Histone deacetylase 3 prepares brown adipose tissue for acute thermogenic challenge

Abstract

Brown adipose tissue is a thermogenic organ that dissipates chemical energy as heat to protect animals against hypothermia and to counteract metabolic disease. However, the transcriptional mechanisms that determine the thermogenic capacity of brown adipose tissue before environmental cold are unknown. Here we show that histone deacetylase 3 (HDAC3) is required to activate brown adipose tissue enhancers to ensure thermogenic aptitude. Mice with brown adipose tissue-specific genetic ablation of HDAC3 become severely hypothermic and succumb to acute cold exposure. Uncoupling protein 1 (UCP1) is nearly absent in brown adipose tissue lacking HDAC3, and there is also marked downregulation of mitochondrial oxidative phosphorylation genes resulting in diminished mitochondrial respiration. Remarkably, although HDAC3 acts canonically as a transcriptional corepressor, it functions as a coactivator of oestrogen-related receptor α (ERRα) in brown adipose tissue. HDAC3 coactivation of ERRα is mediated by deacetylation of PGC-1α and is required for the transcription of Ucp1, Ppargc1a (encoding PGC-1α), and oxidative phosphorylation genes. Importantly, HDAC3 promotes the basal transcription of these genes independently of adrenergic stimulation. Thus, HDAC3 uniquely primes Ucp1 and the thermogenic transcriptional program to maintain a critical capacity for thermogenesis in brown adipose tissue that can be rapidly engaged upon exposure to dangerously cold temperature.

Extended Data Figure 1. Ablation of HDAC3 in adipose tissue depots

(a) Immunoblot analysis of interscapular BAT, inguinal WAT, and epididymal WAT of Adipoq-Cre HDAC3 KO and control littermates, or Ucp1-Cre HDAC3 KO and control littermates maintained at 22°C (n= 2, all groups) demonstrating tissue-specific conditional knockout of HDAC3. (b) Interscapular BAT mass, (c) Relative BAT mitochondrial number, and (d) Total body mass from Adipoq-Cre HDAC3 KO and Ucp1-Cre HDAC3 KO versus control littermates maintained at 22°C (n= 13 Adipoq-Cre, n= 9 control; n= 9 Ucp1-Cre, n= 10 control). (e) Representative hematoxylin and eosin (H&E) staining of inguinal white adipose from 10–12 week old Adipoq-Cre HDAC3 KO, Ucp1-Cre HDAC3 KO, Ucp1 KO, or control mice housed at 22°C. Scale bars, 100μm. Data are represented as mean ± s.e.m.

Extended Data Figure 2. BAT HDAC3 is required for cold-mediated induction of Ucp1 expression and HDAC3 expression is not altered by acute cold

a–b BAT Ucp1 mRNA levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C in (a) Adipoq-Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (b) Ucp1-Cre HDAC3 KO versus control (n= 5, 5, per temperature). c–d iWAT Ucp1 mRNA levels following 3 h exposure to 4°C, versus control littermates maintained at 22°C in (c) Adipoq-Cre HDAC3 KO versus control (n= 5, 5, per temperature) and (d) Ucp1-Cre HDAC3 KO versus control (n= 5, 5, per temperature). (e) BAT HDAC3 mRNA expression levels following a 3 h exposure to 4°C (from 22°C) versus control littermates maintained at 22°C (n= 5, 5, per temperature). (f) BAT HDAC3 protein levels following 3 h acute cold exposure at 4°C (from 22°C) versus control littermates maintained at 22°C. VCL, vinculin. NS, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as determined by a two-way ANOVA and Holm-Sidak’s post-test (a–d) or two-tailed Student’s t-test (e). Data are represented as mean ± s.e.m.

