BCL6 regulates brown adipocyte dormancy to maintain thermogenic reserve and fitness

Abstract

Brown adipocytes provide a metabolic defense against environmental cold but become dormant as mammals habituate to warm environments. Although dormancy is a regulated response in brown adipocytes to environmental warmth, its transcriptional mechanisms and functional importance are unknown. Here, we identify B cell leukemia/lymphoma 6 (BCL6) as a critical regulator of dormancy in brown adipocytes but not for their commitment, differentiation, or cold-induced activation. In a temperature-dependent manner, BCL6 suppresses apoptosis, fatty acid storage, and coupled respiration to maintain thermogenic fitness during dormancy. Mechanistically, BCL6 remodels the epigenome of brown adipocytes to enforce brown and oppose white adipocyte cellular identity. Thus, unlike other thermogenic regulators, BCL6 is specifically required for maintaining thermogenic fitness when mammals acclimate to environmental warmth.

Keywords: acclimation; brown fat; metabolism; thermogenesis; transcription.

Fig. 1.

BCL6 is required to maintain thermogenic capacity of dormant BAT. (A) Immunoblotting for BCL6 and lamin B1 protein in nuclear extracts of iBAT of Bcl6^f/f, Bcl6^f/fUcp1^Cre, and Bcl6^f/fAdipoq^Cre mice (n = 2 to 3 per genotype). (B) Gross morphology of iBAT isolated from Bcl6^f/f, Bcl6^f/fUcp1^Cre, Bcl6^f/fAdipoq^Cre, and Bcl6^f/fMyf5^Cre mice bred and housed at 22 °C. (C and D) Core temperature measurements (C) and survival curves (D) of Bcl6^f/f and Bcl6^f/fUcp1^Cre female mice bred at 22 °C and subjected to 4 °C cold challenge (n = 4 to 5 per genotype). (E and F) Core temperature measurements (E) and survival curves (F) of Bcl6^f/f and Bcl6^f/fUcp1^Cre female mice bred at 30 °C and subjected to 10 °C cold challenge (n = 8 to 10 per genotype). The P value for E was calculated using the Mann–Whitney U test at the 3 h time point. (G and H) Norepinephrine-stimulated changes in oxygen consumption rate (VO₂) in Bcl6^f/f and Bcl6^f/fUcp1^Cre female mice housed at 30 °C (G, n = 6 per genotype) or 22 °C (H, n = 4 to 6 per genotype). The arrow indicates the time of norepinephrine (NE) injection. (I) Norepinephrine-stimulated changes in oxygen consumption rate (VO₂) in Bcl6^f/f and Bcl6^f/fUcp1^Cre male mice initially housed at 22 °C (until 5 wk of age) followed by housing at 30 °C for 6 wk (n = 3 to 5 per genotype). (J and K) Representative infrared images (J) and quantified thermographic measurements of interscapular surface temperature (K) for Bcl6^f/f and Bcl6^f/fUcp1^Cre male mice subjected to 4 °C cold challenge from 30 °C (n = 5 to 6 per genotype). (L) Immunoblotting for phosphorylated PKA substrates, β3-adrenergic receptor (ADRB3), and HSP90 in whole-cell extracts of iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre male mice that were housed at 30 °C and injected with CL-316,243 (1 mg/kg) or vehicle (Veh) for 30 min. Data are presented as mean ± SEM.

Fig. 2.

