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Histone propionylation is a mark of active chromatin

Abstract

Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function.

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Figure 1: Histone lysine propionylation and butyrylation sites.
Figure 2: HATs can have propionylation and butyrylation activities.
Figure 3: Histone propionylation is sensitive to propionyl-CoA metabolism.
Figure 4: Histone acylations are specifically enriched at active promoter regions.
Figure 5: Correlation between histone acylations and gene expression.
Figure 6: Histone acylations differentially bind the BAF remodeling complex.
Figure 7: Histone propionylation stimulates transcription.

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Acknowledgements

We thank K. Bloom and M. Bennett (Children's Hospital Philadelphia) for Acads knockout tissues; A. Guenzel and M. Barry (Mayo Clinic) for Pcca knockout and gene-therapy-treated tissues; L. Arrigoni and J. Pospisilik for help with liver chromatin preparation; L. Tora (Institut de Genetique et de Biologie Moleculaire et Cellulaire, IGBMC) for baculoviruses expressing GCN5 and PCAF and for anti-GCN5 antibody; P. Laurette and I. Davidson (IGBMC) for antibodies against (P)BAF complex subunits; P. Eberling for peptide synthesis; S. Knapp (University of Frankfurt) for PFI-3 inhibitor; D. Widmann for initial analyses of data; and members of the Schneider laboratory for helpful discussions and reagents. M.C.-T. acknowledges support from the Helmholtz Association's Initiative and Networking Fund and from the University of Groningen (Rosalind Franklin Fellowship). Work by G. Meszaros and R.R. was supported by a European Research Council (ERC) starting grant (ERC-2011-StG, 281271-STRESSMETABOL). Work in the Schneider laboratory was supported by the Agence Nationale de Recherche (CoreAc), the DFG through SFB 1064, the Epigenomics of Common Diseases EpiTrio project and the Helmholtz Gesellschaft. Sequencing was performed by the IGBMC Microarray and Sequencing platform, a member of the 'France Génomique' consortium (ANR-10-INBS-0009).

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Contributions

A.F.K. and R.S. conceived the project. A.F.K. characterized antibodies, performed most of the ChIP, knockdown and peptide pulldown experiments and analyzed data. A.N. contributed to antibody characterization and knockdown experiments, and performed GAL4 recruitment ChIP and luciferase assays. L.Z.S. performed in vitro transcription experiments with the help of R.M. S.L.G. and D.A.G. performed analyses of ChIP and RNA-seq data. F.R. and G. Mittler performed and analyzed histone-modification mass spectrometry experiments. M.P.B. and M.V. performed and analyzed peptide-pulldown mass spectrometry experiments. G. Meszaros, H.F.M. and R.R. contributed to animal fasting and liver cross-linking experiments. A.T. and M.C.-T. performed ChIP–seq data analyses. A.F.K., S.D. and R.S. wrote the manuscript.

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Correspondence to Robert Schneider.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mass Spectrometric identification and analysis of histone propionylation and butyrylation.

Acid-extracted histones from HeLa cells were separated by C8 reversed phase chromatography, run on a 4-20% gradient Tris-glycine gel and bands cut out and digested either with trypsin or ArgC. Peptides were then analyzed via LC-MS. (A) Tandem spectra (MS/MS) of H3 peptide (10-17) showing the presence of propionylation (mass shift: 56.03 Da) at K14 (H3K14pr). (B) MS/MS of H3 peptide (9-17) showing butyrylated (mass shift = 70.04 Da) K14 (H3K14bu). (C) Tandem spectra (MS/MS) of double modified H3 peptide (9-17) acetylated (42.01 Da) at K9 and propionylated at K14 (H3K9acK14pr). (D) same as C but with butyrylated K14 (H3K9acK14bu).. Note: In C, prominent unannotated peaks (in black) correspond to fragment ions originating from a co-eluting isobaric peptide isomer, which contains the alternative combinatorial modification pattern – H3K9prK14ac. Supporting spectra are available upon request.

Supplementary Figure 2 Characterization of antibodies specific for histone propionylation and butyrylation.

(A) Immunoblotting using H3K14pr (left), and H3K14bu (right) antibodies on acid-extracted histones from untreated and 10 mM sodium butyrate (NaBu) – treated HeLa cells. (B-C) Peptide competition with anti-H3K14pr (B) and anti-H3K14bu (C) antibodies. Antibodies were pre-incubated with either with un, ac, pr, or bu H3K14 peptides before being used to probe blots with acid extracted histones. For (A-C) Ponceau is shown as loading control and the position of each core histone is indicated. (D-E) Peptide dot blot assays with anti-H3K14pr (D), anti-H3K14bu (E) antibodies and histone H3 peptides unmodified (un), acetylated (ac), propionylated (pr) or butyrylated (bu) at lysines 9, 14, 18 and 23. Peptide sequences are given in Supplementary Table S1. (F) Immunoblotting for H3K14pr and H3K14bu in different human and mouse cell lines. Anti-H3K14bu and anti-H3K14pr antibodies were used to probe acid extracts from indicated cell lines. (G) Immunoblotting of H3K14pr and H3K14bu using acid-extracted histones from five different mouse tissues. For (F,G) Ponceau is shown as loading control. (H) Immunofluorescence staining of mouse fibroblasts (MEFs) using anti-H3K14bu antibody. DAPI was used to stain the DNA. Scale bar represents 5 μm.

