Brown adipose tissue (BAT) adaptively transfers energy from glucose and fat into heat by inducing a gene network that uncouples mitochondrial electron transport. However, the innate transcription factors that enable the rapid adaptive response of BAT are unclear. Here, we identify estrogen-related receptor gamma (ERRγ) as a critical factor for maintaining BAT identity. ERRγ is selectively expressed in BAT versus WAT, in which, in the absence of PGC1α, it drives a signature transcriptional network of thermogenic and oxidative genes in the basal (i.e., thermoneutral) state. Mice lacking ERRγ in adipose tissue (ERRγKO mice) display marked downregulation of BAT-selective genes that leads to a pronounced whitening of BAT. Consistent with the transcriptional changes, the thermogenic capacity of ERRγKO mice is severely blunted, such that they fail to survive an acute cold challenge. These findings reveal a role for ERRγ as a critical thermoneutral maintenance factor required to prime BAT for thermogenesis.

Here, using a combination of biochemical, genetic, and genomic approaches, we uncover an innate or pre-demand function for BAT ERRγ referred to as “priming.” Essentially, transcriptional priming by ERRγ allows the establishment of the appropriate chromatin template needed to facilitate the rapid response to cold exposure. Thus, loss of ERRγ in BAT results in decreased expression of BAT signature genes under thermoneutral (TN) conditions, an unusual “whitening” in the appearance of the BAT organ, and an inability of knockout (KO) mice to survive acute cold exposure.

The estrogen-related receptor gamma (ERRγ, encoded by Esrrg) is an orphan nuclear receptor (NR) and a member of the subfamily of estrogen-related receptors that also includes ERRα and ERRβ (). In muscle, ERR isoforms variously contribute to aspects of mitochondrial biogenesis, mitochondrial maintenance, vascularity, energy expenditure, and mitochondrial uncoupling. Although the activity of ERRα is dependent on PGC1α/β induction, ERRγ is sufficient to orchestrate oxidative functions in highly energetic tissues such as type 1 muscle fibers and pancreatic beta cells (). In muscle, ERRγ functions to maintain a highly oxidative basal state even in the absence of exercise, and transgenic expression is sufficient to promote type 1 fiber-type switching (). In the pancreas, ERRγ is required for beta cell maturation and glucose-stimulated insulin secretion (GSIS), and its loss, by targeted knockout, leads to glucose intolerance (). Although ERRγ is also highly expressed in BAT, its contribution to BAT identity or function is not known.

Two distinguishable thermogenic adipocytes have been described in rodents with distinct developmental and anatomical features: classic brown adipocytes located in dedicated BAT depots and beige adipocytes, which reside mainly in subcutaneous WAT. Adult human BAT has been shown to have characteristics of both rodent classic brown adipocytes and beige adipocytes, so understanding both of these cell types is critical (). In contrast to beige adipocytes that arise postnatally in response to external cues such as chronic cold exposure, brown adipocytes express relatively high levels of thermogenic genes prior to an adaptive challenge and thus remain “primed” for thermogenesis, even in the basal (i.e., non-stimulated) state (). Although transcription factors and co-regulators, such as PPARγ coactivator 1-alpha (PGC1α), have been shown to mediate adaptive changes in BAT in response to cold, how thermogenic capacity is established and maintained prior to challenge and prior to PGC1 induction is less understood ().

Brown adipose tissue (BAT) and white adipose tissue (WAT) differ in their gene expression signatures, morphologies, and physiological functions (). In contrast to WAT, BAT expresses high levels of genes involved in fatty acid oxidation and thermogenesis, is rich in mitochondria, and has numerous small lipid droplets compared with the unilocular droplets of WAT (). Because the major function of WAT is to store and release lipid, it is well equipped to adapt to fluctuations in nutrient availability. BAT, on the other hand, is specialized to burn lipids and glucose in response to the need for extra heat, such as reduced ambient temperature or arousal from hibernation. Although foundational links among WAT, peroxisome proliferator-activated receptor gamma (PPARγ), nutrient storage, and adiposity have been extensively studied, much less is known about BAT. Indeed, with so much focus on WAT and the obesity epidemic, it has only recently been appreciated that adult humans can display cold-activated browning and that relative BAT mass inversely correlates with obesity ().

