Metabolic regulation of gene expression by histone lactylation

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 diseases^1,2. 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.

Extended Data Figure 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–A4: Dots contain 1, 4, 16, and 64 ng, respectively, of a peptide library containing a lactylated lysine residue. B1–B4: Dots contain 64 ng of a peptide library containing an unmodified (K), acetylated (Kac), propionylated (Kpr), and butyrylated (Kbu) lysine residue, respectively. C1–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, where C is cysteine, × 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 2-fold or 10-fold 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, HeLa and MDA-MB-231 cells, respectively, treated with indicated chemicals. k, MS/MS spectra of an isotopically labeled histone Kla peptide identified from MCF-7 cells cultured with 10 mM isotopic (¹³C₃) sodium L-lactate for 24 hours. a–k represent three independent experiments.

Extended Data Figure 2.. Histone Kla is modulated by glycolysis pathway.

a–c, A549 (a), HeLa (b), and MEF (c) cells were cultured with indicated doses of glucose for 24h, without pyruvate. Histone Kla and Kac were analyzed by immunoblots using indicated antibodies. d, MS/MS spectra of a ¹³C₆-glucose labeled histone Kla peptide and its unlabeled 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 6h, 12h, 24h, and 48h, with or without 10mM DCA. i–k, Histone Kla and Kac levels were analyzed by immunoblots using whole cell lysates from MCF-7, HepG2 and MEF cells exposed to 25 mM glucose for indicated time points. l, m, SILAC-MS/MS quantification of histone Kla and Kac marks from MCF-7 cells, comparing Rotenone (10 nM, 24h) vs DMSO (l), DCA (10mM, 24h) vs PBS (m). SILAC ratio was normalized to protein abundance. Each dot in the scatter dot plot represents one identified peptide from core histone. Graphs show 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 Welch’s t test (Two tailed). a–d, i–k, Data represent three independent experiments. e–h, Data represent two independent experiments.

Extended Data Figure 3.. Histone Kla is induced by hypoxia.

a–d, Antibody specificity was analyzed by dot blot assay. un: unmodified lysine; ac: acetyl lysine; pr: propionyl lysine; bu: butyryl lysine; hib; 2-hydroxyisobutyryl lysine; bhb: β-hydroxybutyryl lysine; cr: crotonyl lysine; succ: succinyl lysine. The Kla library was the same as the one used in Extended Data Fig. 1f. 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 RAW264.7 cells in response to hypoxia (1% oxygen) at the indicated time. i, j, Intracellular lactate (i) and histone Kla levels (j) were measured in MCF-7 cells comparing normoxia, hypoxia (1% oxygen, 24hrs), hypoxia in the presence of 10mM oxamate or 10mM DCA. k, l, Intracellular lactate (k) and histone Kla levels (l) were measured comparing LDHA−/−, LDHB−/−, or LDHA−/− & LDHB−/− with wildtype HepG2 cells. Graphs show mean with s.e.m. from three biological independent samples; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. a–d, g, h, k, and l represent three independent experiments.

Extended Data Figure 4.. Histone Kla is induced during M1 macrophage polarization.

a–f, Quantitative proteomic analysis of histone extracts from M0 and M1 macrophages (BMDM) cultured in the presence of U-¹³C₆ glucose for 3h, 6h, 12h, and 24h, or with 10uM GNE-140 (LDHA/B inhibitor) for 24h. g, Histone Kla and Kac levels were analyzed by immunoblots 24h-post LPS/IFNγ activation, with or without replenishing fresh media (containing LPS/IFNγ or not) every 4h. h, BMDM cells were stimulated with PBS (M0), LPS/IFNγ (M1), and interleukin-4 (M2) for 24 hours, respectively. Intracellular lactate was measured using a lactate colorimetric kit. Graphs show mean with 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 10-fold excess of corresponding peptides. k, H3K18la antibody specificity was shown by full immunoblot using total lysate from MCF-7 cells with or without 10mM sodium L-lactate treatment for 24h. 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 (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 study, representing data from single experiment. a–f, Data represent two independent experiments. g, i–k, Data represent three independent experiments.

