Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1,2,3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4,5,6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.
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Data from ChIP-seq and RNA-seq experiments have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database under accession numbers GSE106495 and GSE117910. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE48,49 partner repository with the dataset identifier PXD008106. Additional raw data files are available at https://chorusproject.org under project no. 1513. We declare that the data supporting findings for this study are available within the article and Supplementary Information (Supplementary Fig. 1). Related data are available from the corresponding author upon reasonable request. No restrictions on data availability apply.
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We thank R. Cagan (ISMMS), J. Coplan (SUNY Downstate) and C. Tamminga (UTSW) for providing Drosophila, macaque and human samples, respectively, for analysis of H3K4me3Q5ser (Fig. 1e), and G. Johnson (University of Rochester Medical Center) for wild-type and catalytically dead TGM2 constructs. This work was supported by grants from the National Institutes of Health: DP1 DA042078 (I.M.), R01 MH116900 (I.M.), R21 DA044767 (I.M.), P50 MH096890 (I.M.), R37 GM086868 (T.W.M.), P01 CA196539 (T.W.M. and B.A.G.), R01 GM110174 (B.A.G.), R21 DA040837 (B.A.G.), R01 CA129325 (R.G.R.), R01 CA204639 (R.G.R.), T32 DA007135 (R.M.B.), as well as awards from: MQ Mental Health Research Charity, MQ15FIP100011 (I.M.), Alfred P. Sloan Foundation, Fellowship in Neuroscience (I.M.), the JPB Foundation (F.H.G.), the Bob and Mary Jane Engman Foundation (F.H.G.), the Volkswagen Foundation (N.A.) and the National Natural Science Foundation of China (31430020 and 31621092, H.L.).
Nature thanks Tatiana Kutateladze and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Structures of 5-HT and 5-PT in transamidation reactions. b, Immunofluorescence of intracellular 5-PT in HeLa cells (bottom) after exogenous application of the molecule (versus vehicle, top). Intracellular 5-PT was imaged in fixed cells after chemical labelling with Alexa Fluor 488 azide. DAPI was used as a nuclear co-stain. Results confirmed in at least two independent experiments. Scale bars, 500 μm. c, Cell versus lysate donor competition assays indicating that application of excess 5-HT to live HeLa cells, but not to processed lysates, before chemical labelling and 5-PT-based pulldowns results in loss of H3 signal after immunoprecipitation. Input and immunoprecipitate western blots are shown. d, Cellular fractionation analysis identifying H3 serotonylation post-immunoprecipitation in HeLa cell chromatin, but not in soluble nuclear or cytosolic fractions. Input and immunoprecipitate western blots are shown. H3 results confirmed in at least three independent experiments.
a, Western blot analysis of TGM2 expression across mouse brain and peripheral tissues (including blood) validate TGM2 as a ubiquitously expressed enzyme in mammals. Whereas two isoforms of TGM2 exist and are expressed differentially across tissues, the functions of these two variants remain unknown (n = 1 per brain region or organ tissue). b, Bioorthogonal labelling of 5-PT-modified H3 from HeLa cell extracts in the absence or presence of the transglutaminase inhibitor cystamine (4 mM). Input and immunoprecipitate western blots are shown. Results confirmed in at least two independent experiments. c, Quantitative PCR with reverse transcription (RT–qPCR) analysis of TGM genes comparing HeLa and HEK 293T cells (n = 1 per cell type). Insert western blot validating that TGM2 is differentially expressed across cell types. d, Bioorthogonal labelling of 5-PT-modified H3 from mock (non-transfected) versus nuclear expression of TGM2 in HEK 293T cell extracts. Catalytically dead TGM2(W241A) was used as a negative control. Input and immunoprecipitate western blots are shown. Results confirmed in at least two independent experiments. e, Recombinant histones (H3 versus H4) and reconstituted nucleosomes, as well as fibrinogen (positive control) and BSA (negative control), were subjected to radioactive serotonylation (5-3HT) reactions in vitro (n = 3), in the absence or presence of cystamine (one-way ANOVA: fibrinogen, F2,6 = 33.47, ***P = 0.0006; Dunnett’s post hoc test comparing +TGM2 and –TGM2, P < 0.001; BSA, F2,6 = 1.000, P = 0.4219; H3, F2,6 = 130.0, ****P < 0.0001; Dunnett’s post hoc test comparing +TGM2 and –TGM2, P < 0.001; H4, F2,6 = 0.7345, P = 0.5184; nucleosomes, F2,6 = 88.52, ****P < 0.0001; Dunnett’s post hoc test comparing +TGM2 and –TGM2, P < 0.001, and comparing +TGM2/+cystamine and –TGM2, P < 0.05). f, LC–MS/MS analysis of a TGM2-transamidated H3 peptide (H31–15), identifying Gln5 as a substrate for serotonylation. The y and b series indicate peptide fragments at amide bonds. Results confirmed in at least two independent experiments. g, TGM2 monoaminylation assays examining MDC transfer to recombinant wild-type H3 versus H3(Q5A), indicating Q5 as a dominant reactive amino acid substrate for H3. Coomassie staining of loading is shown. H3 results confirmed in at least three independent experiments. Data are mean ± s.e.m.
