Abstract
The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases1,2. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous ‘lactate clock’ in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.
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Data availability
The ChIP–seq and RNA–seq data have been made available at the Gene Expression Omnibus (GEO) repository under the accession number GSE115354. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE31 partner repository with the dataset identifier PXD014870. All other data are available from the authors upon reasonable request.
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Acknowledgements
HEK293T p300 knockout cells were provided by X. Li. We thank S. Khochbin for brainstorming and critical reading of this manuscript. We thank K. Delaney and all other members of the Zhao and Becker laboratories for discussions and technical support. This work was supported by the University of Chicago, Nancy and Leonard Florsheim family fund (Y.Z.), NIH grants R01GM115961, R01DK118266 (Y.Z.), R01DK102960, R01HL137998 (L.B.), R01CA129325, R01DK071900 (R.G.R.), and NSF1808087 (Y.G.Z.).
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Contributions
Y.Z. conceived the project and developed the general ideas and research strategy. D.Z., L.B. and Y.Z. designed the experimental approach and composed the manuscript. D.Z. performed most of the experiments. Z.T. and R.G.R. carried out in vitro chromatin-based transcription experiments. Y.W., H.H., W.L., J.D., L.D., S.K., S.L. and M.P.-N. contributed to mass spectrometry-related experiments and analysis; R.H., Z.Y. and B.R. performed the library construction and next-generation sequencing for ChIP–seq and RNA-seq; M.H. and Y.G.Z. synthesized l-lactyl-CoA. H.H. and D.Z. analysed ChIP–seq and RNA-seq data. G.Z. provided all primary BMDM cell cultures. D.C. and H.A.S. carried out the bacterial infection experiments; C.C. carried out TAM experiments.
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Competing interests
Y.Z. is a co-founder, board member, and advisor to PTM Bio Inc. L.B. is a co-founder and CSO of rMark Bio Inc., and a founder and CEO of Onchilles Pharma Inc.
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Peer review information Nature thanks Luke O’Neill, Kathryn Wellen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 Validation of histone lysine lactylation.
a, c, e, Extracted ion chromatograms from HPLC–MS/MS analysis of histone Kla peptides derived from cultured cells (in vivo), the synthetic counterparts, and their mixtures. b, d, MS/MS spectra of histone Kla peptides derived from in vivo, the synthetic counterparts, and their mixtures. f, g, Antibody specificity tests by dot blot and competition assay. f, Dot blot was carried out with a pan anti-Kla antibody and the following peptide libraries. A1, A2, A3 and A4: dots contain 1, 4, 16 and 64 ng, respectively, of a peptide library containing a lactylated lysine residue. B1, B2, B3 and B4: dots contain 64 ng of a peptide library containing an unmodified (K), acetylated (Kac), propionylated (Kpr) and butyrylated (Kbu) lysine residue, respectively. C1, C2, C3 and C4: dots contain 64 ng of a peptide library containing a β-hydroxybutyrylated (Kbhb), 2-hydroxyisobutyrylated (Khib), crotonylated (Kcr) and malonylated (Kma) lysine residue, respectively. The libraries contained a mixture of CXXXKXXXX peptides, in which C is cysteine, X is a mixture of all 19 amino acids except for cysteine, and K is lysine with or without the indicated modifications. g, Competition was carried out by incubating the pan anti-Kla antibody with a twofold or tenfold excess of the indicated peptide libraries before the dot blot assay. h–j, Exogenous lactate boosts histone Kla levels. Immunoblot analysis of histone Kla and Kac from human MCF-7 cells treated with indicated doses of l-lactate (h), and from human HeLa (i) and MDA-MB-231 (j) cells treated with 25 mM sodium chloride, sodium lactate or sodium acetate. k, MS/MS spectra of an isotopically labelled histone Kla peptide identified from MCF-7 cells cultured with 10 mM isotopic (13C3) sodium l-lactate for 24 h. Data in a–k represent three independent experiments.