BCL6 maintains uncoupled respiration in dormant BAT. (A) Oxygen consumption rate of iBAT isolated from Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C and exposed to 4 °C for 15 min (n = 5 to 6 per genotype). (B) Immunoblotting for UCP1 and HSP90 in whole-cell extracts of iBAT of male 8-wk-old mice housed at 22 °C or 30 °C (n = 3 to 4 per genotype and temperature). (C) Immunoblotting for subunits of mitochondrial complexes I to V and HSP90 in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 22 °C or 30 °C (n = 3 to 4 per genotype and temperature). (D) Genome browser track showing BCL6 binding near the promoter of the Atp5g1 gene in iBAT isolated from Bcl6^f/f and Bcl6^f/fUcp1^Cre (negative control) mice housed at 30 °C. Kb, kilobases. (E) Quantitative RT-PCR measurement of Atp5g1 mRNA in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 to 6 per genotype). AU, arbitrary units. (F) ATP synthase activity in mitochondria isolated from iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 to 5 per genotype). (G) Body mass of male Bcl6^f/f and Bcl6^f/fUcp1^Cre mice fed a high-fat diet at 30 °C starting at the age of 10 wk (n = 13 per genotype, data pooled from multiple experiments). (H) Body composition analysis by dual-energy X-ray absorptiometry (DEXA) of male Bcl6^f/f and Bcl6^f/fUcp1^Cre mice fed a high-fat diet at 30 °C (n = 14 per genotype, data pooled from multiple experiments). (I and J) Glucose (I) and insulin (J) tolerance tests in male Bcl6^f/f and Bcl6^f/fUcp1^Cre mice fed a high-fat diet at 30 °C (n = 14 to 15 per genotype, data pooled from multiple experiments). Data are presented as mean ± SEM.

Fig. 3.

BCL6 regulates survival of dormant brown adipocytes. (A) Venn diagrams showing differentially expressed genes in iBAT of Bcl6^f/fUcp1^Cre mice (fold change ≥ 1.5, adjusted P value < 0.05). Genes up-regulated (Left) and down-regulated (Right) in iBAT of Bcl6^f/fUcp1^Cre mice that were housed at different ambient temperatures. (B) Gene ontology enrichment analysis of up-regulated genes in iBAT of Bcl6^f/fUcp1^Cre mice housed at 30 °C. Enriched biological processes and corresponding P values are shown. (C) Heat map of up-regulated genes in the “apoptotic process” gene ontology category in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice (n = 4 per genotype and temperature). The list of genes is provided in SI Appendix, Table S1. (D) Pie chart showing the distribution of BCL6 binding sites across the genome of iBAT. TTS, transcription termination site. (E) Heat map of BCL6-regulated genes (fold change ≥1.5) in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C that are nearest to a BCL6 binding site. The list of genes is provided in SI Appendix, Table S3. (F) Genome browser tracks showing BCL6 binding sites near proapoptotic genes Bmf and Egln3 in iBAT isolated from Bcl6^f/f and Bcl6^f/fUcp1^Cre (negative control) mice housed at 30 °C. (G) Quantitative RT-PCR measurement of Bmf and Egln3 mRNAs in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 to 6 per genotype). (H) Quantification of terminal deoxynucleotidyl transferase dUTP nick end labeled (TUNEL⁺) apoptotic cells in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre male mice housed at 22 °C or 30 °C for 1 wk (n = 3 to 5 per genotype). (I) BAT mass in 8-wk-old Bcl6^f/f and Bcl6^f/fUcp1^Cre male mice bred at 22 °C or 30 °C (n = 6 to 10 per genotype and condition). sBAT = subscapular BAT. (J) DNA content in adipocyte fraction of iBAT from Bcl6^f/f and Bcl6^f/fUcp1^Cre mice bred at 30 °C (n = 4 to 5 per genotype). Data are presented as mean ± SEM.

Fig. 4.

BCL6 regulates fatty acid metabolism in dormant BAT. (A) Gene ontology enrichment analysis and corresponding P values for the down-regulated genes in iBAT of Bcl6^f/fUcp1^Cre mice housed at 30 °C. (B and C) Heat maps of genes involved in β-oxidation of fatty acids (B) and acyl-CoA thioesterase (ACOT) genes (C) (n = 4 per genotype and temperature). (D) Genome browser track showing BCL6 binding site near the promoter of Acot1 gene in iBAT isolated from Bcl6^f/f and Bcl6^f/fUcp1^Cre (negative control) mice housed at 30 °C. (E) Quantitative RT-PCR measurement of Acot1, Acot3, and Acot4 mRNAs in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 to 6 per genotype). (F and G) Measurement of ACOT activity in cytosolic (F) and mitochondrial (G) fractions of isolated iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 5 per genotype). ACOT activity is expressed as micromoles of CoA produced per minute per microgram of protein. (H) Hematoxylin and eosin staining of representative iBAT sections from Bcl6^f/f and Bcl6^f/fUcp1^Cre mice bred and housed at 22 °C. (Scale bar, 100 μm.) (I) A model depicting changes in fatty acid metabolism in dormant BAT of Bcl6^f/fUcp1^Cre mice. Data are presented as mean ± SEM.

