Abstract
Emerging evidence suggests that epigenetic regulation is dependent on metabolic state, and implicates specific metabolic factors in neural functions that drive behaviour1. In neurons, acetylation of histones relies on the metabolite acetyl-CoA, which is produced from acetate by chromatin-bound acetyl-CoA synthetase 2 (ACSS2)2. Notably, the breakdown of alcohol in the liver leads to a rapid increase in levels of blood acetate3, and alcohol is therefore a major source of acetate in the body. Histone acetylation in neurons may thus be under the influence of acetate that is derived from alcohol4, with potential effects on alcohol-induced gene expression in the brain, and on behaviour5. Here, using in vivo stable-isotope labelling in mice, we show that the metabolism of alcohol contributes to rapid acetylation of histones in the brain, and that this occurs in part through the direct deposition of acetyl groups that are derived from alcohol onto histones in an ACSS2-dependent manner. A similar direct deposition was observed when mice were injected with heavy-labelled acetate in vivo. In a pregnant mouse, exposure to labelled alcohol resulted in the incorporation of labelled acetyl groups into gestating fetal brains. In isolated primary hippocampal neurons ex vivo, extracellular acetate induced transcriptional programs related to learning and memory, which were sensitive to ACSS2 inhibition. We show that alcohol-related associative learning requires ACSS2 in vivo. These findings suggest that there is a direct link between alcohol metabolism and gene regulation, through the ACSS2-dependent acetylation of histones in the brain.
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Acknowledgements
We thank the Metabolomics Core of the Diabetes Research Center (DRC) for providing the mass spectrometry quantification of metabolites; J. D. Rabinowitz (the Princeton Metabolomics Core director) and C. Jang for advice; and the Neurons R Us core of the Mahoney Institute for Neurological Sciences for preparations of primary hippocampal neurons. We especially acknowledge J. Whetstine for the suggestion to test whether the administration of alcohol to a pregnant female mouse leads to histone acetylation in the gestating fetal brain. G.E. was supported by The Brody Family Medical Trust Fund Fellowship in Incurable Diseases of The Philadelphia Foundation. This work was supported by NIH P01AG031862 and NIH R01AA027202.
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P.M. and S.L.B. developed the primary hypothesis; P.M., G.E. and S.L.B. designed the project; P.M. and G.E. performed most of the experiments; P.M. planned the ethanol-d6-labelling mass spectrometry, which was performed together with R.N., G.E. and S.S. with support from B.A.G.; G.E. and S.S. performed the acetate-d3-labelling mass spectrometry; the RNA-seq was performed in vivo by P.M. and in vitro by G.E., with support from R.N; R.N. conducted the ChIP–seq; G.D. analysed all ChIP–seq and RNA-seq datasets; G.E., S.I.L. and D.C.A. performed the behavioural characterization with support from E.A.H.; P.M. performed the labelling experiments for metabolomic analysis with support from E.J.N.; the fetal alcohol labelling was done by G.E., with support from S.L.R.; P.M., G.E. and S.L.B. wrote the manuscript. All authors reviewed the manuscript and discussed the work.
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Extended data figures and tables
Extended Data Fig. 1 Ethanol-derived acetyl groups are rapidly incorporated into histone acetylation in the brain.
a, Mass spectrometry analysis of serum acetate shows the rapid increase in levels of acetate in mice that were injected with alcohol, at 30 min after injection (n = 3 for saline, n = 4 for acetate group). Data are mean ± s.e.m.; P = 0.0258 (two-tailed unpaired t-test). b, Ethanol-d6 is readily metabolized and thus labels blood acetate pools. Acetate-d3 was detected by mass spectrometry (n = 4 per group). Data are mean ± s.e.m.; P = 0.0016 (two-tailed unpaired t-test). c, Incorporation of the ethanol-d6 label into histone acetylation in the cortex shows a similar pattern to the hippocampus. The axis of the Arachne plot represents the percentage of the third isotope for the acetylated peptide, corresponding to the d3-labelled form; the natural relative abundance of that isotope is apparent in the ‘none’ and ‘saline 1 h’ treatment groups d, Histone acetylation is relatively independent of alcohol metabolism in skeletal muscle, a tissue in which the expression of ACSS2 is low. e, f, Mass spectra (representative examples from three biological replicates) showing the relative abundance of deuterated histone H4-triacetyl peptide (amino acids 4–17) in the hippocampus of wild-type mice at baseline and 4 h after injection of ethanol-d6. Increases of the M+1 (blue lines), M+2 (green lines) and M+3 (red lines) species are shown in e and indicate a major increase of M+3. The contribution of singly (orange), doubly (grey) and triply (yellow) deuterated peptides to the isotopic distribution is shown in f. The relative abundance of the M+3 species is increased by about sixfold at 4 h after injection of ethanol-d6, and is overwhelmingly due to the triply deuterated peptides; by contrast, the contribution of singly and doubly deuterated peptides to the M+3 species is minimal. The experiment was performed with three biological replicates per group. g, The relative abundance of the first four isotopes of each of the seven peptides in the untreated samples corresponds to the theoretical isotopic distribution of the peptides (calculated using enviPat30; samples not treated with ethanol-d6; n = 20). Data are mean ± s.d. h, Natural abundance-corrected contribution of M+1, M+2 and M+3 species to the labelling of histone acetylation in the liver and hippocampus after intraperitoneal injection of ethanol-d6 (calculated using FluxFix31; n = 3 per group). Data are mean ± s.d.