Extended Data Fig. 2 Histone Kla is modulated by the glycolysis pathway.
a–c, A549 (a), HeLa (b) and mouse embryonic fibroblast (MEF) (c) cells were cultured with indicated doses of glucose for 24 h, without pyruvate. Histone Kla and Kac were analysed by immunoblots using indicated antibodies. d, MS/MS spectra of a U-13C6-glucose labelled histone Kla peptide and its unlabelled counterpart from MCF-7 cells. e–h, Quantitative proteomic analysis of histone extracts from MCF-7 cells cultured in the presence of U-13C6 glucose for 6 h, 12 h, 24 h and 48 h, with or without 10 mM DCA. i–k, Histone Kla and Kac levels were analysed by immunoblots using whole-cell lysates from MCF-7, HepG2 and MEF cells exposed to 25 mM glucose for the indicated times. l, m, SILAC–MS/MS quantification of histone Kla and Kac marks from MCF-7 cells, comparing rotenone (10 nM, 24 h) versus DMSO treatment (l), and DCA (10 mM, 24 h) versus PBS treatment (m). SILAC ratio was normalized to protein abundance. Each dot in the scatter dot plot represents one identified peptide from core histone. Data are mean ± s.e.m. l, Kac: 1.121 ± 0.05084, n = 31; Kla: 1.599 ± 0.139, n = 25. m, Kac: 1.038 ± 0.03813, n = 49; Kla: 0.6627 ± 0.06376, n = 24. Statistical significance was determined using two-tailed Welch’s t-test. Data in a–d, i–k represent three independent experiments. Data in e–h represent two independent experiments
Extended Data Fig. 3 Histone Kla is induced by hypoxia.
a–d, Antibody specificity was analysed by dot blot assay. ac, acetyl lysine; bhb, β-hydroxybutyryl lysine; bu, butyryl lysine; cr, crotonyl lysine; hib, 2-hydroxyisobutyryl lysine; la, lactyl lysine; pr, propionyl lysine; succ, succinyl lysine; un, unmodified lysine. Kla library contains a mixture of CXXXKlaXXXX peptides, in which C is cysteine, X is a mixture of all 19 amino acids except for cysteine, and Kla is lactyl lysine. e, f, SILAC-MS/MS quantification of histone Kla and Kac marks from MCF-7 cells, comparing hypoxic (1% oxygen for 24 h) and normoxic conditions. SILAC ratio was normalized to protein abundance. g, h, Immunoblots of histone Kla and Kac from human HeLa and mouse RAW 264.7 cells in response to hypoxia (1% oxygen) at the indicated time. i, j, Intracellular lactate levels (i) and histone Kla levels (j) were measured in MCF-7 cells comparing normoxia, hypoxia (1% oxygen, 24 h) and hypoxia in the presence of 10 mM oxamate or DCA. k, l, Intracellular lactate levels (k) and histone Kla levels (l) were compared in LDHA−/−, LDHB−/−, LDHA−/−LDHB−/− or wild-type (WT) HepG2 cells. Data are mean and s.e.m. from three biological independent samples; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data in a–d, g, h, k and l represent three independent experiments.
Extended Data Fig. 4 Histone Kla is induced during M1 macrophage polarization.
a–f, Quantitative proteomic analysis of histone extracts from M0 and M1 macrophages (BMDMs) cultured in the presence of U-13C6-glucose for 3, 6, 12 and 24 h, or with 10 μM GNE-140 (LDHA/B inhibitor) for 24 h. g, Histone Kla and Kac levels were analysed by immunoblots 24 h after activation by LPS and IFNγ, with or without replenishing fresh media (containing LPS and IFNγ or not) every 4 h. h, BMDM cells were stimulated with PBS (M0), LPS plus IFNγ (M1), and IL-4 (M2) for 24 h. Intracellular lactate was measured using a lactate colorimetric kit. Data are mean and s.e.m. from three biological independent samples; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. i, j, Antibody specificity was evaluated by ChIP–qPCR. Competition was carried out by pre-incubating the indicated antibodies with a tenfold excess of corresponding peptides. k, H3K18la antibody specificity was shown by full immunoblot using total lysate from MCF-7 cells with or without 10 mM sodium l-lactate treatment for 24 h. l, H3K18la and H3K18ac are enriched in promoter regions. The promoter was defined as regions ± 2 kb around known transcription start sites. m, n, H3K18la and H3K18ac correlate with steady-state mRNA levels. The average ChIP signal intensity (read count per million mapped reads) for indicated antibodies is shown for genes with different expression levels (the top 25%, the second 25%, the third 25%, and the bottom 25% of RNA-seq counts). o, p, IGV tracks for Arg1 and Crem from ChIP–seq analysis, representing data from single experiment. Data in a–f represent two independent experiments. Data in g, i–k represent three independent experiments.