Extended Data Figure 5.. Histone Kla specific genes are associated with late activated M2-like gene expression.

a, b, Heatmaps showing expression kinetics of total genes (a) and H3K18la-specific genes (b) during M1 macrophage polarization. N=4 biological replicates. The color key represents log2 transformed fold change relative to the mean of each row. Arrows next to the heatmaps refer to late activated genes (16–24h) 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 FC>=1 and H3K18ac log2 FC<=0.5) and late gene activation. d, Gene Ontology analysis (biological processes) of H3K18la-specific genes. Statistical significance was determined by the modified Fisher Exact P-Value (EASE score) using DAVID Bioinformatics Resources 6.8, n=1223 genes. e-j, BMDM cells were infected with indicated gram-negative bacteria for 24 hours. Intracellular lactate (e) and histone Kla levels (f) were measured 24h after bacterially 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 analyzed by RT-qPCR for indicated time points after bacterially challenge. k, Activities of iNOS and ARG1 were analyzed by immunoblots and commercialized kits from BMDMs activated by the indicated stimuli. Graphs show mean with s.e.m. from three biological replicates. f and k represent three independent experiments.

Extended Data Figure 6.. Histone Kla levels are positively correlated with Arg1 expression in tumor associated macrophages.

Tumor-associated macrophages (TAM) and Peritoneal macrophages (PMs) purify were confirmed by flow cytometry using CD11b (ThermoFisher Scientific, 25–0112) and F4/80 (ThermoFisher Scientific, 17–4801) antibodies (a). Data were quantified by FlowJo v.10.4.1. Histone Kla and Kac levels were analyzed by immunoblots (b), intracellular lactate was measured using a lactate colorimetric assay kit (c), and gene expression of Arg1 and Vegfa were analyzed by RT-qPCR (d, e) from FACS-sorted peritoneal macrophages (PM), TAMs within the tumor from LLC and B16 tumors. c–e, Graphs show mean with s.e.m. n=5 biological independent samples; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. a and b represent five independent mice.

Extended Data Figure 7.. Decreased lactate production lowered histone Kla levels and Arg1 expression during M1 polarization.

a, b, Genotyping of Ldha^fl/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 analyzed using a lactate colorimetric assay kit and global histone Kla levels (i) were measured by immunoblots 24h-post M1 polarization. Inhibitors were treated 30min after M1 polarization. Gene expression was analyzed by RT-qPCR at indicated time points after M1 polarization (j–m). Graphs show mean with s.e.m. from three biological replicates. g, statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. a, b, c and i represent three independent experiments.

Extended Data Figure 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. Results are shown as mean ± s.e.m. from four biological replicates. RPKM: Reads Per Kilobase of transcript per Million mapped reads (RPKM). e, Number of lactate-activated H3K18la-specific genes at indicated times are shown in a Venn diagram. f, Gene Ontology analysis (biological processes) of LA-induced H3K18la-specific genes at 16 and 24 h post-M1 polarization. Statistical significance was determined by the modified Fisher Exact P-Value (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 Holm-Sidak method. h, H3K18la occupancy at Vegfa promoter was analyzed 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 post-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. Graphs show mean with s.e.m. i, Data represent three independent experiments.

Extended Data Figure 9.. Histone Kla directly stimulates gene transcription from recombinant chromatin in vitro.

a, Protocol for assembly, modification and transcription of chromatin templates. b, P300 catalyzes histone lactylation in a p53-dependent manner. c, Histone lactylation directly stimulates p53-dependent transcription from recombinant chromatin. d, H3 and H4 KR mutations eliminate p300-dependent transcriptional activation by p53. Recombinant chromatin was assembled with wildtype or H3KR, H4KR, H2AKR or H2BKR mutant histones as indicated. e, HEK293T cells were transfected with vector or FLAG-p300 plasmid. At 48-hr post-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) where 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. UV detection wavelength was fixed at 214 and 254 nm. k, MALDI-Mass spectrometry analysis of L-lactyl-CoA. b–g, i–k, Data represent three independent experiments.

Figure 1.. Identification and validation of histone Kla.

a, Illustration of Kla structure. b, MS/MS spectra of a lactylated histone peptide (H3K23la) derived from MCF-7 cells (in vivo), its synthetic counterpart, and their mixture. b-ion refers to the amino-terminal parts of the peptide and y-ion refers to the carboxy-terminal parts of the peptide. Data represent two independent experiments. c, Illustration of histone Kla sites identified in human and mouse cells.

Figure 2.. Lactate regulates histone Kla.