Supplementary Figure 3 Histone acetyltransferases can propionylate and butyrylate histones.

(A) In vitro histone acylation assay using full length Flag-tagged GCN5 or PCAF (purified from insect cells), unlabeled acyl-coA donors (acetyl-, propionyl- and butyryl-coA) and calf thymus histones as substrate. Histones were resolved by SDS-PAGE. The indicated antibodies used to probe the membranes ponceau staining as loading control are shown. (B) Time course histone propionylation assay on histone octamers using Flag-tagged GCN5. Same volumes of reaction were spotted on nitrocellulose membrane at each time point and immunoblotting done with anti-H3K14pr antibody. (C) Quantification of the H3K14pr signal in (B). (D) siRNA knockdown of GCN5 and PCAF in HeLa cells. Anti-PCAF and anti-GCN5 antibodies are used to confirm depletion and anti-tubulin as loading control. (E) Immunoblotting to verify the depletion of p300 upon siRNA-mediated knockdown with tubulin as loading control. *Non-specific band. (F) Quantification of H3K9ac, H3K14pr and H3K14bu signals normalized to H4 in GCN5 and/or PCAF knockdown samples (see Fig.2B). Error bars represent standard deviation of three independent knockdown experiments. (G) Quantification of H3K18ac, H3K14pr and H3K14bu signals normalized to H4 in p300 and/or CBP knockdown samples (see Fig.2C). Error bars represent standard deviation of at two independent knockdown experiments. (H-I) RT-qPCR results assessing the efficiency of siRNA-mediated knockowns for p300/CBP (H), GCN5/PCAF (I) respectively. Error bars indicate standard deviation of two technical replicates of a representative experiment. (J) Immunoblotting for GCN5 and PCAF following their double knockdown in HeLa cells for ChIP experiment in Fig.2E. (K) Quantification of GCN5 and PCAF signals in (J) normalized to stain-free total protein staining. Error bars represent standard deviation of three technical replicates. (L) Quantification of H3K14pr signals from wild type (Wt), Pcca−/− and Pcca−/− gene therapy treated mice livers (see Fig. 3D). Error bars represent standard deviation of three different livers. (M) Quantification of H3K14bu signals from Wt and Acads−/− livers (see Fig. 3E). Error bars represent standard deviation of three technical replicates.

Supplementary Figure 4 H3K14 is propionylated and butyrylated in mouse livers.

Dialyzed (PBS) acid-extracted histones from mouse livers were separated on a 16% Tris-glycine gel and bands cut out and digested with trypsin. Peptides were then analyzed via LC-MS. (A) Tandem spectra (MS/MS) of H3 peptide (10-17) showing the presence of propionylation (mass shift: 56.03 Da) at K14 (H3K14pr). (B) MS/MS of H3 peptide (9-17) showing butyrylation (mass shift = 70.04 Da) at K14 (H3K14bu).

Supplementary Figure 5 H3K14pr and H3K14bu are histone marks linked to active transcription.

(A). Overlap of H3K14pr and H3K14bu with H3K4me3 in refed and fasted livers. Target genes were defined with the presence of a peak ±1kb of a TSS. (B) Immunoprecipitation of purified native HeLa mononucleosomes with indicated antibodies followed by immunoblotting to detect co-occuring modifications. Input represents 2%. (C) Top five enriched Gene Ontology (GO) biological process terms for H3K14pr-specific genes in livers from refed mice. (D) Top five enriched GO biological process terms for H3K14bu-specific genes in livers from refed mice. (E) Spearman correlation heatmap of histone acylations with other active (H3K4me3, H3K9bhb) (Xie, Z., et al. Mol Cell, 2016. 62(2)) and repressive (H3K9me3, H3K27me3) (Sugathan, A. and D.J. Waxman. Mol Cell Biol, 2013. 33(18)) marks as well as RNA pol II (ser5phospho) (Koike, N., et al., Science, 2012. 338(6105)). AL, ad libitum; ST, starved; R, Refed; F, Fasted (F-G) Top ten GO biological process terms associated with "triple-acylated" genes in the refed (F) and fasted (G) state. See Fig. 5B-C.

Supplementary Figure 6 Histone acylations bind the BAF complex differentially

(A) Euclidean clustering heatmap of proteins identified by mass spectrometry as enriched in affinity purifications with H3K14 peptides. Purifications were performed using HeLa nuclear extract and biotinylated H3 peptides unmodified (un), acetylated (ac), propionylated (pr) or butyrylated (bu) at K14. BAF/PBAF complex subunits binding to H3K14ac and H3K14pr are marked on the right. (B) Coomassie-stained gel of purified GST-tagged 2nd bromodomain of PBRM1 (GST-PBRM1(2)) used for the direct binding experiment in Fig. 6D-E.

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Kebede, A., Nieborak, A., Shahidian, L. et al. Histone propionylation is a mark of active chromatin. Nat Struct Mol Biol 24, 1048–1056 (2017). https://doi.org/10.1038/nsmb.3490

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