Extended Data Fig. 5 Histone Kla-specific genes are associated with late activated M2-like gene expression.
a, b, Heat maps showing expression kinetics of total genes (a) and H3K18la-specific genes (b) during M1 macrophage polarization. n = 4 biological replicates. The colour key represents log2-transformed fold change relative to the mean of each row. Arrows next to the heatmaps refer to late activated genes (16–24 h) from H3K18la-specific or total genes used for contingency test. c, Contingency table analysis (Fisher’s exact tests) shows the relation between specific H3K18la enrichment (H3K18la log2-transformed fold change ≥ 1 and H3K18ac log2-transformed fold change ≤ 0.5) and late gene activation. d, Gene Ontology analysis (biological processes) of H3K18la-specific genes. Statistical significance was determined by modified Fisher’s exact test (EASE score) using DAVID bioinformatics resources 6.8; n = 1,223 genes. e–j, BMDM cells were infected with indicated Gram-negative bacteria for 24 h. Intracellular lactate (e) and histone Kla levels (f) were measured 24 h after bacterial challenge. e, n = 3 biological replicates; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. g–j, Gene expression was analysed by RT–qPCR at indicated time points after bacterial challenge. k, Activities of iNOS and ARG1 were analysed by commercialized kits from BMDMs activated by the indicated stimuli. Data are mean and s.e.m. from three biological replicates. Data in f and k represent three independent experiments.
Extended Data Fig. 6 Histone Kla levels are positively correlated with Arg1 expression in TAMs.
a, The purity of TAMs and peritoneal macrophages (PMs) was confirmed by flow cytometry using CD11b and F4/80 antibodies. b–e, Data were quantified by FlowJo v.10.4.1. Histone Kla and Kac levels were analysed by immunoblots (b), intracellular lactate was measured using a lactate colorimetric assay kit (c), and gene expression of Arg1 and Vegfa were analysed by RT–qPCR (d, e) from FACS-sorted peritoneal macrophages and TAMs within the tumour from LLC and B16 tumours. Data in c–e are mean and s.e.m. n = 5 biological independent samples; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data in a and b represent five independent mice.
Extended Data Fig. 7 Decreased lactate production lowered histone Kla levels and Arg1 expression during M1 polarization.
a, b, Genotyping of Ldhafl/fl × LysM-Cre+/− mice. c, Genotype validation by LDHA immunoblot analysis. d–g, Gene expression analysis of cytokines by RT–qPCR at indicated time points after M1 polarization. h–m, Intracellular lactate levels (h) were analysed using a lactate colorimetric assay kit and global histone Kla levels (i) were measured by immunoblots 24 h after M1 polarization. Inhibitors were treated 30 min after M1 polarization. Gene expression was analysed by RT–qPCR at indicated time points after M1 polarization (j–m). Data are mean and s.e.m. from three biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data in a–c and i represent three independent experiments.
Extended Data Fig. 8 Exogenous lactate activates M2-like gene expression through histone Kla.
a–d, Exogenous lactate (LA) does not interfere with gene expression of inflammatory cytokines. Data are mean ± s.e.m. from four biological replicates. e, Number of lactate-activated H3K18la-specific genes at indicated times are shown in a Venn diagram. f, Gene Ontology analysis (biological processes) of lactate-induced H3K18la-specific genes at 16 and 24 h after M1 polarization. Statistical significance was determined by modified Fisher’s exact test (EASE score) using DAVID bioinformatics resources 6.8; n = 112 genes. g, Vegfa was induced by exogenous lactate during M1 macrophage polarization; n = 4 biological replicates; statistical significance was determined using multiple t-tests corrected using the Holm–Sidak method. h, H3K18la occupancy at the Vegfa promoter was analysed by ChIP–qPCR at indicated time and treatment; data represent three technical replicates from pooled samples. i–m, HIF1a is not required for histone Kla-mediated Arg1 induction during M1 polarization. i, Immunoblot of HIF1a at indicated time points after M1 polarization. j, Illustration of genomic loci targeted by Arg1 and Vegfa ChIP–qPCR primers. HRE indicates regions containing the putative HIF1a binding motif ‘ACGTG’. k–m, ChIP–qPCR analysis of HIF1a binding to indicated genomic locations; data represent three technical replicates from pooled samples. Data are mean and s.e.m. Data in i represent three independent experiments.
