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• W2010364183 abstract "PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program. PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program. ▴ High-throughput cDNA screening identified TLE3 as a promoter of adipocyte differentiation ▴ TLE3 is induced by PPARγ and participates in a feed-forward adipogenic loop ▴ TLE3 facilitates PPARγ action on the promoters of adipocyte target genes ▴ TLE3 blocks β-catenin action and antagonizes Wnt signaling during adipogenesis Adipocytes are specialized cells that store excess energy in the form of triglycerides and also serve an endocrine function, secreting adipokines that influence systemic energy homeostasis (Halaas et al., 1995Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Weight-reducing effects of the plasma protein encoded by the obese gene.Science. 1995; 269: 543-546Crossref PubMed Scopus (4103) Google Scholar, Steppan et al., 2001Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. The hormone resistin links obesity to diabetes.Nature. 2001; 409: 307-312Crossref PubMed Scopus (3780) Google Scholar, Yamauchi et al., 2001Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (3886) Google Scholar). The formation of adipocytes is dependent on peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteins (C/EBPs), transcription factors that coordinately regulate genes involved in lipid metabolism (Freytag et al., 1994Freytag S.O. Paielli D.L. Gilbert J.D. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells.Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (378) Google Scholar; Tontonoz et al., 1994bTontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1939) Google Scholar). Ectopic expression of PPARγ programs fibroblasts to differentiate into adipocytes (Tontonoz et al., 1994cTontonoz P. Hu E. Spiegelman B.M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor.Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3007) Google Scholar). Many of the genes characteristic of the differentiated adipocyte are direct targets of PPARγ and/or C/EBPα (Christy et al., 1989Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (454) Google Scholar, Dalen et al., 2004Dalen K.T. Schoonjans K. Ulven S.M. Weedon-Fekjaer M.S. Bentzen T.G. Koutnikova H. Auwerx J. Nebb H.I. Adipose tissue expression of the lipid droplet-associating proteins S3-12 and perilipin is controlled by peroxisome proliferator-activated receptor-gamma.Diabetes. 2004; 53: 1243-1252Crossref PubMed Scopus (158) Google Scholar, Schoonjans et al., 1996Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. PPARalpha and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene.EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (986) Google Scholar, Tontonoz et al., 1994bTontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer.Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1939) Google Scholar, Tontonoz et al., 1995Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. PPAR gamma 2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene.Mol. Cell. Biol. 1995; 15: 351-357Crossref PubMed Google Scholar). PPARγ is also the therapeutic target of the thiazolidinedione antidiabetic drugs that promote lipid storage and adipokine production in adipose tissue (Lehmann et al., 1995Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma).J. Biol. Chem. 1995; 270: 12953-12956Crossref PubMed Scopus (3374) Google Scholar). Cell-specific gene regulation is driven by DNA-binding factors working in concert with cofactors (Roeder, 2005Roeder R.G. Transcriptional regulation and the role of diverse coactivators in animal cells.FEBS Lett. 2005; 579: 909-915Crossref PubMed Scopus (251) Google Scholar). Several cofactors have been identified that interact with PPARγ and facilitate its action (Cho et al., 2009Cho Y.W. Hong S. Jin Q. Wang L. Lee J.E. Gavrilova O. Ge K. Histone methylation regulator PTIP is required for PPARgamma and C/EBPalpha expression and adipogenesis.Cell Metab. 2009; 10: 27-39Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, Ge et al., 2002Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Transcription coactivator TRAP220 is required for PPAR gamma 2-stimulated adipogenesis.Nature. 2002; 417: 563-567Crossref PubMed Scopus (261) Google Scholar, Gelman et al., 1999Gelman L. Zhou G. Fajas L. Raspé E. Fruchart J.C. Auwerx J. p300 interacts with the N- and C-terminal part of PPARgamma2 in a ligand-independent and -dependent manner, respectively.J. Biol. Chem. 1999; 274: 7681-7688Crossref PubMed Scopus (192) Google Scholar, Grøntved et al., 2010Grøntved L. Madsen M.S. Boergesen M. Roeder R.G. Mandrup S. MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis.Mol. Cell. Biol. 2010; 30: 2155-2169Crossref PubMed Scopus (52) Google Scholar, Qi et al., 2003Qi C. Surapureddi S. Zhu Y.J. Yu S. Kashireddy P. Rao M.S. Reddy J.K. Transcriptional coactivator PRIP, the peroxisome proliferator-activated receptor gamma (PPARgamma)-interacting protein, is required for PPARgamma-mediated adipogenesis.J. Biol. Chem. 2003; 278: 25281-25284Crossref PubMed Scopus (51) Google Scholar). Interestingly, however, few if any of these factors are regulated components of the differentiation program, i.e., their expression does not change during differentiation. Rather, they act as constitutive factors to permit PPARγ-dependent transcription. In line with this constitutive role, increasing the expression of most PPARγ coactivators above the basal state does not enhance adipogenesis. Expression of PPARγ coactivator-1α (PGC-1α) is highly regulated in brown adipose tissue (BAT) and promotes the expression of genes important for thermogenesis (Puigserver et al., 1998Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis.Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (2860) Google Scholar). However, PGC-1α is not believed to play an important role in the development of WAT, and therefore the question of whether coactivators may be regulated components of the white adipose tissue differentiation program remains to be addressed. The Wnt signaling pathway is a major physiological inhibitor of adipogenesis that is responsible for maintaining preadipocytes in an undifferentiated state (Ross et al., 2000Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Inhibition of adipogenesis by Wnt signaling.Science. 