the remodeling rates measured are ATP-dependent. Data are presented as the mean of individual MN values at each time point (n = 3). c, Denaturing gel analysis of histone mutant octamers used to generate individual MNs for follow-up sliding assays. A subset of octamers, also utilized for follow-up dimer exchange and thermal stability assays, were already depicted in Extended Data Fig. 4f. d, Native gels of assembled MNs used in sliding assays. e, Representative gel images (all assays performed in triplicate) showing a time-course of DNA cleavage during ACF sliding in REA assays. f, Observed rate constants for dimer-tetramer interface mutant MNs do not exhibit statistically significant differences relative to wild-type (p > 0.05) in a two-sample, unequal variances t-test. Data are presented as the fit kobs parameter ± s.d. (n = 3). ns, not significant. Source data

Extended Data Fig. 8 Globular domain mutations alter gene expression and impair cell differentiation.

a, Western blot analysis of acid extracted histones from C3H10T1/2 cells stably expressing HA-tagged histone mutants shows mutant incorporation into chromatin. A standard curve of mixtures of murine H2B (mH2B) and tagged murine H2B (mH2B-F-HA) was used to estimate the relative expression of mutant histones, which ranged from 1–17% of the total H2B protein. b, Representative immunofluorescence images of tagged histone mutants expressed in C3H10T1/2 cells. Scale bar, 50 µm. c, Principal component analysis (PCA) of gene expression data from histone mutant cell lines. PCA distinguishes wild-type from mutant cell lines, as well as individual mutations from each other. d, Expression of select cancer-associated genes representing the dysregulated molecular pathways in histone mutant-expressing cell lines as assessed by RT-qPCR (relative to wild type). Data presented are the mean ± s.e.m. (n = 3) and values from mutant cell lines exhibit significant differences relative to wild-type (p < 0.05) in a two-sample t-test, unless otherwise noted. ns, not significant. nd, not detected. e, Mutant and wild-type H2B cell lines were induced to undergo chondrocyte differentiation for 7 days. Representative images are shown after Alcian Blue staining, as well as the quantified Alcian Blue staining of cell extracts, reported as the mean ± s.d. (n = 8). An asterisk marks mutant cell lines that cause a significant reduction in staining compared to both the parent line and to cells expressing wild-type H2B (p < 0.05, unequal variances t-test). Source data

Extended Data Fig. 9 Gene ontology analysis of cells expressing globular domain mutations.

a, Gene ontology (GO) analysis for individual histone mutants showing the top ten upregulated and downregulated KEGG pathways. The dotted line shows the statistical cutoff (Benjamini FDR < 0.05) used for selecting which GO groups to display in Fig. 5b. b, Quantitative analysis showing the relative gene expression values (mutant to wild-type histone) from the significantly regulated GO groups presented in Fig. 5b.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Tables 7 and 8.

Reporting Summary

Supplementary Table 1

Histone mutants represented in the barcoded MN and humanized yeast libraries.

Supplementary Table 2

Humanized yeast phenotypes (no phenotype, impaired growth or no growth) for each histone mutant included in the library.

Supplementary Table 3

Raw data and subsequent calculations from Nap1-mediated dimer exchange experiment on the barcoded MN library.

Supplementary Table 4

Raw data and subsequent calculations from ACF remodeling experiment on the barcoded MN library.

Supplementary Table 5

RNA-sequencing data for cells expressing mutant and wild-type histones.

Supplementary Table 6

Gene ontology analysis of significantly up- and downregulated genes from cells expressing mutant histones.

Source data

Source Data Fig. 3

Unprocessed gels and/or blots.

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Bagert, J.D., Mitchener, M.M., Patriotis, A.L. et al. Oncohistone mutations enhance chromatin remodeling and alter cell fates. Nat Chem Biol 17, 403–411 (2021). https://doi.org/10.1038/s41589-021-00738-1

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