We next challenged ERRγKO mice with acute cold exposure. Although the initial response to cold exposure was similar between genotypes, the high metabolic rate needed to fend off cold dropped precipitously in ERRγKO mice ( Figure 4 B). Accordingly, although control mice maintained their body temperature upon exposure to acute cold, ERRγKO mice reached life-threatening hypothermia after only 3 hr exposure ( Figure 4 C). Furthermore, thermographic imaging revealed that BAT surface temperature was rapidly decreased in ERRγKO mice upon exposure to cold ( Figures 4 D and S4 I). Importantly, the inability of ERRγKO mice to maintain their body temperature severely compromised their survival. Although ∼80% of control mice withstood ≥6 hr of acute cold exposure, all of the ERRγKO mice died, indicating the key survival role of ERRγ in this type of acute cold challenge ( Figure 4 E).

These results led us to consider the possibility that ERRγ might be required to direct the many changes needed for TN mice to activate BAT and respond to an acute cold challenge. To initially explore this notion, the thermogenic response of mice to a norepinephrine (NE) injection was monitored by adaptive changes in V. Although control mice increased their metabolic rate upon NE injection by 2,400 ml/kg/hr, ERRγKO mice managed only a 1,200 ml/kg/hr increase, indicating impaired thermogenic capacity at TN ( Figure 4 A). A similar blunted VOresponse was observed in response to the β3 agonist CL316,243 without any change in RER ( Figures S4 G and S4H). As catecholamine signaling is not compromised in ERRγKO mice ( Figures 3 H and 3I), we attribute their impaired thermogenic capacity to reduced basal expression of oxidative and thermogenic genes.

To determine the physiological consequence of the loss of ERRγ in BAT at TN, mice were maintained on standard chow or challenged with a high-fat diet (HFD). Over a period of 14 weeks, the weight gain of ERRγKO mice was similar to control mice on both diets ( Figure S4 A), and no differences in body composition, serum parameters, or insulin and glucose tolerance were seen ( Figures S4 B–S4F). Furthermore, with the exception of BAT, the gross appearance and weights of the major metabolic organs in ERRγKO mice were indistinguishable from control mice (data not shown). Notably, these findings indicate that loss of BAT identity due to lack of ERRγ does not predispose mice to diet-induced obesity or insulin resistance when housed at TN. Additionally, there were no changes in browning of WAT ( Figures S3 F and S3G), significant differences in RER, VO, physical activity, or food intake between WT and ERRγKO mice upon gradual acclimation to cold ( Figures S3 H–S3K).

Interestingly, although genes important for BAT function were downregulated, expression of the β3 adrenoreceptor Adrb3 was significantly upregulated in ERRγKO BAT ( Figure 2 A). To explore whether this change was functionally relevant, catecholamine signaling was compared in wild-type (WT) and ERRγKO mice. Although cAMP levels were similar in BAT lysates from the two genotypes after treatment with the β3 agonist CL316,243, increased levels of hormone-sensitive lipase (HSL) phosphorylation (p-HSL) were found in ERRγKO BAT, indicating enhanced catecholamine sensitivity ( Figures 3 H, 3I, and S3 E). Taken together, these findings suggest that ERRγKO mice may upregulate catecholamine signaling as a compensatory mechanism for their defective basal thermogenic program.

The marked decrease in Ucp1 and unexpected increase in Lep expression in ERRγKO mice suggested that ERRγ is required to maintain BAT functionality under TN conditions () ( Figure 3 A). We attribute these changes to the regulation of BAT-selective genes, as ERRγ deficiency did not affect the expression of and was not found on the promoters of WAT-selective genes at TN ( Figure S3 A; data not shown). These findings indicate that ERRγ is required for maintaining the robustness of the brown adipose phenotype by promoting the expression of key BAT-selective genes. In addition to these transcriptional changes, ERRγKO BAT was visibly smaller and paler than in control flox/flox mice ( Figures 3 B and S3 B), and increases in lipid droplet size and triacylglycerol (TAG) levels were observed ( Figures 3 C–3E). Furthermore, although mitochondria number and size were unchanged ( Figures S3 C–S3D), the percentage of uncoupled respiration was reduced in mitochondria isolated from ERRγKO BAT ( Figure 3 F), consistent with the lower levels of UCP1 ( Figure 2 E). Moreover, fatty acid metabolism was compromised in ERRγKO BAT mitochondria, as evidenced by reduced palmitate-fueled relative to glutamate-fueled oxygen consumption rate (OCR) ( Figure 3 G), in agreement with the observed changes in gene expression ( Figures 2 A and 2B).

