Epigenetic dynamics of the thermogenic gene program of adipocytes

Abstract

Brown adipose tissue (BAT) is a metabolically beneficial organ capable of burning fat by dissipating chemical energy into heat, thereby increasing energy expenditure. Moreover, subcutaneous white adipose tissue (WAT) can undergo so-called browning/beiging. The recent recognition of the presence of brown or beige adipocytes in human adults has attracted much attention to elucidate the molecular mechanism underlying the thermogenic adipose program. A number of key transcriptional regulators critical for the thermogenic gene program centering on activating the UCP1 promoter, have been discovered. Thermogenic gene expression in brown adipocytes rely on coordinated actions of a multitude of transcription factors, including EBF2, PPARγ, Zfp516 and Zc3h10. These transcription factors probably integrate into a cohesive network for BAT gene program. Moreover, these transcription factors recruit epigenetic factors, such as LSD1 and MLL3/4, for specific histone signatures to establish the favorable chromatin landscape. In this review, we discuss advances made in understanding the molecular mechanism underlying the thermogenic gene program, particularly epigenetic regulation.

Introduction

Adipose tissue has a central role in controlling mammalian metabolic homeostasis. White adipose tissue (WAT) is specialized to store excess calories in the form of triglycerides, while brown adipose tissue (BAT), evolutionarily, functions as a mechanism to combat hypothermia via non-shivering thermogenesis [1]. Classic brown adipocytes contain a high density of mitochondria that constitutively express Uncoupling protein 1, UCP1, to dissipate chemical energy as heat by uncoupling fuel combustion from ATP production [1, 2]. In addition, so-called beige adipocytes in WAT depot are recruited upon β₃-adrenergic activation [3–5]. Thus, several human studies demonstrated the emergence of thermogenic tissues in adults after chronic cold exposure even in subjects who initially lacked detectable BAT or BAT-like depots [6–9]. Based on several cross-sectional studies, human BAT or BAT-like tissue is inversely correlated with body mass index and visceral fat [7, 10, 11], and BAT activity is correlated with positive metabolic outcomes. These findings owe considerable attention as a promising avenue to combat obesity and associated metabolic diseases.

One of the major advances in the field of BAT biology has been identification of essential transcription factors and cascades, which are involved in brown and beige adipose development. As in most biological processes, BAT, especially beige fat, rely heavily on environmental cues for its full activation of the thermogenic gene program. Therefore, it is imperative to understand interactions between genetic components and environment factors. Epigenetic changes encompass various modifications to both DNA and histones. This review is focused on recent advances in the molecular mechanism underlying the epigenetic regulation of thermogenesis of brown/beige adipocytes.

Origin of brown and beige adipocytes

Brown adipocytes derive prenatally from the sub-population of dermomyotome precursors expressing Myf5, En1 and Pax7 [3, 12, 13]. These dermomyotome precursor cells differentiate into brown preadipocytes which express Early B cell factor-2 (EBF2), a brown preadipocyte commitment marker, localizing in interscapular regions of rodents and human infants [11]. Ablation of EBF2 in mice results in a complete loss of BAT function as UCP1 was barely detectable, while expression of white fat selective genes was increased [14]. EBFs have been reported to directly bind and activate Peroxisome proliferator activated receptor γ (PPARγ) and CCAAT-enhancer-binding proteinα (C/EBPα) promoters for adipogenesis [11]. Importantly, EBF2 was shown to facilitate binding of PPARγ to BAT-selective targets, such as PR domain containing 16 (PRDM16) to determine BAT identify [14].

In contrast to brown adipocytes, beige adipocytes develop postnatally within WAT depots upon appropriate environmental cues such as cold exposure and feeding. The origin of precursor cells of beige adipocytes is more complicated as beige adipocytes may not include one cell type, but encompass multiple types of cells that can emerge in WAT in response to the external stimuli [15, 16]. Thus, beige adipocytes may derive from heterogeneous precursors including progenitor cells expressing Sma, Myh11, PDGFRα, or PDGFRβ [17–20](reviewed in [21]). Moreover, it is still controversial whether beige adipocytes arise from precursors via de novo differentiation and/or from transdifferentiation of existing white adipocytes [22–25].