Extended Data Fig. 2 Dynamics of ethanol- and acetate-induced heavy-label incorporation.
a, b, Relative abundance of deuterated histone acetylation in dHPC, vHPC, cortex, liver and muscle at 8 h (a) and 24 h (b) after intraperitoneal injection of ethanol-d6. c, 13C-labelled ethanol, introduced by intraperitoneal injection, readily labels histone acetylation in the hippocampus (percentage increase over natural abundance of 13C acetyl groups in saline-injected mice; n = 1). d, In contrast to heavy ethanol–d6, a non-labelled ethanol control does not increase the natural abundance of heavy histone acetylation in the hippocampus. e, Histone acetylation is relatively independent of alcohol metabolism in skeletal muscle. Relative abundance of deuterated histone acetylation in skeletal muscle tissue at 30 min and 4 h in wild-type mice, and 30 min in dHPC ACSS2-knockdown mice. f, g, Heavy acetate, introduced by intraperitoneal injection, readily labels histone acetylation in the dHPC (f) and the cortex (g) (n = 2 at 30 min; n = 3 per group at other time points). Data are mean ± s.e.m. h, Levels of acetate measured by mass spectrometry in hippocampal tissue after injections of acetate and ethanol (n = 3 per group). Data are mean ± s.e.m.; P = 0.0335 for acetate versus saline; P = 0.0285 for ethanol versus saline (two-tailed unpaired t-test).
Extended Data Fig. 3 Metabolite labelling in hippocampal tissue 30 min after intraperitoneal injection of ethanol-d6.
a–f, Mass spectrometry quantification of metabolite labelling in hippocampal tissue at 30 min after intraperitoneal injection of ethanol-d6. The ethanol-d6 label was incorporated into the acetate pool in the hippocampus (a). By contrast, ethanol-d6 did not contribute to the glucose (b) or 3-hydroxybutyrate (d) pools in the hippocampus, and only minimally to the lactate pool (c). Labelling was observed in the hippocampal glutamine (e) and citrate or isocitrate (f) pools.
Extended Data Fig. 4 Representative H3K9ac and H3K27ac dHPC ChIP–seq tracks in control and ethanol-treated wild-type and ACSS2-knockdown mice.
a-c, ChIP-seq for H3K9ac and H3K27ac in untreated and ethanol-treated wild-type and ACSS2-knockdown mice. Genome-browser track views show the Cep152 locus (chr2: 125,603,000–125,626,000) (a), Uimc1 locus (chr5: 55,064,000–55,089,000) (b) and Nsmaf locus (chr4: 6,425,000–6,464,000) (c). The experiment was performed with three independent biological replicates per group.
Extended Data Fig. 5 Epigenetic and transcriptional changes in the dHPC of control and ethanol-treated wild-type and ACSS2-knockdown mice.
a–d, Decile plots of genes that are enriched in H3K9ac (a) and H3K27ac (b) show correlation with mRNA expression levels in hippocampus, in wild-type mice 1 h after injection with ethanol. By contrast, in ACSS2-knockdown mice, the correlation between histone H3K9 acetylation (c) and H3K27 acetylation (d) and alcohol-related mRNA expression is largely lost (n = 16,553 genes (population) arranged into ten equal-sized deciles by ChIP–seq enrichment of acetylation). Box plots as in Fig. 2. e, f, GO analysis on H3K9ac (e) and H3K27ac (f) peaks that are induced by ethanol in wild-type but not ACSS2-knockdown mice (n = 332 H3K9ac peaks and n = 480 H3K27ac peaks). GO enrichment analysis was performed using a modified Fisher’s exact test (EASE) with the FDR corrected by the Yekutieli procedure; −log10 transformations of nominal P values are shown.