a, Representative semisynthesis of serotonylated histone H3 comprising (i) native chemical ligation between the serotonylated H31–13 α-thioester and a truncated histone H314–135, followed by (ii) cysteine alkylation to give K14 thialysine. b, c, RP-HPLC and mass spectrometry characterization of semisynthetic histone proteins, including H3K4me3 (b) and H3Q5Ser (c). Purified proteins were eluted from a C18 RP-HPLC column using a gradient of 0–73% solvent B (0.1% TFA in 9:1 acetonitrile:water) in solvent A (0.1% TFA in water) detecting absorption at 214 nm. Mass spectra of purified proteins were deconvoluted (inset) and observed versus calculated masses are shown. Results confirmed in at least three independent experiments. d, e, Validation of octamer (d) and mononucleosome (e) assembly post-semisynthesis of unmodified, K4me3 and Q5ser proteins. Results confirmed in at least three independent experiments. f, TGM2 monoaminylation assays on unmodified versus H3K4me3 nucleosomes using biotin cadaverine in place of 5-HT (n = 3, two-tailed Student’s t-test; t4 = 0.500, P = 0.64). H4 is provided as a loading control. g, MLL1-complex methyltransferase assays on unmodified versus Q5ser mononucleosmes (−SAM/−MLL1, +SAM/−MLL1 and +SAM/+MLL1; n = 3, two-tailed Student’s t-test, +/+ versus +/+; t4 = 0.3100, P = 0.76). Data are mean ± s.e.m.
a, Synthesis of peptide antigens on 2-Cl trityl resin by (i) iterative Fmoc solid-phase peptide synthesis incorporating Fmoc–Glu(OAII)-OH at position 5 and either Fmoc–Lys(Boc)-OH or Fmoc–Lys(Me3)-OH at position 4, (ii) followed by Pd(0) deallylation, (iii) 5-HT coupling and (iv) acidolytic cleavage from the resin and global deprotection. Side-chain protecting groups are omitted for clarity. b, Peptide dot blot titrations testing the H3Q5ser antibody’s reactivity against unmodified versus Q5ser peptides; note that linear signal was observed only with the Q5ser peptide. Direct blue staining was used to control for peptide loading. c, Western blot analysis of TGM2 serotonylation assays on unmodified mononucleosomes, which reveals that the H3Q5ser antibody only detects signal when the nucleosomes have been transamidated with 5-HT. Direct blue staining was used to control for protein loading. d, Peptide competition western blot analysis of lysates from RN46A-B14 cells indicating the specificity of the H3Q5ser antibody. e, Peptide dot blot titrations testing the H3K4me3Q5ser antibody’s reactivity against various peptides; note that linear signal was observed only with the K4me3Q5ser peptide. Direct blue staining was used to control for peptide loading. f, Western blot analysis of TGM2 serotonylation assays on monomeric EPL K4me3 H3, revealing that the H3K4me3Q5ser antibody only detects signal when the K4me3 histone has been transamidated with 5-HT. Direct blue staining was used to control for protein loading. g, Peptide competition western blot analysis of lysates from RN46A-B14 cells indicating the specificity of the H3K4me3Q5ser antibody in vivo; note that a minimum of 20 μg protein was loaded for all other comparisons made throughout the study. h, Peptide competition western blot analysis of lysates from hPSC-derived 5-HT neurons indicating the specificity of the H3K4me3Q5ser antibody in human cells. For experiments in b–h, similar results were confirmed in at least two independent experiments or assays.