Extended Data Figure 3. HDAC3 is neither induced nor required for brown adipogenesis, but required for cell-autonomous Ucp1 expression

(a) Gene expression spanning differentiation of cultured wild type primary brown adipocytes (n= 5 replicates per time point). (b) Depletion of HDAC3 in Day 8 cultured mature brown adipocytes following addition of 2μm 4-hydroxytamixofen (4-OHT) during Days 0–2 of differentiation to Rosa26-CreER-positive (HDAC3 KO) and Rosa26-CreER-negative (Control) cells derived from littermates (n= 3, 3). (c) Adipocyte-specific gene expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (d) Assessment of lipid accumulation (evaluated by Oil Red-O staining) in cultured HDAC3 KO versus control primary brown adipocytes. (e) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control (n= 3, 3). (f) UCP1 protein expression in cultured primary brown adipocytes following depletion of HDAC3 versus control. (n= 3, 3). (g) Ucp1 mRNA expression in cultured primary brown adipocytes following depletion of HDAC3 versus control and treated with vehicle (ethanol) or isoproterenol (1μm) for 3 h (n= 4 per group). VCL, vinculin. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as determined by a two-tailed Student’s t-test (b, c, e) or by a two-way ANOVA and Holm-Sidak’s post-test (g). Data are represented as mean ± s.e.m.

Extended Data Figure 4. HDAC3 is required for expression of mitochondrial OXPHOS and TCA cycle genes

(a) Bioinformatic extension of identified Gene Ontology categories (Fig 2c.) to all oxidative phosphorylation and TCA cycle genes as retrieved by HUGO gene nomenclature database. Asterisk designates gene expression change in RNA-seq dataset with an FDR <0.01. b–c, RT-qPCR verification of gene expression changes highlighted in Figure 2d, in (b) Adipoq-Cre HDAC3 KO vs. control littermates at 29°C (upper, n= 9, 6) and 22°C (lower, n= 9, 7) (c) Ucp1-Cre HDAC3 KO vs. control littermates at 29°C (upper, n= 5, 6) and 22°C (lower, n= 5, 7). *P<0.05, **P<0.01, ***P<0.001, as determined by a two-tailed Student’s t-test. Data are represented as mean ± s.e.m.

Extended Data Figure 5. Metabolic studies of Adiponectin-Cre and Ucp1-Cre HDAC3 KO mouse models

a–b NMR analysis of body composition, (a) Adipoq-Cre mice versus control littermates (n= 8, 11), (b) Ucp1-Cre mice versus control littermates (n= 7, 9). c–n, CLAMS metabolic cage analysis. c–d Oxygen consumption (VO₂), (c) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5), (d) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). e–f ANCOVA VO₂ analysis (linear regression analysis of total body mass and oxygen consumption) (e) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5), (f) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). g–h Respiratory Exchange Ratio (RER), (g) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5), (h) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). i–j Heat measurements (kcal/hr), (i) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5). (j) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). k–l Food Intake, (k) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5), (l) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). m–n Physical activity, (m) Adipoq-Cre HDAC3 KO vs. control littermates (n= 6, 5), (n) Ucp1-Cre HDAC3 KO vs. control littermates (n= 6, 6). P-values shown in italics. CLAMS data is graphed as rolling averages. NS, not significant, *P<0.05 as determined by a two-tailed Student’s t-test (a–d, g–n) or ANCOVA (e, f). Data are represented as mean ± s.e.m.

Extended Data Figure 6. Effect of high fat diet on Adiponectin-Cre and Ucp1-Cre HDAC3 KO mouse models

12-week old weight-matched HDAC3 KO and control littermates were fed high-fat diet (HFD) for 12 weeks (a) Weekly body weights, (n= 8 Adipoq-Cre, n= 10 control), (b) Body composition analysis by NMR, (n= 8 Adipoq-Cre, n= 10 control), (c) Weekly body weights, (n= 7 Ucp1-Cre, n= 7 control), (d) Body composition analysis by NMR, (n= 7 Ucp1-Cre, n= 7 control). (e) RT-qPCR of BAT HDAC3 mRNA expression following 12 weeks HFD versus regular chow fed controls (n= 7, 5, respectively). Data are represented as mean ± s.e.m.