Transcriptional regulation of the thermogenic gene program

It is clear that gene programs controlling cellular functions are regulated by a web of coordinated transcriptional events, integrating at chromatin. Thus, a network of transcription factors that directly bind to promoter or enhancer regions along with their recruited epigenetic factors must work together to fine-tune thermogenesis in response to varying environmental conditions. Moreover, adaptive thermogenesis relies on a coordinated signaling cascade in response to cold [26]. In particular, increased UCP1 transcription has been shown to be dependent on β₃-adrenergic receptor-PKA-p38 MAPK pathway by phosphorylating Activating transcription factor 2 (ATF2) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), both of which bind the −2.5 kb enhancer region of the UCP1 gene [26–28](reviewed in [29]). Recently, zinc finger CCCH-type containing 10 (Zc3h10), a BAT enriched, cold induced transcription factor, that binds near the −4.6 kb UCP1 promoter region was shown to be another downstream target of p38 MAPK for activation of UCP1 and other thermogenic genes [30].

Much attention has also fallen on cofactors of thermogenesis that are recruited by transcription factors, most notably, PRDM16 and PGC1α. PGC1α is a key regulator of mitochondrial biogenesis and is highly expressed in brown/beige adipocytes with their abundant mitochondria in which UCP1 resides and functions for thermogenesis [31]. Importantly, PRDM16 was first identified to be required for BAT gene program [32]; Since PRDM16 does not bind DNA, PRDM16 relies on transcription factors, such as Zfp516 that binds the UCP1 promoter region of −70 bp, as well as those that bind the −2.5 kb UCP1 enhancer region, such as C/EBPβ, PPARγ and C-terminal binding protein (CtBP) [33–36]. PRDM16 was shown also to interact with a Mediator subunit, MED1 that may bridge promoter and enhancer regions of BAT-enriched genes with RNA Pol II and the transcriptional machinery [32, 37]. Key transcription factors that activate the thermogenic gene program are described below (Table 1).

Table 1.

Histone modifications of thermogenic genes

Histone modification	Histone modifiers	Histone modifying enzymes	Histone marks	Effect	Effectors and interacting proteins	loss- and gain-of-function refs.
Histone acetylation	HAT	Gcn5/PCAF	H3K9ac	Activation	PPARγ, PRDM16	[65, 66]
	HDAC	HDAC1	H3K27	Repression	UCP1, PGC1α, PRC1/2	[67, 68, 102, 103]
		HDAC3	PGC1α	Repression	PGC1a, ERRα	[67, 69–71]
		HDAC9	N/A	Repression	N/A	[72]
		HDAC11	H3K27	Repression	BRD2	[74]
		SIRT1	PGC1α	Activation	PPARγ, PGC1α	[75, 104]
		SIRT3	PGC1α	Activation	PGC1α	[76, 77]
		SIRT6	PGC1α	Activation	ATF2	[78]
Histone methylation	HMT	MLL3/4	H3K4me1/2	Activation	PPARγ, C/EBPα, C/EBPβ, CBP/p300	[81–83]
		G9a	H3K9me2	Repression	PPARγ, C/EBPβ	[80, 105]
		EHMT1	H3K9me2/3	Activation	PRDM16	[32, 40, 79, 106]
	KDM	JMJD1A	H3K9me1/2	Activation	PPARγ, PGC1α SWI/SNF	[84–86]
		LSD1	H3K9me1/2	Activation	Zfp516, PRDM16, Nrf1	[49, 87–89, 97]
		JMJD3	H3K27	Activation	Rreb1	[92]
		UTX	H3K27me2/3	Activation	CBP	[68, 91]

PPARγ

PPARγ is a master regulator of adipocyte differentiation [38, 39]. Interestingly, treatment of PPARγ agonists, such as thiazolidinedione (TZD) in white adipocytes has been shown to induce browning to obtain brown adipocyte-like characteristics [40]. The treatment of PPARγ agonists in mice increases expression of brown adipose-selective markers, including UCP1 and CideA in inguinal WAT, and induces mitochondrial biogenesis with increased expression of PGC1α. Gray et al. showed that mice expressing dominant-negative PPARγ (P465L PPARγ) had impaired activation of thermogenic genes, such as UCP1 and PGC1α, in response to cold with reduced thermogenic capacity [41].