Fig. 5.

BCL6 reinforces brown and opposes white adipocyte–specific enhancers to maintain cellular identity. (A) Heat map of nuclear encoded brown and white adipocyte–specific genes (BATLAS) in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice at 22 °C and 30 °C. The list of genes is provided in SI Appendix, Table S4. (B) Heat maps showing H3K27ac peaks that are up-regulated or down-regulated in iBAT of Bcl6^f/fUcp1^Cre mice housed at 30 °C. H3K27ac peaks in heat maps represent an average of 3 biological replicates, and the amplitude of each peak center (±3 kb) is represented in color as indicated. (C) Venn diagram showing the overlap of BCL6-regulated enhancers at 30 °C with brown and white adipocyte–specific enhancers. The numbers indicate the number of enhancers in each field. (D and E) Pie charts showing the percentage of BCL6-regulated brown (D) and white (E) adipocyte–specific enhancers that are positively or negatively regulated by BCL6 at 30 °C. (F and G) Representative H3K27ac genome browser tracks for brown adipocyte–specific genes Ucp1 and Elovl3 (F), fatty acid metabolism genes Acot1, Acot3, and Acot4 (F), white adipocyte–specific genes Ccdc80 and Lep (G), and ATP synthesis gene Atp5g1 (G). The effects of temperature and BCL6 are highlighted for each category. Data are presented as mean ± SEM.

Fig. 6.

BCL6 regulates brown adipocyte enhancers by direct and indirect mechanisms. (A and B) De novo sequence motif discovery at hyperacetylated (A) and hypoacetylated (B) H3K27 sites in iBAT of Bcl6^f/fUcp1^Cre mice housed at 30 °C; PWM = position weight matrix. For the top enriched motif in B, a list of the top 7 matches from a database of known transcription factor binding site motifs and corresponding match scores are listed. (C) Genome browser tracks for visualization of the Lep, Ccdc80, and Nnat loci highlighting BCL6 and H3K27ac ChIP-seq data. (D) Percentage of H3K27 acetylation sites in iBAT (all sites or only sites that are hyperacetylated or hypoacetylated in Bcl6^f/fUcp1^Cre mice at 30 °C) that are bound by ERRγ or ERRα. (E) Genome browser tracks for visualization of the Ucp1, Slc27a2, Hadha, Hadhb, and Acaa2 loci highlighting BCL6, ERRα, ERRγ, and H3K27ac ChIP-seq data. (F) Quantitative RT-PCR measurement of Esrra, Esrrg, and Esrrb mRNA in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 to 6 per genotype). (G) Venn diagram showing the overlap between BCL6 and ERRα binding sites in iBAT at 30 °C. (H) ChIP-seq analysis of ERRα recruitment to binding sites in iBAT of Bcl6^f/f and Bcl6^f/fUcp1^Cre mice housed at 30 °C (n = 4 per genotype). Percentages of total binding sites with increased, decreased, or unchanged enrichment in Bcl6^f/fUcp1^Cre mice are indicated. Data are presented as mean ± SEM.

Fig. 7.

A model for regulation of brown adipocyte dormancy by BCL6. (A) During adaptation to cold, norepinephrine (NE) released by the sympathetic nervous system (SNS) activates β-adrenergic signaling in brown adipocytes, which supports their survival and increases their thermogenic capacity. Survival is mediated by the action of ERK1/2, while fatty acid oxidation (FAO), mitochondrial biogenesis, and mitochondrial uncoupling (UCP1) are stimulated at the transcriptional level by peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and the coactivator protein PGC-1α. (B) In contrast, during adaptation to warmth, when sympathetic tone is minimal and brown adipocytes become dormant, BCL6 reinforces survival and preserves thermogenic capacity. BCL6 promotes survival by repressing proapoptotic genes, such as Bmf and Egln3, and maintains reserve thermogenic capacity by repressing genes involved in hydrolysis of acyl-CoAs (Acot1, Acot3, Acot4) and mitochondrial coupling (Atp5g1), as well as white adipocyte–specific genes (Nnat, Lep, Ccdc80) and potentially by stimulating the activity of ERRs to promote expression of genes involved in FAO and uncoupled respiration (Ucp1).