We next investigated if ERRγ binding affects the accessibility of chromatin by performing assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in BAT from ERRγKO and flox/flox control mice that had been acclimated to TN. Notably, we observed higher chromatin accessibility at promoter proximal regions (±5 kb of TSS) of ERRγ-regulated genes compared to non-ERRγ-regulated genes ( Figure 2 F). Additionally, the accessibility was reduced upon ERRγ knockout, suggesting that ERRγ further induces the open chromatin environment ( Figure 2 F, left). Although we observed less ATAC-seq signal around non-ERRγ-regulated genes, no changes were observed upon loss of ERRγ ( Figure 2 F, right). Taken together, these findings indicate that ERRγ acts to prime TN BAT for thermogenesis by promoting DNA accessibility around key genes needed for the rapid response to cold exposure.

A complementary story was recently published identifying a role for HDAC3 in establishing a transcriptional landscape in BAT required for the rapid response to acute cold challenge (). Dating mining of RNA-seq from HDAC3 KO BAT () and comparison with RNA-seq from ERRγKO BAT (both performed at TN) revealed commonly downregulated genes in HDAC3KO and ERRγKO BAT, including the key genes UCP1 and Cox7a1, raising the logical possibility of cooperation between HDAC3 and ERRγ in BAT ( Figure S2 F). However, the majority of the downregulated genes were unique to the specific model, consistent with predominantly parallel and possibility complementary regulatory pathways.

To explore the mechanisms through which ERRγ controls the expression of metabolic genes in the basal state, we determined its genome-wide binding sites in BAT of control mice acclimated to TN. ERRγ-bound regions identified by ChIP-seq were significantly enriched in ERRγ motifs, consistent with direct DNA binding ( Figure 2 C). In addition, bound regions were enriched in CEBPA and EBF motifs, suggesting co-localization of ERRγ with other important regulators of thermogenesis ( Figure 2 C) (). Although the majority of binding sites were in intergenic and intronic regions, as reported for many transcription factors ( Figure S2 D), we found ERRγ directly bound to promoter regions of key BAT genes downregulated in ERRγKO mice, including Ucp1 and Fabp3 ( Figure 2 D). Accordingly, we found a ∼50% reduction in UCP1 protein in ERRγKO BAT ( Figure 2 E) supporting a central role for ERRγ in BAT function. GO analysis of the subset of genes bound by ERRγ (±1 kb of transcription start site [TSS]) and downregulated in ERRγKO BAT at TN revealed the same pathways and processes as those identified when all downregulated genes in ERRγKO BAT were analyzed, supporting a direct role for ERRγ in regulating the expression of these key genes involved in mitochondrial function at TN ( Figures S2 E and 2 B).

Interestingly, loss of ERRγ led to a reduction in BAT Pparα expression at TN ( Figure 2 A, bottom). As PPARα controls genes involved in fatty acid use and thermogenesis in BAT (), we explored whether the downregulation of BAT signature genes in ERRγKO mice was secondary to decreased PPARα expression. Comparative gene expression analysis of BAT from Pparα-null mice under TN conditions revealed that PPARα and ERRγ control largely unique gene sets, with minimal overlap in downregulated genes ( Figures S2 B and S2C). These findings indicate that the reduction in Pparα expression is not causative in the altered ERRγKO gene signature.

In order to investigate the function of ERRγ in BAT metabolism, ERRγKO mice and control flox/flox littermates were housed under chronic cold acclimated conditions (4°C; see Figure 2 A, top), mild cold stress (22°C, room temperature [RT]), or at thermoneutrality (30°C, TN), and their BAT gene signatures were compared using RNA sequencing (RNA-seq). Two hundred ninety-one genes were dysregulated in ERRγKO mice under chronic cold conditions (149 up- and 142 downregulated) and 71 genes at RT (34 up- and 37 downregulated) ( Figure S2 A). Strikingly, 380 expression changes were observed between mice housed at TN, at which 156 and 224 genes were up- and downregulated, respectively ( Figure S2 A). Notably, there was minimal overlap in dysregulated genes among the three temperature conditions ( Figure S2 A). We next examined the expression of genes important for BAT identity and thermogenesis, lipid metabolism, and oxidative phosphorylation in BAT from ERRγKO mice relative to control flox/flox littermates. Indeed, we found that the majority of these key BAT genes were dysregulated at TN, while there were minimal changes under cold or RT conditions ( Figure 2 A, bottom). In addition, Gene Ontology (GO) analysis of all significantly downregulated genes in ERRγKO BAT under TN conditions identified mitochondrion, respiratory chain, translation (the majority of genes were involved in mitochondrial translation), and fatty acid metabolic process as the predominant dysregulated pathways and processes ( Figure 2 B).