LC–MS/MS analysis of endogenous and synthetic (N-terminally labelled with D5) H3Q5ser or H3K4me3Q5ser peptides in RN46A-B14 cells (post-differentiation, top) and brain (mouse DRN, bottom) following immunoprecipitation using our H3Q5ser and H3K4me3Q5ser antibodies, respectively. a, Liquid chromatography–mass spectrometry (LC–MS, MS1) chromatograms showing elution profiles for the samples including the endogenous peptide versus a synthetic peptide standard. Top to bottom: (i) total ion current chromatogram depicting the summed intensity of all signals; (ii) base peak chromatogram showing the intensity of the top signal; (iii) extracted ion chromatogram of the m/z of the endogenous peptide; (iv) extracted ion chromatogram of the m/z value of the synthetic, heavy-labelled peptide. b, MS/MS (MS2) spectrum (aligned by m/z, and magnified where indicated on either side of the precursor ion) of the H3Q5ser and H3K4me3Q5ser peptides obtained by HCD fragmentation. b+ and y+ fragment ions were annotated manually, and the top insets show good coverage. Results confirmed in at least two independent experiments (RN46A-B14 cells pre- versus post-differentiation and/or in mouse brain).
a, Immunofluorescence of H3K4me3Q5ser in brain (DRN) with or without peptide competition as indicated. DAPI was used as a nuclear co-stain and a secondary-antibody-only control is also included. Scale bars, 100 μm. b, Immunofluorescence of H3K4me3Q5ser in DRN (no peptide block) revealing a euchromatic distribution pattern in the nucleus with near-total exclusion from DAPI-rich chromocentres typical of neurons. Scale bars, 10 μm. c, Immunofluorescence images of H3K4me3Q5ser in DRN, counterstained with DAPI, NeuN (a neuronal cell marker) and TPH2 (a marker of serotonergic neurons). Merged images reveal that H3K4me3Q5ser is not only expressed in serotonergic neurons (NeuN+TPH2+, yellow circles) but also in non-serotonergic neurons (NeuN+TPH2–, white circles) and in non-neuronal cells (NeuN–TPH2–, red circles). Aq, aqueduct. Scale bars, 100 μm. d, Western blot validation of TPH2 knockout (KO) in DRN of TPH1 and TPH2 KO mice. Direct blue was used to control for loading. e, Immunofluorescence validation of 5-HT depletion in DRN of TPH1 and TPH2 knockout mice versus wild-type littermate controls. Scale bars, 100 μm. For all immunofluorescence experiments (a–e), results were confirmed in at least two independent experiments. f, Western blot validation that TPH1 and TPH2 knockout results in loss of H3K4me3Q5ser signal in DRN (n = 11 wild type versus n = 7 TPH1 and TPH2 knockout, two-tailed Student’s t-test; t16 = 3.425, **P = 0.0035); no effects on H3K4me3 or total H3 expression were observed. Data are mean ± s.e.m.