PPARγ increases UCP1 gene expression by directly binding to the −2.5 kb enhancer region of the UCP1 promoter. Genome-wide binding analyses revealed that PPARγ binds to other brown adipose-selective genes also [32]. It has also been reported that SIRT1 dependent deacetylation of PPARγ is necessary to recruit PRDM16 to PPARγ for thermogenic gene expression in white adipocytes [42]. Moreover, PPARγ interacts with PGC1α to activate thermogenic genes. In addition, expression of PGC1α can be increased rapidly in response to β₃-adrenergic stimulation, which is mediated by PKA-p38 MAPK pathway to enhance its stability and transcription. PGC1α enhances expression of several thermogenic genes involved in adaptive thermogenesis, as well as mitochondrial biogenesis such as Nuclear respiratory factor-1 (Nrf1) [28, 41, 43–45].

IRF4

Interferon regulatory factor 4 (IRF4) is one of key transcriptional drivers of the thermogenic gene program. IRF4 is a cold-inducible transcription factor that activates the UCP1 promoter by binding at the −1.3 kb region. Mice with IRF4-deficiency in UCP1⁺ cells had a reduced thermogenic gene expression, resulting in cold intolerance. Accompanied with a reduced thermogenic capacity and energy expenditure, these mice were obese and insulin resistant. IRF4 and PGC1α induce each other’s expression. Moreover, it has been shown that IRF4 binds to an interferon-stimulated response element (ISRE) of the UCP1 promoter and that IRF4 directly interacts with PGC1α to fully activate the UCP1 promoter [46]. Moreover, IRF4 was reported to regulate metabolism. IRF4 expression was reported to be induced in both brown and white adipocytes in the fasted state when, due to the absence of insulin, FoxO1 remains in the nucleus to activate the IRF4 promoter [47]. IRF4 also participates in regulation of lipolysis by inducing expression of ATGL and HSL. Thus, IRF4 can help to generate fatty acid substrates that can fuel the mitochondrial activity and also can bind UCP1 for its activation.

Zfp516

Zinc finger protein 516 (Zfp516), a cold-inducible, BAT-enriched transcription factor, has been identified by high-throughput screening of transcription factors that can activate −5.5 kb UCP1 promoter. In this regard, previous studies showed that the −4.5 kb UCP1 was sufficient to drive BAT-specific and regulation expression in transgenic mice [48]. Through a series of luciferase assays and ChIP-qPCR, Dempersmier et al. showed that Zfp516 functions by binding to a proximal region of the UCP1 promoter for transcriptional activation [33]. Zfp516 also binds the PGC1α promoter region for activation. Overexpression of Zfp516 in adipose tissue in mice increased expression of thermogenic genes such as UCP1, PGC1α, Elongation of very long chain fatty acids protein 3 (Elovl3) and Cytochrome c oxidase subunit 8B (Cox8b), as well as induction of drastic browning in inguinal WAT (iWAT). Thus, Zfp516 transgenic mice had enhanced thermogenic capacity and energy expenditure, resulting in protection against diet-induced obesity. Importantly, Zfp516 represses the myogenic program and drives the BAT gene program to promote brown adipocyte differentiation, similar to PRDM16 overexpression. Indeed, Zfp516 directly interacts and recruits PRDM16. Zfp516 directly interacts also with a histone demethylase, Lysine-specific histone demethylase 1 (LSD1), to recruit LSD1 to promoter regions of thermogenic genes for transcriptional activation [49].