Errγ is highly expressed in BAT versus WAT under basal, TN conditions, similar to the profile observed in human adipocyte cell lines ( Figures 1 A and 1B ) (). Notably, at TN, Errγ is found in the mature adipocyte fraction but not in the stromal vascular fraction (SVF), suggestive of a role in brown adipocyte function ( Figure 1 C). Consistent with this notion, we find ERRγ to be induced late during differentiation of the PAZ-6 human brown adipose cell line () ( Figure 1 D). Furthermore, Errγ expression in BAT positively correlates with important BAT genes, including Ppargc1a, Cox7a1, and Ucp1 in 37 diverse strains of mice from the BXD genetic reference population () ( Figure 1 E). However, in contrast to the marked induction upon chronic cold exposure of Ppargc1a and Esrra (encoding ERRα), Errγ expression was unaffected, consistent with a role in BAT priming ( Figure 1 F) (). In order to investigate the role of ERRγ in BAT function, we generated ERRγ adipose conditional KO (ERRγKO) mice and confirmed its selective deletion in BAT ( Figures 1 G and S1 A). Consistent with this, ERRγ protein was not detectable in BAT from ERRγKO mice ( Figure 1 H). ERRγKO mice were born at a normal Mendelian ratio and exhibited no obvious phenotypic abnormalities under standard animal housing conditions ( Figures S1 B–S1F).

Discussion

This study uncovers a mechanism for maintaining BAT structure and functional readiness in the TN state. Surprisingly, in TN mice with ERRγ deficiency, the BAT depot whitens, shrivels, and functionally disintegrates, resulting in a physically lethal hypothermia if exposed to sudden cold. The intrinsic transcriptional activity of ERRγ allows it to prime TN BAT in the absence of inducible drivers such as PGC1α. By maintaining thermogenic genes in an accessible state, BAT remains robust and ready for thermogenesis, in turn allowing a TN host to respond quickly to acute cold exposure.

BAT is inherently different than WAT, even under basal conditions. Our studies here identify ERRγ as an important factor in maintaining its gene expression signature, morphology, and physiological function. Understanding how these fundamental differences between BAT and WAT are maintained is important to understand BAT biology. Indeed, we show that ERRγ drives a transcriptional program that includes key genes necessary for proper mitochondrial function in BAT, including genes involved in FA use, uncoupling, mitochondrial translation, and oxidative phosphorylation. Mice lacking ERRγ in BAT have dysfunctional BAT mitochondria and are unable to appropriately respond to an acute cold challenge ( Figure 4 F). We found that ERRγ is required to maintain DNA accessibility around key BAT genes at TN. ERRγ likely allows the establishment of the appropriate chromatin template needed to facilitate the rapid response to cold exposure, effectively priming BAT for thermogenesis.

A logical extension of this work is the suggestion that thermogenesis may not be a single or monolithic state but may occur in diverse ways to anticipate different challenges. For example, the adaptive induction of PGC1α allows this cofactor to become gradually induced under progressive cold challenge (such as the cool state of RT or a gradual reduction from RT to life-threatening cold (e.g., 4°C). In this context, ERRα dormant isoform exploits PGC1α induction at RT and below to enhance mitochondrial biogenesis and thermogenic capacity in an adaptive fashion. In contrast, sudden cold exposure from the TN state apparently depends on a baseline thermogenic capacity, which is highly compromised in ERRγKO mice. This supports the argument that there are mechanistically two distinct types of thermogenic challenge and that the differing dependences on ERRα, ERRβ, and ERRγ and their need for PGC1α exploits this potential. This duality holds some analogy to the roles of ERRs and PGCs in skeletal muscle in which ERRγ is intrinsically high in the mitochondria-rich and highly vascularized type I muscle fiber, while ERRα dominates in the PGC1α-dependent type II fiber.

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Seale P. Prdm16 is required for the maintenance of brown adipocyte identity and function in adult mice. Interestingly, loss of ERRγ in BAT does not predispose mice to systemic metabolic dysregulation on a HFD. This finding is reminiscent of mice lacking PRDM16 in BAT (Myf5-Cre PRDM16f/f mice). PRDM16 has been shown to be a critical component of the transcriptional network that drives and maintains BAT identity (). Similar to ERRγKO mice, Myf5-Cre PRDM16f/f mice do not gain more weight than WT mice despite a severely blunted thermogenic capacity. Interestingly, brown adipocytes derived from PRDM16-knockout BAT have decreased expression of Errγ as well as BAT-selective ERRγ target genes, including Ucp1, Coxa1, and Pparα. It is possible that some PRDM16 effects are mediated through ERRγ and also that these factors may synergize to maintain BAT identity. Further studies, including comparison of binding sites between ERRγ and PRDM16, as well as other BAT transcription factors at TN, will be important to unravel the mechanistic hierarchy of factors that cooperate to drive transcription in brown adipocytes.