Extended Data Fig. 9 Histone Kla directly stimulates gene transcription from recombinant chromatin in vitro.
a, Protocol for assembly, modification and transcription of chromatin templates. b, P300 catalyses histone lactylation in a p53-dependent manner. c, Histone lactylation directly stimulates p53-dependent transcription from recombinant chromatin. d, H3 and H4 lysine-to-arginine (KR) mutations eliminate p300-dependent transcriptional activation by p53. Recombinant chromatin was assembled with wild-type or H3KR, H4KR, H2AKR or H2BKR mutant histones as indicated. e, HEK293T cells were transfected with vector or Flag-tagged p300 plasmid. At 48 h after transfection, whole-cell lysates were prepared and immunoblotted with indicated antibodies. f, g, Immunoblots of histone Kla and Kac levels in HCT116 (f) and HEK293T cells (g) in which p300 was genetically deleted. h–k, Quality control of synthesized l-lactyl-CoA. h, Illustration of l-lactyl-CoA structure. i, j, HPLC analysis of the synthesized l-lactyl-CoA. The UV detection wavelength was fixed at 214 and 254 nm. k, MALDI-mass spectrometry analysis of l-lactyl-CoA. Data in b–g and i–k represent three independent experiments.
Supplementary information
Supplementary Figure
Supplementary Figure 1 contains gel source data with marker size indications. The corresponding figures are indicated.
Supplementary Table
Supplementary Table 1: Histone Kla and Kac peptides from U-13C6-glucose labeling experiment identified by MS/MS, related to Fig. 2. Histones were extracted from MCF-7 cells cultured in the presence of 25mM U-13C6-glucose, digested with trypsin, and analyzed by HPLC/MS/MS. A list of 13C labeled and unlabeled histone Kla and Kac peptides were shown. See details in Methods.
Supplementary Table
Supplementary Table 2: H3K18la specific genes from ChIP-seq analysis, related to Fig. 3. A list of genes with promoters marked by exclusively elevated H3K18la (defined as H3K18la-log2[M1/M0] ≥1 and H3K18ac-log2[M1/M0] ≤0.5) in ChIP-seq analysis were shown.
Supplementary Table
Supplementary Table 3: H3K18la and H3K18ac shared genes from ChIP-seq analysis, related to Fig. 3. A list of genes with promoters marked by elevated in both H3K18la and H3K18ac (H3K18la-log2[M1/M0] ≥1 and H3K18ac-log2[M1/M0] ≥0.5) in ChIP-seq analysis were shown.
Supplementary Table
Supplementary Table 4: Total RNA-seq genes, related to Fig. 3. A list of total differentially expressed genes from RNA-seq were shown, n=4 independent biological replicates in each condition. Differential expression analysis was implemented using edgeR version 3.16.5 with FDR cutoff 0.05 (Benjamini-Hochberg).
Supplementary Table
Supplementary Table 5: Lactic acid activated H3K18la specific genes at 16-24h related to Fig. 4. A list of genes with promoters marked by exclusively elevated H3K18la (defined as H3K18la-log2[M1/M0] ≥1 and H3K18ac-log2[M1/M0] ≤0.5) and were induced by lactic acid at 16-24h were shown.
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Zhang, D., Tang, Z., Huang, H. et al. Metabolic regulation of gene expression by histone lactylation. Nature 574, 575–580 (2019). https://doi.org/10.1038/s41586-019-1678-1
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DOI: https://doi.org/10.1038/s41586-019-1678-1
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