Zc3h10

Zc3h10 is a recently reported another BAT-enriched, cold-induced transcription factor that activates the UCP1 promoter [30]. In fact, Zc3h10 has been reported as a RNA binding protein for pri-miRNA processing [50]. However, Yi et al. show that Zc3h10 activates the UCP1 promoter by directly binding to the −4.6 kb region via its bZIP domain [30]. Zc3h10 also binds and activates Mitochondrial transcription factor A (Tfam) and Nrf1, that are known for mitochondrial biogenesis. Transgenic mice overexpressing Zc3h10 in adipose tissue showed an enhanced thermogenic capacity and energy expenditure, protected from diet-induced obesity. Moreover, transgenic mice overexpressing Zinc finger-mutant Zc3h10, which cannot bind RNA, still showed the same effect, clearly demonstrating Zc3h10 functioning as a transcription factor for the thermogenic gene program. Ablation of Zc3h10 in UCP1⁺ cells in mice resulted in decreased thermogenic gene expression, severe cold intolerance and reduced energy expenditure, leading to adiposity. Via a combination of ChIP and luciferase assays along with in vitro and in vivo phosphorylation studies, Yi et al. showed that phosphorylation of Zc3h10 at S126 by p38 MAPK is required for Zc3h10 binding to the distal UCP1 promoter region for transcriptional activation. Nonphosphorylatable S126A-Zc3h10 mutant could not bind to the target sites for transcriptional activation. It is plausible that, upon cold exposure, Zc3h10 that binds the −4.6 kb region, along with ATF2 and PGC1α that bind at the −2.5 kb enhancer region of the UCP1 promoter, are all phosphorylated by p38 MAPK to work together in a cooperative manner to activate UCP1 transcription [27, 28].

Epigenetic factors for histone modification for thermogenesis

As with most complex diseases, obesity results from gene and environment interactions. Thus, temperature and diet may influence the thermogenic gene program through epigenetic events [51]. A major epigenetic event involves modifications of histones which are the building blocks of nucleosome composed of histone octamer core (2 copies of H2A, H2B, H3 and H4) wrapped around by DNA. Because N-terminal histone tails are protruded from the histone octamer core, they serve as major sites of post-translational modification including acetylation, methylation, phosphorylation and ubiquitination. Histone modification is mediated by a balance of various writers that deposit specific histone marks and erasers that remove histone modifications. Readers or effector proteins can recognize and bind specific histone marks for their recruitment to chromatin for chromatin remodeling [52]. Therefore, histone modifications are important drivers of controlling gene expression as it alters chromatin accessibility and transcriptional activity through the combined actions of epigenetic enzymes.

In brown adipose biology, histone acetylation and methylation are most well-studied among epigenetic modifications (reviewed in [53, 54]). Histone acetylation is catalyzed by histone acetyltransferases (HATs) using an intermediary metabolite, acetyl-CoA. The transfer of acetyl group to lysine residue decreases the positive charge, thereby weakening the electrostatic interaction between histones and DNA thus loosening chromatin compaction [55]. Lysine acetylation is associated with gene transcription as readers of lysine acetyl marks, such as Bromodomain protein 4 (BRD4). BRD4 recognizes histone acetylation and recruits Positive transcription elongation factor (P-TEFb) to facilitate RNA Pol II activity in the proximal promoter region [56, 57]. Conversely, histone deacetylation is a gene repressive mark and is catalyzed by histone deacetylases (HDACs). Histone methylation mainly occurs on amino groups of specific lysine that may be mono-, di-, or trimethylated; whereas, the terminal guanidinyl group of arginine residues may be mono- or dimethylated [53]. Histone methylation does not alter the electrostatic charge of histone proteins but rather affects DNA accessibility. While H3K4me3 is an important active mark that recruits TFIID and pre-initiation complex [58–60], not all cells depend on H3K4me3 for the transcription initiation. Instead, the recruitment of TFIID can be achieved through removal of the repressive H3K27me3 mark in response to different cues [61]. Furthermore, H3K9ac is highly associated with gene transcription at active promoters, and H3K9ac can induce the release of Pol II from the pause to activate elongation through recruitment of the elongation complex [62]. In contrast, H3K9me2 is suggested to be a gene repressive mark and associated with heterochromatin formation [63]. In short, H3K4me2/3, H3K9ac and H3K27ac are well-recognized hallmarks of transcription activation, whereas H3K9me2/3 and H3K27me2/3 represent gene repression (reviewed in [64]). Moreover, first discovered in embryonic stem cells, “bivalent” chromatin bears both active mark, H3K4me3, and repressive mark, H3K27me3, at genes that are inactive but poised for transcription [61]. Regardless, the interplay between transcription factors and epigenetic modifiers is crucial for epigenetic dynamics. Histone modifications are context dependent, and some of the recent advances in histone modifiers in thermogenic gene regulation are discussed below.