Extended Data Fig. 7 H3K4me3Q5ser positively correlates with gene expression in developing mouse brain.
a, RT–qPCR analysis of Tph2 gene expression in embryonic mouse brain (E9.5 (n = 6) versus E17.5 (n = 8), two-tailed Student’s t-test; t12 = 3.100, **P = 0.0092). Actb was used as a normalization control. b, Quantitative western blot analysis of H3K4me3 and H3K4me3Q5ser expression in embryonic mouse brain at E9.5 versus E17.5 (two-tailed Student’s t-test, two-sided, n = 3 independent biological replicates per age; K4me3: t4 = 3.168, *P = 0.0339; K4me3Q5ser: t4 = 4.277, *P = 0.0129). H3 was used as a loading control. c, MACS v.2.1.1-based peak-calling for H3K4me3 versus H3K4me3Q5ser ChIP-seq data in embryonic mouse brain at E17.5 (FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons; n = 3 independent biological replicates per age). d, Analysis of H3K4me3Q5ser differential enrichment comparing E17.5 and E9.5 embryonic mouse brain using diffReps (FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons; n = 3 independent biological replicates per age). Pie chart indicates distribution of genic differential enrichment events for the mark comparing promoter and gene body regions. e, Overlap of differential enrichment events at protein-coding genes for H3K4me3 and H3K4me3Q5ser versus differential gene expression during mouse brain development indicates significant associations between the two marks, as well as positive correlations with gene expression. f, Odds-ratio analysis (using Fisher’s exact tests) of overlapping genes displaying differential H3K4me3Q5ser enrichment versus differential gene expression (n = 3 independent biological replicates per age, FDR <0.05 cut-off applied after adjusting for multiple comparisons) indicating positive correlations between mark enrichment and permissive gene expression during development. Insert numbers indicate respective P values for associations, followed by the number of protein-coding genes overlapping per significant category. Data are mean ± s.e.m.
Extended Data Fig. 8 Functional validations of the role of H3 serotonyl in permissive gene expression.
a, Peptide dot blot titrations testing the ability of the H3K4me3 antibody to detect the mark in the absence or presence of Q5ser; note that Q5ser does not occlude antibody recognition of the mark. Results confirmed in at least two independent experiments. b, Peptide competition qChIP validation of H3K4me3Q5ser enrichment at two target loci (Actb and Vim) identified via ChIP-seq in RN46A-B14 cells (undifferentiated, n = 1 per group). c, Overlap of differential enrichment events at protein-coding genes for H3K4me3 and H3K4me3Q5ser versus differential gene expression in response to RN46A-B14 cell differentiation indicates positive correlations between H3K4me3Q5ser gene expression in the absence of significant changes in H3K4me3 enrichment (FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons. n = 3 independent biological replicates per differentiation state per antibody). d, Recombinant methylation assays (MLL1-complex-mediated) on wild-type H3 versus H3(Q5A), followed by western blot for H3K4me3, indicating that the Q5A mutation does not affect methylation capacity at K4. Direct blue was used to control for protein loading. Results confirmed in at least two independent experiments. e, Odds-ratio analysis (using Fisher’s exact tests) of overlapping genes displaying differential H3K4me3Q5ser enrichment (or not) versus differential gene expression (DEx, n = 5 (H3.3(WT)) versus n = 6 (H3.3(Q5A)) independent biological replicates per virus), FDR <0.05 cut-off applied after adjusting for multiple comparisons) comparing differentiated RN46A-B14 cells expressing either H3.3(WT) or H3.3(Q5A). Insert numbers indicate respective P values for associations, followed by the number of protein-coding genes overlapping per significant category. f, Heat map of RNA-seq data comparing undifferentiated and differentiated (n = 5 H3.3(WT) versus n = 6 H3.3(Q5A)-expressing) RN46A-B14 cells using normalized RNA expression values (averaged between replicates) to generate z-scores for each row. The genes that displayed differential enrichment during differentiation are represented, along with altered gene expression (see Fig. 3), and along with opposing regulation in the context of H3.3(Q5A). These genes were found to significantly enrich for pathways associated with axon guidance signalling via KEGG analysis (see Supplementary Table 28). g, RT–qPCR analysis of candidate gene expression in RN46A-B14 cells (n = 5 DMSO versus n = 6 LDN 27219/Tgm2i; one-tailed Student’s t-test; Iws1: t9 = 2.559, *P = 0.0154; Sema3e: t8 = 3.982, **P = 0.0020; Robo1: t9 = 3.344, **P = 0.0043; Srgap1: t8 = 3.312, **P = 0.0053; Nrp1: t9 = 2.452, *P = 0.0183; Zmat3: t9 = 3.820, **P = 0.0020; Actg1: t8 = 7.836, ****P < 0.0001; Reln: t9 = 2.209, *P = 0.0273; Qser1: t8 = 2.513, *P = 0.0181; Ppp3ca: t8 = 2.418, *P = 0.0210; Arid5b: t9 = 1.754, *P = 0.0567; Pten: t9 = 1.936, *P = 0.0424). Actb was used as a normalization control. h, Immunofluorescence of RN46A-B14 cells infected during differentiation with lentiviruses expressing either wild-type H3.3–HA or H3.3(Q5A)–HA. Results confirmed at least three independent coverslips per viral treatment. Scale bars, 20 μm. i, Neurite outgrowth analysis examining RN46A-B14 cellular length post-differentiation (n = 54 H3.3(WT) versus n = 44 H3.3(Q5A)-expressing cells; two-tailed Student’s t-test, t96 = 4.664, ****P < 0.0001). Data are mean ± s.e.m.