Extended Data Fig. 6 Transcriptional changes in primary hippocampal neurons that were treated with supraphysiological levels of acetate.
a, Structure of ACSS2i (C20H18N4O2S2). b, RNA-seq showing differentially regulated genes in primary hippocampal neurons that were treated with 5 mM acetate (n = 4 replicates per group; volcano plot of likelihood ratio test (two-sided) in DESeq2; FDR controlled for multiple hypothesis testing). c, d, GO analysis of genes that are significantly upregulated (c; n = 3,613 genes) and significantly downregulated (d; n = 3,987 genes) after treatment with 5 mM acetate. GO analysis was performed with GOrilla, using a minimal hypergeometric test. e, RNA-seq in primary hippocampal neurons that were isolated from C57/Bl6 mouse embryos and treated with acetate (5 mM) in the presence or absence of ACSS2i. Of the 3,613 acetate-induced genes, 2,107 are not upregulated in the presence of ACSS2i (n = 3,613 induced genes (population) or 3,613 randomly sampled genes (population); P < 2.2 × 10−16 (two-sided Mann–Whitney rank-sum test)). Box plots as in Fig. 2. f, Acetate-induced genes in primary hippocampal neurons are shown in blue (n = 3,613). Of these genes, 2,107 (non-overlapping with orange) were sensitive to ACSS2i, and 1,506 were also induced in the presence of ACSS2i (overlapping with orange). GO enrichment analysis was performed using a modified Fisher’s exact test (EASE) with the FDR corrected by the Yekutieli procedure; −log10 transformations of nominal P values are shown.
Extended Data Fig. 7 Representative RNA-seq tracks in control and acetate-treated primary hippocampal neurons in the presence or absence of ACSS2i.
a–d, Genome-browser track views showing examples of gene upregulation after treatment with acetate in hippocampal neurons, and diminished induction after treatment with ACSS2i (n = 4 per cohort). RNA-seq track views show the Slc17a7 locus (chr7: 45,162,500–45,179,000) (a), the Ccnjl locus (chr11: 43,525,000–43,595,000) (b), the Cpne7 locus (chr8: 123,152,500–123,137,500) (c) and the Ndufv3 locus (chr17: 31,523,000–31,534,000) (d).
Extended Data Fig. 8 Transcriptional changes that are induced by acetate in primary hippocampal neurons relate to in vivo ACSS2 peaks and in vivo changes in gene expression that are induced by ethanol.
a, Cumulative number of ACSS2 peaks near the transcription start site of acetylated ACSS2i sensitive genes, indicating that the majority of ACSS2-binding events occur over or proximal to the gene promoter. b, GO analysis for the 830 overlapping genes between the in vivo RNA-seq and ex vivo hippocampal-neuron RNA-seq (n = 830 genes (population)). GO enrichment analysis was performed using a modified Fisher’s exact test (EASE) with the FDR corrected by the Yekutieli procedure.
Extended Data Fig. 9 Behavioural importance of ACSS2 expression in the dHPCand heavy-label incorporation in the fetal brain.
a, Representative image showing virus localization to the dHPC, and western blot (n = 4 mice) showing ACSS2 levels in the dHPC of wild-type and ACSS2-knockdown mice (for gel source data, see Supplementary Fig. 1). b, Quantification of the levels of ACSS2 protein in the dHPC and cortex of wild-type and dHPC ACSS2-knockdown mice (n = 4 mice). Data are mean ± s.e.m.; P = 0.0001 and q = 0.0001 for ACSS2-knockdown versus wild-type mice (dHPC); P = 0.2666 and q = 0.1347 for ACSS2-knockdown versus wild-type mice (cortex) (multiple t-test). c, ACSS2 is required for alcohol-induced associative learning. Mean time (seconds per minute) spent in unconditioned and ethanol-conditioned chambers following ethanol-induced CPP training in wild-type (n = 8) and dHPC ACSS2-knockdown mice (n = 10). Bar graphs represent mean ± s.e.m. and data points correspond to individual mice. d, Incorporation of ethanol-d6 into histone acetylation in the fetal brain. Data represent the second of two pools of embryos (n = 4 per pool) from maternal ethanol-d6 injection. The axes of the Arachne plots represent the percentage of the third isotope for the acetylated peptide, corresponding to the d3-labelled form.
Supplementary information
Supplementary Figure
Supplementary Figure 1: Uncropped Western blots. Cortical and dorsal hippocampal ACSS2 and GAPDH scans in WT and ACSS2 KD animals (corresponding to Extended Data Fig 9.a) are shown. Top panel shows enhanced chemoluminescence (ECL) only. Bottom panel shows composite images with molecular weights indicated.
Supplementary Table
Supplementary Table 1: Heavy labeling pattern following d6-EtOH injections. M+1, M+2, and M+3 labeling in liver, cortex and hippocampus is shown for all histone peptides assessed in this study (data corresponding to Fig. 1b, Fig. 1c and Extended Data Fig. 1c). For each isotope, fold change (FC) and statistical significance (P value from unpaired Student’s t-test) are shown. Red and green colors indicate the largest fold changes and smallest P values, respectively.
Supplementary Table
Supplementary Table 2: Raw mass spectrometry data. Raw, unprocessed data are shown for each mass spectrometry and metabolomics experiment included in this manuscript. Excel sheets are named after the corresponding main or extended data figures.
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Mews, P., Egervari, G., Nativio, R. et al. Alcohol metabolism contributes to brain histone acetylation. Nature 574, 717–721 (2019). https://doi.org/10.1038/s41586-019-1700-7
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DOI: https://doi.org/10.1038/s41586-019-1700-7
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