Histone acetyltransferases

A well-studied histone acetyltransferase, Gcn5 is a highly conserved HAT with its mammalian paralogue, PCAF, and acetylates H3K9. Both PCAF and H3K9ac are enriched at transcription start sites of many active genes. Gcn5/PCAF are enriched at promoter regions of PPARγ and PRDM16 to increase their expression for brown adipogenesis [65, 66]. Jin et al. reported that Gcn5/PCAF has multiple functions in BAT cell differentiation. In Gcn5/PCAF double knockout brown preadipocytes, induction of PPARγ as well as its downstream target genes such as aP2, was severely impaired, as transcription elongation of PPARγ was hindered. In addition, Gcn5/PCAF double ablation prevented PRDM16 expression. This was due to blocking of the recruitment of Pol II onto the PRDM16 promoter, causing severe impairment in brown adipogenesis [66]. In this regard, prolonged IBMX treatment could bypass the need for Gcn5/PCAF by inducing C/EBPβ and likely stimulating C/EBPβ phosphorylation to increase its binding to the PPARγ promoter, but IBMX treatment could not bypass Gcn5/PCAF-dependent PRDM16 expression [66].

Histone deacetylases

Many studies revealed that HDAC activity is critical in regulating thermogenic gene program. Inhibition of Class I selective HDACs in C2C12 cells was shown to promote mitochondrial biogenesis and mitochondrial activity by inducing several key genes for mitochondrial biogenesis, such as Tfam and PGC1α [67]. HDAC1 was reported to negatively regulate the thermogenic gene program through deacetylation of H3K27 [68]. HDAC1 not only decreases H3K27 acetylation but also can physically interact with PRC1/2 complexes including EZH2, SUZ12 and RNF2 to lead to H3K27 trimethylation at UCP1 and PGC1α promoter regions, further compacting chromatin [68]. Thus, overexpression of HDAC1 in brown adipocytes blocks β₃-adrenergic stimulated BAT gene expression, while HDAC1 knockdown in brown adipocytes results in increased H3K27ac and reduced H3K27me3 at promoter regions of UCP1 and PGC1α, leading to increased thermogenic gene expression [68]. β₃-adrenergic stimulation suppresses HDAC1 expression and dissociates HDAC1 from promoter regions of BAT enriched genes thereby increasing H3K27 acetylation for transcriptional activation. Other groups reported different mechanisms by which Class I HDACs can repress the thermogenic gene expression. HDAC3 along with HDAC4, HDAC5 and the nuclear corepressor, NCoR, may be recruited by members of the myocyte enhancer factor 2 (MEF2) family of transcription factors to repress PGC1α transcription [69, 70]. Interestingly, HDAC3 was reported to activate the thermogenic aptitude of brown adipose tissue by deacetylating PGC1α. Ablation of HDAC3 via UCP1-Cre or adiponectin-Cre in mice caused a rapid loss of core body temperature. HDAC3 mediated deacetylation of PGC1α was reported to coactivate an ERRα-driven transcription of UCP1 and PGC1α as well as genes involved in oxidative phosphorylation [71].