Extended Data Figure 7. Transcriptional Role of HDAC3 and ERRα in BAT

(a) Heat map demonstrating correlation of RNA-seq and GRO-seq data. Differentially expressed genes in RNA-seq or GRO-seq data were sorted by log₂FC in RNA-seq. (b) De novo motif enrichment at repressed eRNAs in Adipoq-Cre, HDAC3 KO mice versus control littermates (n= 10, 10; pooled biological replicates/library) maintained at 22°C and ranked by P-value. (c) Endogenous HDAC3 co-immunoprecipitation of ERRα in differentiated mature brown adipocytes. (d–e) RT-qPCRs of BAT Ucp1 eRNA expression and (f) Ucp1 mRNA at 22°C and 29°C in Adipoq-Cre and Ucp1-Cre HDAC3 KO mice versus control littermates, 29°C (n= 9 Adipoq-Cre, 6 control; n= 5 Ucp1-Cre, 6 control) and 22°C (n= 9 Adipoq-Cre, 7 control; n= 5 Ucp1-Cre, 7 control). (g) ChIP-qPCR of ERRα at Ucp1 enhancers in Adipoq-Cre HDAC3 KO versus control littermates (n= 3, 3) adapted to 29°C. (h) RT-qPCR of ERRα and Ucp1 mRNA expression and (i) Ucp1 eRNA expression in ERRα KO BAT versus control littermates (n= 8, 7). (j) RT-qPCR analysis of ERRα and Ucp1 mRNA expression in mature brown adipocytes following siRNA mediated knockdown of ERRα versus scramble 72 h post-transfection (n= 3, 3). *P<0.05, **P<0.01, ***P<0.001 as determined by a two-tailed Student’s t-test (d–e, g–j), two-way ANOVA with Holm-Sidak’s post-test (f). P-values for motif enrichment as determined by binomial test (b). Data are represented as mean ± s.e.m.

Extended Data Figure 8. Role of HDAC3 on PGC-1α acetylation and function

(a) Co-immunoprecipitation of HDAC3 and PGC-1α with ERRα from 293FT cells. (b) Luciferase reporter assay of transcription driven by the major Ucp1 enhancer (−6 kb) following transfection of ERRα, PGC-1α, GCN5, and/or HDAC3 (n= 3 replicates per condition). (c–d) Primary brown pre-adipocytes from Rosa26-CreER-positive HDAC3^F/F and HDAC3^F/F control littermates transduced with MSCV retroviruses: Control, PGC-1α WT, or non-acetylatable PGC-1α R13 mutant, and treated with 2μm 4-OHT during days 0–2 of differentiation to deplete HDAC3, and studied at Day 8 of differentiation. (c) Immunoblot analysis of exogenous PGC-1α expression in primary brown adipocytes (n= 2 replicates pooled per lane). (d) RT-qPCR analysis of Ucp1 and Fasn expression in control and HDAC3 KO primary brown adipocytes following transduction with MSCV-Control (n= 4 control, 3 HDAC3 KO), MSCV-PGC-1α WT (n= 4 control, 4 HDAC3 KO), or MSCV-PGC-1α R13 (non-acetylatable mutant) (n= 3 Control, 4 HDAC3 KO). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, as determined by a one-way ANOVA and a Tukey’s post-test (b, d). Data are represented as mean ± s.e.m.

Extended Data Figure 9. HDAC3 and ERRα activate Ppargc1β enhancers and transcription

