White and brown adipocytes have shared but distinct transcriptional programs that facilitate long-term lipid storage and thermogenesis, respectively. The transcriptional determinants that specify white versus brown fat-selective gene expression are incompletely understood. We have shown here that adipose subtype-selective gene expression is accomplished in part through differential recruitment of the PPARγ cofactors TLE3 and Prdm16. TLE3, which is preferentially expressed in white fat, inhibits the expression of brown-selective Prdm16 target genes while promoting the expression of white-selective genes. Expression of TLE3 in vivo favors the “whitening” of brown adipose tissue and the suppression of adaptive thermogenesis, whereas the loss of TLE3 leads to the “browning” of inguinal WAT. The interaction of Prdm16 with TLE3 and PPARγ is mutually exclusive and TLE3 antagonizes thermogenic gene expression by inhibiting the co-occupancy of Prdm16 and PPARγ on adipocyte promoters. Although PPARγ is well known to interact with a variety of cofactors, to our knowledge this is the first study to directly correlate differential promoter occupancy of PPARγ coactivators with cell type-selective gene expression.
Although the vast majority of adipocyte genes are expressed at similar levels in brown and white adipocytes, a subset is preferentially expressed in one adipocyte type or the other. PPARγ drives a transcriptional cascade common to both white and brown adipocytes, regulating the expression of shared markers of the differentiated phenotype, for example aP2, CD36, Plin, and adiponectin (Tontonoz and Spiegelman, 2008
). But PPARγ also promotes the expression of brown-selective markers such as UCP1 and Elovl3, and white-selective markers such as Ephx1. The mechanistic basis for the tissue-selective actions of PPARγ on brown and white adipocyte promoters is still unclear.
Several auxiliary factors have been identified that work in concert with PPARγ to facilitate brown fat-selective gene expression. The first to be characterized was PGC-1α, a cold-inducible coactivator of PPARγ that drives the expression of UCP1 in brown adipose tissue (Puigserver et al., 1998
). PGC-1α promotes the expression of cold-inducible genes and those linked to mitochondrial oxidative metabolism, but has little effect on the expression of a number of other BAT-selective genes (Uldry et al., 2006
). In addition to promoting adaptive thermogenesis, PGC-1α also plays important roles in oxidative metabolism and mitochondrial biogenesis in heart and skeletal muscle (Lehman et al., 2000
; Wu et al., 1999
). More recently, Prdm16 was identified as a critical factor that drives the entire brown fat-selective transcriptional program through direct interaction with PPARγ (Seale et al., 2008
). Prdm16 is highly enriched in BAT and its forced expression in white preadipocytes or myoblasts promotes a lineage switch to brown fat (Seale et al., 2008
; Seale et al., 2007
). Mice overexpressing Prdm16 in adipose tissue show increased expression of BAT-selective transcripts in certain white adipose tissue depots, such as inguinal WAT (Seale et al., 2011
), and Prdm16 also suppresses the expression of certain WAT-selective transcripts (Kajimura et al., 2008
By comparison, relatively little is known about factors that specify white adipocyte-selective gene expression. It has been unclear whether white fat is simply a “default” transcriptional program that is executed by PPARγ in the absence of Prdm16 and PGC-1α, or whether there are white fat-selective counterparts to these brown-selective cofactors. Our data suggest that TLE3 is one such factor, enforcing an opposing transcriptional program to Prdm16.
We initially identified TLE3 as an adipogenic coregulator in a high-throughput cDNA screen (Villanueva et al., 2011
). TLE3 is induced early in the course of adipocyte differentiation and its expression is directly regulated by PPARγ. TLE3 and PPARγ act synergistically to drive adipocyte gene expression and differentiation, and colocalize on PPAR response elements within adipogenic promoters. PPARγ likely employs some common cofactors to promote gene expression common to both WAT and BAT, perhaps including p300, SRCs and mediator complex, and we initially suspected that TLE3 would promote the expression of all PPARγ target genes equally. Unexpectedly, however, we found that TLE3 exerts differential effects on BAT- and WAT-selective gene expression. Brown adipose tissue from aP2-TLE3 Tg mice showed histological features of white fat and impaired adaptive thermogenesis, and these changes correlated with an impaired capacity to induce thermogenic gene expression during cold challenge. Conversely, mice lacking TLE3 showed enhanced expression of thermogenic genes in both WAT and BAT and improved cold-tolerance. These findings suggest that TLE3 plays a specific role in helping to define the differential brown, white and perhaps beige adipocyte gene programs.
During brown preadipocyte differentiation, the induction of genes involved in lipid biosynthesis and lipid droplet formation occurs prior to the induction of most BAT-selective transcripts such as UCP-1. TLE3 is also induced early during the course of brown adipocyte differentiation, suggesting that TLE3 may be help to establish the lipid-storage and lipid-synthesis capacity common to all adipocytes, and may prevent the induction in brown fat transcripts during early differentiation. During the later stages of brown preadipocyte differentiation, TLE3 expression declines, allowing the brown fat program to proceed. This observation is consistent with the relatively low level of expression of TLE3 in mature BAT compared to mature WAT.
Together with earlier work (Kajimura et al., 2008
), our data suggest that the mechanistic basis for the differential actions of PPARγ in the WAT and BAT gene expression programs involves the formation of cell-type selective transcriptional complexes on the promoters of key adipocyte genes. Prdm16 binds directly to PPARγ, and studies have established that the ability of Prdm16 to interact with PPARγ and C/EBPβ is critical for its ability to drive BAT development (Kajimura et al., 2009
; Seale et al., 2008
). TLE3 is also present in PPARγ-containing transcriptional complexes and can be localized to PPREs by ChIP, although the two proteins do not appear to interact directly (Villanueva et al., 2011
). We have shown that the choice of PPARγ to form complexes with Prdm16 and TLE3 is mutually exclusive. Prdm16 binds to TLE3, and TLE3 competes for the interaction between Prdm16 and PPARγ. Elevated TLE3 expression antagonizes thermogenic gene expression by competing for the occupancy of Prdm16 on adipocyte promoters. Conversely, the loss of TLE3 expression in BAT provides a more permissive context for the actions of Prdm16. It is likely that in cell types where both TLE3 and Prdm16 are present, such as beige adipocytes in WAT depots, PPARγ-TLE3 and PPARγ-Prdm16 complexes exist in equilibrium, and the precise balance between these complexes may help to determine the level of activation for particular promoters. The Prdm16-C/EBP interaction, which does not appear to be affected by TLE3, is also likely to be an important determinant in the activation of certain adipocyte genes.
Overall, these studies highlight an intricate balance between overlapping transcriptional programs that specifies adipose cell-type specific gene expression. Ultimately, a better understanding of the mechanisms by which PPARγ controls the lipid storage and thermogenic gene programs may aid in the development of pharmacologic modulators of adipocyte phenotype. Optimization of PPARγ agonists for the their ability to affect the balance between TLE3 and Prdm16 recruitment may facilitate the therapeutic manipulation of thermogenic gene expression and energy expenditure in humans.