Ablation of a Class II HDAC, HDAC9 has been shown to enhance beige adipogenesis, and the HDAC9 global knockout mice had increased energy expenditure and adaptive thermogenesis, particularly upon high fat diet feeding (HFD)[72]. In this regard, HFD feeding was reported to decrease thermogenic gene expression with diminished expression of Fibroblast growth factor 21 (FGF21), an autocrine/paracrine factor that can induce thermogenic genes, while HDAC9 ablation blunted this decrease in obese mice [73]. However, how HDAC9 ablation specifically increases beige adipogenesis still remains to be studied. In addition, a Class IV HDAC, HDAC11 was reported to function as a repressor of the thermogenic gene program. Global knockout of HDAC11 in mice stimulated BAT development, beiging of WAT, and enhanced thermogenic activity through its association with BRD2, a member of the BET family of acetyl histone binding proteins [74].

Unlike other classes of deacetylases which mainly function on histone residues, Class III deacetylases, SIRT1 and SIRT3 are implicated to act on PGC1α. Activation of cAMP/PKA pathway causes SIRT1 phosphorylation, increasing its catalytic activity to deacetylate PGC1α [42, 75–77]. Mice overexpressing SIRT1 had increased BAT activity, fatty acid oxidation and energy expenditure. Recently, cold-inducible, SIRT6 was identified to stimulate the thermogenic gene program by interacting with phosphorylated ATF2 for PGC1α transcription [78]. Overall, these studies demonstrate evidence that various HDACs are critical regulators of the thermogenic gene program.

Histone methyltransferases

In the search of how PRDM16 complex controls the cell fate between muscle and brown fat cells, Euchromatic histone N-lysine methyltransferase 1, EHMT1 is known to be histone methyltransferase that catalyzes H3K9 di- or tri-methylation (H3K9me2/3). Ohno et al. reported EHMT1 as a part of the PRDM16 complex to control BAT cell fate [79]. EHMT1 is highly enriched in BAT and in cultured BAT cells, which correlates with PRDM16 expression. They showed that ablation of EHMT1 in brown adipocytes induced muscle differentiation by decreasing H3K9me2/3 at muscle gene promoter regions. Interestingly, these authors also showed that EHMT1 promoted expression of BAT-enriched genes by stabilization of PRDM16. Thus, ablation of EHMT1 in adipose tissue in mice caused impairment of thermogenesis bringing obesity and insulin resistance.

Other histone methyltransferases, such as G9a and Mixed-lineage leukemia proteins (MLL3/4), also play roles in regulating thermogenesis. G9a directly represses PPARγ by catalyzing H3K9me2, while G9a deletion enhances chromatin opening to allow binding of an adipogenic transcription factor C/EBPβ at the PPARγ promoter region [80]. MLL3/4, H3K4me1/2 methyltransferases, are known to function at the enhancer regions for cell type specific gene expression [81, 82]. MLL3/MLL4 are required for recruitment of CBP/p300, H3K27 acetyltransferases that are found to be enriched on active enhancers during brown adipogenesis. Through RNA-seq and ChIP-along with FAIRE-seq for chromatin opening, Lai at al. showed that MLL3/4 are required for superenhancer formation by MLL3/4 priming followed by H3K27 acetylation by CBP/p300, thereby sequentially shaping the dynamic enhancer landscapes during adipogenesis [81]. In BAT, more than 200 genes related to metabolism were altered, suggesting an important role of H3K4 methylation status [83].