(a) Genome browser tracks of the Ppargc1β locus highlighting GRO-seq and ChIP-seq data from HDAC3 KO and control BAT (y-axis scales, normalized reads, reads per million) demonstrating co-binding of HDAC3, ERRα, and NCoR at functional enhancers. (b) BAT PGC-1β mRNA levels in Adipoq-Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7). (c–d) RT-qPCR of eRNAs found at HDAC3 and ERRαenhancers in Adipoq-Cre HDAC3 KO BAT versus control littermates (29°C: n= 9, 6; 22°C, n= 9, 7) and Ucp1-Cre HDAC3 KO BAT versus control littermates (29°C: n= 5, 6; 22°C, n= 5, 7). (e) RT-qPCR analysis of Ucp1 mRNA expression in mature brown adipocytes following combinatorial siRNA knockdown of Pgc-1α, Pgc-1β and/or ERRα versus scramble siRNA (n= 5 replicates per condition). Statistical analysis performed amongst groups transfected with siRNAs. (f) Quantification of Ucp1 and Pgc-1a nascent gene body transcription (GRO-seq) at 22°C and 29°C in Adipoq-Cre HDAC3 KO BAT versus control littermates (n= 10, 10, pooled biological replicates per library). *P<0.05, **P<0.01, ***P<0.001, as determined by a two-tailed Student’s t-test (b–d), one-way ANOVA and a Holm-Sidak’s post-test (e) or an exact test (performed in EdgeR). Data are represented as mean ± s.e.m.

Figure 1. HDAC3 controls BAT thermogenesis

(a, b) Effect of acute cold exposure from standard housing at 22°C to 4°C on Adipoq-Cre HDAC3 knockout (KO) mice versus control littermates (n= 15, 8), Ucp1-Cre HDAC3 KO mice versus control littermates (n= 15, 7), and Ucp1^−/− mice (n= 15): a, core body temperature; b, survival. (c) Oxygen consumption rates of anesthetized Adipoq-Cre HDAC3 KO mice versus control littermates (n= 12, 5), Ucp1-Cre HDAC3 KO mice versus control littermates (n= 6, 5), and Ucp1^−/− mice (n= 5) following injection of 1 mg kg⁻¹ norepinephrine. (d) Mitochondrial respiration of purified BAT homogenates from Adipoq-Cre HDAC3 KO mice versus control littermates (n= 5, 6), and Ucp1-Cre HDAC3 KO mice versus control littermates (n= 6, 5), following brief acclimation to thermoneutrality. Mitochondria were provided palmitoylcarnitine and pyruvate substrates. UCP1-dependent respiration was assessed upon addition of guanosine diphosphate (GDP), and coupled respiration rates of complex I, II, and IV were determined in the presence of adenosine diphosphate (ADP). (e) Representative hematoxylin and eosin staining of interscapular brown adipose from 10–12 week old mice at 22°C. Scale bars, 100μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as analyzed by two-way analysis of variance (ANOVA) and a Tukey’s post-test (a,c), log-rank (Mantel-Cox) test (b), or two-tailed Student’s t-test (d). Data are represented as mean ± s.e.m.

Figure 2. HDAC3 is required for expression of UCP1 and OXPHOS genes in BAT

(a) Scatterplot of RNA-seq data showing HDAC3 regulated BAT genes from Adipoq-Cre HDAC3 KO versus control littermates (n= 4, 4) adapted to thermoneutrality (fold-change > 1.5 up (red) or down (blue) and FDR < 0.01). RPKM, reads per kilobase per million. (b) Immunoblot from BAT of Adipoq-Cre HDAC3 KO, control littermates, or Ucp1^−/− adapted to 29°C or maintained at 22°C (n= 3, 3; n= 3, 3; n= 2). (c) Gene ontology (GO) and pathway analysis of down-regulated genes identified by RNA-seq and selected by Enrichr combined score (KP, KEGG Pathway; BP, Biological Process; CC, Cellular Component). (d) Heat map depicting down-regulated genes identified in GO analysis.