a, Western blot quantifications related to Fig. 4a (Student’s t-tests, two-sided) of modified H31–10 peptide immunoprecipitations from HeLa nuclear extracts (TAF2: n = 3 per peptide, t4 = 2.724, *P = 0.05; TAF3: n = 3 per peptide, t4 = 2.920, *P = 0.04; TAF5: n = 3 per peptide, t4 = 3.685, *P = 0.02; TAF7: n = 3 per peptide, t4 = 8.885, ***P = 0.0009; TBP: n = 4 per peptide, t6 = 5.383, **P = 0.0017). b, Modified H31–10 peptide immunoprecipitations against purified TFIID from Flag-tagged TBP expressing soluble HeLa nuclear extracts (left, silver stain of the purified complex), followed by western blots (right). Inputs are provided (n = 1 per protein examined). c, Gene plot (ngs.plot) of TAF3–TFIID enrichment (fold change (expressed in log2) versus input) comparing signals pre- and post-differentiation (n = 3 independent biological replicates per differentiation states). d, IGV genome browser tracks of the Prrg4 locus for H3K4me3, H3K4me3Q5ser and TAF3–TFIID (versus DNA input) in RN46A-B14 cells pre- and post differentiation (example locus was chosen on the basis of MACS v.2.1.1- and diffReps-based statistical comparisons). Odds-ratio analysis (using Fisher’s exact tests) of overlapping genes displaying differential H3K4me3 or H3K4me3Q5ser enrichment (or those genes containing differential sites for both or neither of the two marks, FDR <0.05, FC >1.2) versus TAF3 peaks (e; n = 3 independent biological replicates per differentiation state, MACS v.2.1.1, FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons; normalized to respective DNA inputs) or differential TAF3–TFIID enrichment (f; n = 3 independent biological replicates per differentiation state, diffReps, FDR <0.05, FC >1.2 cut-offs applied after adjusting for multiple comparisons) post-differentiation. Insert numbers indicate respective P values for associations, followed by the number of protein-coding genes overlapping per significant category. g, Model of the effect of Q5ser on K4me3-mediated gene expression in vivo; our data suggest that the presence of combinatorial H3K4me3Q5ser alters interactions with certain K4me3 reader proteins, such as the TFIID complex, to potentiate or stabilize permissive gene expression in mammalian cells.
This file contains uncropped western blots and other gels, as well as images/quantification for each of the human PSC lines used to generate 5-HT neurons.
This file contains Supplementary Tables 1-34, containing processed ChIP- and RNA-seq data from cells and brain, as well as proteomic binding data and qPCR/qChIP primer sets.
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Farrelly, L.A., Thompson, R.E., Zhao, S. et al. Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature 567, 535–539 (2019). https://doi.org/10.1038/s41586-019-1024-7
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Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced transcriptional and behavioral plasticity in male rats