Histone demethylases

H3K9 methylation has been implicated to regulate BAT development and the thermogenic gene program. Jumonji domain containing 1A, JMJD1A (also called Jhdm2a or KDM3A), activates thermogenic gene expression by catalyzing demethylation of H3K9me1/2 and is associated with lower H3K9me2 at the UCP1 enhancer and promoter regions. Using a hypomorphic mouse model for Jhdm2a lacking the catalytic JmjC domain, Okada et al. showed that Jhdm2a deficiency in mice caused hyperlipidemia and insulin insensitivity [84]. BAT of these mice had lower mitochondrial number, leading to lower oxygen consumption [85]. Furthermore, β₃-adrenergic dependent phosphorylation of JMJD1A facilitated the recruitment of PGC1α and PPARγ to the promoter regions of UCP1 and fatty acid oxidative genes. Upon cold exposure, JMJD1A is phosphorylated at S265 by PKA, which then interacts with the chromatin remodeling complex, SWI/SNF to allow thermogenic gene transcription [86].

Another H3K9 demethylase that regulates the BAT gene program is lysine-specific demethylase 1 (LSD1). Sambeat et al. showed that through a direct interaction with Zfp516, LSD1 is recruited to the proximal promoter region of UCP1, as well as the PGC1α promoter region. LSD1, in turn, demethylates H3K9me1/2 for transcriptional activation [49]. LSD1 ablation in UCP1⁺ cells in mice impairs BAT development and compromises thermogenic capacity. LSD1 ablation in adipose tissues via adiponectin-Cre also impairs the thermogenic gene program and prevents browning of WAT, resulting in adiposity. Furthermore, LSD1 was shown to be necessary for Zfp516 dependent-browning of WAT as induction of thermogenic gene expression was significantly reduced when adipose specific Zfp516 overexpressing mice were crossed with adipose-specific LSD1 knockout mice [49]. Duteil et al. reported that LSD1 was found to interact with Nrf1 to drive mitochondrial biogenesis to support the maintenance of BAT function [87] and to repress WAT-selective genes through interaction with the CoREST complex in BAT [88]. Zeng et al. also detected promotion of the thermogenic gene program by LSD1. However, they reported that this was via repressing a key glucocorticoid-activating enzyme, HSD11B1, that can promote lipid accumulation. They also described that LSD1 interaction with PRDM16 suppressed white fat-selective genes, through demethylation of H3K4me1/2 [49, 89].

Brown adipocytes show lower repressive H3K27me3 at the promoter regions of thermogenic genes [90]. In fact, UTX, a H3K27me2/3 demethylase was reported to be induced upon β₃-adrenergic stimulation to induce thermogenic genes in BAT and WAT. UTX is recruited to promoter regions of UCP1 and PGC1α to decrease H3K27 methylation and to interact with CBP/p300 that, in turn, increases H3K27 acetylation, thereby switching from repression to activation of thermogenic genes [91]. Overexpression of another H3K27me3 demethylase, JMJD3 also was shown to increase thermogenic gene expression in BAT and WAT upon β₃-adrenergic stimulation. JMJD3 was reported to be recruited to the promoter regions of UCP1 and CideA through a transcription factor, Rreb1 in promoting BAT gene expression and browning of WAT [92].

Regulation of epigenetic modifications

Various external cues, such as cold, nutrient availability and stress, are particularly relevant to affect beige adipogenesis with its recruitability. Cold is a potent stimulus of thermogenesis in both brown and beige adipocytes. Upon cold exposure, catecholamines are released from sympathetic nerve endings, activating β₃ -adrenergic receptors to increase intracellular cAMP concentration which then activates PKA-p38 MAPK pathway. p38 MAPK can also be activated by cardiac natriuretic peptides upon cold exposure[93]. p38 MAPK phosphorylates ATF2 which triggers transcriptional activation of UCP1 and PGC1α [27]. Through quantitative phosphoproteomics analyses, Casein Kinase 2 (CK2) was identified recently to be a negative regulator of thermogenesis. CK2 is preferentially activated in white adipocytes, and inhibition of CK2 promotes cAMP stimulated thermogenesis [94]. The authors reported that CK2 phosphorylates and inactivates class I HDACs. Pharmacological inhibition of CK2 in mice promoted an induction of beige adipocytes, protecting mice from diet-induced obesity and insulin resistance. Likewise, expression of several of histone modifiers is regulated by cold and β₃ -adrenergic stimulation. Expression of JMJD1a and LSD1 is induced by β₃ -adrenergic stimulation. Furthermore, PKA phosphorylates JMJD1a to form a complex with SWI/SNF chromatin remodeler, thus bringing transcription factor bound-enhancer regions to thermogenic gene promoters in close proximity [86].