Figure 3. HDAC3 functions as an ERRα coactivator in BAT

(a) Average H3K27ac ChIP-seq profiles of Adipoq-Cre HDAC3 KO mice versus control littermates (n= 3, 3; pooled biological replicates/library) at enhancers bound by HDAC3 within 100 kb of transcription start sites of HDAC3 KO-regulated genes by GRO-seq (fold-change > 1.5 or < 0.5). Decreased H3K27ac at HDAC3 sites near repressed genes (N= 1085, P = 1.0×10⁻¹²³) and increased H3K27ac at HDAC3 sites near induced genes (N= 897, P = 9.0×10⁻⁵⁶) upon loss of HDAC3. (b) Scatter plot of enhancer RNAs (eRNAs) measured by GRO-seq in Adipoq-Cre HDAC3 KO mice versus control littermates (n= 10, 10; pooled biological replicates/library) at 29°C, highlighting induced and repressed eRNAs (Red, Fold-change > 2 & Blue, Fold-change < 0.5). (c) Average HDAC3 ChIP-seq profile of control littermates (n= 5, pooled biological replicates/library) at enhancers with repressed or unchanged eRNAs in HDAC3 KO. Significant HDAC3 binding found at HDAC3 KO-repressed eRNAs relative to unchanged eRNAs (P = 2.4×10⁻²¹⁵). (d) De novo motif search at eRNA sites repressed in HDAC3 KO at 29°C (ranked by P-value). (e) Average ERRα ChIP-seq profile in control littermates (n= 5, pooled biological replicates/library) at enhancers with differential eRNAs in HDAC3 KO. Significant ERRα found at HDAC3 KO-repressed eRNAs relative to unchanged eRNAs (P=1.0×10⁻²⁸⁸). (f) Heat map depicting HDAC3, ERRα, and NCoR co-localization at enhancers with repressed eRNAs in HDAC3 KO mice. (g) Genome browser tracks of the Ucp1 super-enhancer locus highlighting GRO-seq (22°C, 29°C), RNA-seq (29°C), and ChIP-seq (29°C) data (y-axis scales in brackets: reads per million (RPM); eRNA tracks feature adjusted y-axis scale). P-values for ChIP-seq or motif search determined by Wilcoxon or binomial test, respectively.

Figure 4. HDAC3 coactivation of ERRα is mediated by PGC-1α deacetylation

(a) Luciferase reporter assay of transcription driven by an identified Ucp1 enhancer (−6 kb), demonstrating effects of ERRα, HDAC3, wild type PGC-1α, and/or a PGC-1α LXXLL mutant (L1/2/3A) unable to interact with ERRα (n= 3 replicates/condition). (b) Immunoblot analysis of PGC-1α lysine acetylation following immunoprecipitation from co-transfected 293FT cells. (c) Immunoblot analysis of an in vitro deacetylation reaction of purified acetylated-PGC-1α by recombinant human HDAC3, with or without trichostatin A (TSA). (d) Genome browser tracks of the Ppargc1α locus highlighting GRO-seq and RNA-seq data, and eRNAs at HDAC3, ERRα, and NCoR co-bound distal enhancers (boxed). (e) Luciferase reporter assay as in (a) for identified Ppargc1α distal enhancer (−38 kb), (n= 3 replicates/condition). (f) BAT Pgc-1α mRNA from Adipoq-Cre HDAC3 KO mice versus control littermates (n= 9, 6) and Ucp1-Cre HDAC3 KO mice versus control littermates (n= 5, 6). (g) Immunoblot of PGC-1α in BAT nuclear extract of Adipoq-Cre HDAC3 KO versus control littermates (n= 5, 5 pooled replicates per lane). (h) BAT HDAC3 deacetylates PGC-1α to co-activate an ERR-driven transcriptional loop of Ppargc1α/β Ucp1, and OXPHOS genes. (i) Flow chart depicting unbiased hierarchical gene clustering of GRO-seq gene transcription by temperature and genotype. Boxplot displays gene cluster requiring HDAC3 coactivator function, where line denotes median, top/bottom of boxes represents first/third quartiles, and whiskers denote 1.5x the interquartile range from first/third quartiles. (j) Heatmap of thermogenic and OXPHOS genes identified in (i). ***P<0.001, ****P<0.0001 as determined by one-way ANOVA with multiple comparisons and a Tukey’s post-test (a, e) or two-tailed Student’s t-test (f). Data are represented as mean ± s.e.m.