Intracellular lactate (a and d) and histone Kla levels (b and c) were measured from MCF-7 cells cultured in different glucose concentrations or different 2-DG concentrations in the presence of 25mM glucose for 24 hours. Lactate was measured by a lactate colorimetric kit; n=3 biological replicates; statistical significance was determined using one-way ANOVA followed by Sidak’s multiple comparisons test. Immunoblots was carried out using acid-extracted histone samples. The pan anti-Kla and anti-Kac immunoblots indicate molecular weights between 10kD and 15kD. e, Regulation of glycolysis and lactate production by diverse metabolic modulators. f, Intracellular lactate levels were measured in MCF-7 cells treated with indicated glycolysis modulators for 24 hours. N=3 biological replicates; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. g-i, Immunoblots of acid extracted histones (Rotenone and DCA) or whole cell lysates (Oxamate) from MCF-7 cells in response to different glycolysis modulators. j, Intracellular lactate levels were measured in MCF-7 cells in response to hypoxia. N=4 biological replicates; statistical significance was determined using unpaired t test (Two-tailed). k, Immunoblots of acid extracted histones from MCF-7 cells under hypoxia (1% oxygen) for indicated time points. a, d, f, j, Graphs show mean with s.e.m. b, c, g, h, i, k, Data represent three independent experiments.

Figure 3.. Elevated Histone Kla during M1 macrophage polarization is associated with M2-like gene activation.

a-c, Bone marrow-derived macrophages (BMDMs) were activated with LPS+IFNγ. Intracellular lactate (a) was measured using a lactate colorimetric kit. N=3 biological replicates; statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Histone acylations were analyzed by immunoblots using whole cell lysates (b, c). ImageJ was used for quantification; n=3 technical replicates. Data represent two independent experiments. d, BMDM cells were stimulated with PBS (M0), LPS+IFNγ (M1), and interleukin-4 (M2) for 24 hours, respectively. Acid-extracted histones were used for immunoblots. e, f, Scatter plot (e) and bar plot (f) showing genes with promoters marked by exclusively elevated H3K18la (H3K18la-log₂[M1/M0] ≥1 and H3K18ac-log₂[M1/M0] ≤0.5, H3K18la-specific), elevated in both H3K18la and H3K18ac (H3K18la-log₂[M1/M0] ≥1 and H3K18ac-log₂[M1/M0] ≥0.5, shared), or exclusively elevated H3K18ac (H3K18ac-log₂[M1/M0] ≥1 and H3K18la-log₂[M1/M0] ≤0.5, H3K18ac-specific). g, h, Heat maps showing gene expression kinetics (using Reads Per Kilobase of transcript per Million mapped reads (RPKM) values from RNA-seq) of exemplar inflammatory genes (g) and H3K18la-specific genes (h). The color key represents log2 transformed fold change relative to gene expression at 0h. N=4 biological replicates. i, j, BMDM cells were infected with indicated Gram-negative bacteria or LPS, respectively. Histone Kla levels were measured by immunoblots (i) at 24h after bacterial challenge. “+” indicates lower dose and “++” indicates higher dose. Gene expression were analyzed by RT-qPCR (j) at indicated time points post bacterial challenge. N=3 biological replicates. k, Protein levels of iNOS and ARG1 were analyzed by immunoblots from BMDMs activated by the indicated stimuli. a, b, c, j, Graphs show mean with s.e.m. d, i, k, Data represent three independent experiments.

Figure 4.. Lactate activates M2-like gene expression through histone Kla.

a–d, Decreased lactate production in LDHA deficient (myeloid specific Ldha^−/−) BMDM cells resulted in lowered histone Kla levels and Arg1 expression during M1 polarization. Intracellular lactate levels were measured using a lactate colorimetric kit (a) and global histone Kla levels were measured by immunoblots (b) 24h-post M1 polarization. c, Gene expression were analyzed by RT-qPCR at indicated time points after M1 polarization. a–c, N=3 biological replicates. d, H3K18la occupancy was analyzed by ChIP-qPCR 24h-post M1 polarization. Data represent three technical replicates from pooled samples. e–h, Exogeneous lactic acid (25 mM) was added to BMDM cells 4 h post-M1 polarization (LPS+IFNγ), and cells were collected at indicated time points post-M1 polarization for intracellular lactate measurement (e), histone Kla immunoblot analysis (f), gene expression analysis (g) and H3K18la occupancy analysis by ChIP-qPCR (h). e, N=3 biological replicates, f, Data represent three independent experiments. g, RPKM: Reads Per Kilobase of transcript per Million mapped reads (RPKM). N=4 biological replicates. h, Data represent three technical replicates from pooled samples. a, c, d, e, g, h, Graphs show mean with s.e.m; statistical significance was determined using Multiple t tests corrected using Holm-Sidak method (a, c, e, g).