Changes in intracellular metabolic availability may serve as additional levels of epigenetic control and chromatin landscape (reviewed in [95]). Each of the histone modifier classes depend on different metabolites as co-factors or co-substrates for posttranslational modifications of histones, and nutrient status is critical in regulating metabolite availability. HATs use acetyl-CoA to transfer the acetyl group to specific lysine residues of histones, whereas SIRTs depend on NAD⁺ in removing the acetyl group. HMTs use S-adenosyl methionine (SAM) to transfer a methyl group, whereas JmjC -domain containing histone demethylases catalyze the reaction in the presence of α-ketoglutarate, a TCA cycle intermediate. LSD1 utilizes FAD as a cofactor for its demethylase activity [96]. For example, as acetyl-CoA and FAD are important metabolites whose levels can change from glucose and fatty acid metabolism; an increase in FAD levels can promote LSD1-mediated transcription, while loss of FAD may attenuate LSD1 activity [97]. Several studies also reported that co-substrates such as butyrate and a-ketoglutarate participate in epigenetic regulation (reviewed in [98]). Recently, accumulating evidence supports that environmental change and nutritional effects, such as maternal n-3 PUFA intake [99], may change epigenetic programming that can be transmitted to subsequent generations. For example, cold exposure of paternal mice may change the DNA methylation pattern of the sperm that enhances thermogenic capacity of the male offspring [100]. Together, it is clear that environmental signals affect gene expression via various signaling pathways and by changes intracellular metabolites, hence altering epigenome. Unraveling such coordinated interplay will help to understand the regulation of chromatin architecture for thermogenic gene transcription.

Concluding remarks and future direction

Brown and beige adipocytes can function by activation of the thermogenic gene program from appropriate environmental cues. Recent studies revealed various transcription factors and epigenetic factors that work together for activation of the thermogenic gene program. However, the precise epigenetic mechanism that interplays with cellular metabolites and ambient temperature, need to be better studied. In addition, although both brown and beige adipocytes are thermogenic, their properties are distinct in that brown adipocytes maintain thermogenic characteristics, while beige adipocytes can be induced to be thermogenic but reversed under changing conditions. Therefore, their origin, development and the spatiotemporal regulation of chromatin architect, leading to thermogenic gene activation may differ [90]. Recently, a unique temperature dependent chromatin plasticity of beige adipocytes was explored, transiting from a brown to a white chromatin state [101]. The beige adipocytes may retain an epigenetic memory of H3K4me1 from prior cold exposure, in the absence of active enhancer mark H3K27ac even at thermoneutrality. These “whitened” beige adipocytes may maintain this cellular memory by an array of poised enhancers for rapid activation in response to cold exposure. Overall, however, how histone modifiers build an epigenetic memory still remain unclear. With recently developed transcriptomic and epigenomic approaches, epigenetic signatures at single-cell resolution may provide better insights into mechanisms specific for each adipocyte type. Moreover, studying human brown and/or beige adipocytes will be imperative for potential future therapeutic targets for obesity and diabetes.

Fig 1. Key transcription factors, coregulators, and epigenetic modifiers of the thermogenic gene program.

Thermogenic gene expression in brown adipocytes rely on coordinated actions of transcription factors, such as PPARγ, Zfp516 and Zc3h10, that directly bind to the promoter or enhancer regions of UCP1. Coregulators, such as PRDM16 and PGC1a are recruited by transcription factors for transactivation of the UCP1 promoter. In addition, transcription factors recruit epigenetic modifiers such as LSD1, HDAC3 including other undiscovered modifiers for histone modification to establish the favorable chromatin landscape of thermogenic genes.