2000; 289: 950-953Crossref PubMed Scopus (1419) Google Scholar). Wnts are secreted glycoproteins that signal through frizzled receptors leading to the inhibition of the Disheveled/axin/GSK3β complex, thereby preventing the targeted degradation of β-catenin (MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (3639) Google Scholar). Accumulation of nuclear β-catenin activates TCF/LEF transcription factors and increases the expression of Wnt target genes (Molenaar et al., 1996Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos.Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1544) Google Scholar). A number of studies have shown that Wnt opposes the actions of PPARγ in adipogenesis (Bennett et al., 2002Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. Regulation of Wnt signaling during adipogenesis.J. Biol. Chem. 2002; 277: 30998-31004Crossref PubMed Scopus (544) Google Scholar, Liu and Farmer, 2004Liu J. Farmer S.R. Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes.J. Biol. Chem. 2004; 279: 45020-45027Crossref PubMed Scopus (164) Google Scholar). Blocking TCF signaling, for example by ectopic expression of a dominant-negative or conditional deletion of β-catenin in mesenchyme, is sufficient to promote differentiation (Arango et al., 2005Arango N.A. Szotek P.P. Manganaro T.F. Oliva E. Donahoe P.K. Teixeira J. Conditional deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a switch to adipogenesis in the myometrium.Dev. Biol. 2005; 288: 276-283Crossref PubMed Scopus (152) Google Scholar, Ross et al., 2000Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Inhibition of adipogenesis by Wnt signaling.Science. 2000; 289: 950-953Crossref PubMed Scopus (1419) Google Scholar). It has also been suggested that the Wnt signaling pathway is downregulated through the action of PPARγ (Moldes et al., 2003Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis.Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (224) Google Scholar). However, the molecular mechanisms by which Wnt blocks adipogenesis, as well as those that serve to integrate the PPARγ and Wnt signaling pathways, remain to be elucidated. Previously, we developed a high-throughput phenotypic screening platform for the identification of modulators of adipogenesis (Waki et al., 2007Waki H. Park K.W. Mitro N. Pei L. Damoiseaux R. Wilpitz D.C. Reue K. Saez E. Tontonoz P. The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression.Cell Metab. 2007; 5: 357-370Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We utilized this approach to identify small molecules that drive differentiation through the induction of PPARγ expression (Park et al., 2010Park K.W. Waki H. Choi S.P. Park K.M. Tontonoz P. The small molecule phenamil is a modulator of adipocyte differentiation and PPARγ expression.J. Lipid Res. 2010; 51: 2775-2784Crossref PubMed Scopus (31) Google Scholar, Waki et al., 2007Waki H. Park K.W. Mitro N. Pei L. Damoiseaux R. Wilpitz D.C. Reue K. Saez E. Tontonoz P. The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression.Cell Metab. 2007; 5: 357-370Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Here we report the adaptation of this strategy for cDNA library screening and the identification of the groucho family member transducin-like enhancer of split 3 (TLE3) as an adipogenic factor. Despite the fact that TLE proteins have been studied primarily as transcriptional repressors, we find that TLE3 is a potent facilitator of PPARγ activity on its target promoters. We further uncover a mechanism for Wnt-dependent inhibition of adipogenesis and demonstrate that TLE3 antagonizes the Wnt pathway during differentiation. These studies identify TLE3 as a dual-function modulator of adipogenesis that augments PPARγ action and inhibits Wnt signaling. We previously validated a phenotype-based high-throughput screen for chemical modulators of adipogenesis (Waki et al., 2007Waki H. Park K.W. Mitro N. Pei L. Damoiseaux R. Wilpitz D.C. Reue K. Saez E. Tontonoz P. The small molecule harmine is an antidiabetic cell-type-specific regulator of PPARgamma expression.Cell Metab. 2007; 5: 357-370Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We modified this approach to screen genome-size cDNA libraries in 384-well format and used it to identify candidate regulators of adipocyte differentiation (Figure S1A). 10T1/2 cells were retrotransfected simultaneously with a luciferase reporter driven by the −5.4 kb aP2 promoter and a collection of 18,292 individually spotted mammalian cDNA expression vectors. The day after transfection, cells were treated with insulin and a PPARγ agonist (rosiglitazone) to induce adipogenic differentiation; luciferase activity was evaluated 4 days later (Figure S1B). The screen was run in duplicate; each plate contained cDNAs encoding PPARγ and C/EBPα as positive controls. Relative intensities were normalized to their respective plate median values, and mean values and standard deviations were calculated for each well from the replicate screens to identify hits. For reconfirmation, a set of 96 cDNAs encoding putative adipogenesis regulators was chosen and reassayed, and luciferase values were normalized to empty vector controls (Figure S1C). A number of cDNAs were identified as activators of aP2-driven luciferase activity in our screen. PPARγ emerged as the most potent activator, and several additional known adipogenic factors were also represented, including C/EBPα, C/EBPδ, early B cell factor 1 (EBF1), and mitogen-activated protein kinase kinase 6 (MAPKK6). Select cDNAs were subsequently evaluated for adipogenic potential by means of stable retroviral transduction of 10T1/2 cells (Figure S1D). TLE3 was chosen for further analysis, as this factor had not previously been linked with adipocyte biology. We reasoned that if TLE3 was a regulated component of the differentiation program, then its expression should change over the course of differentiation. Indeed, analysis of a time course of 10T1/2 and 3T3-L1 differentiation revealed that TLE3 mRNA expression rose during differentiation and was further enhanced by treatment of the cells with PPARγ agonist (Figures 1A and 1B ). A strong increase in TLE3 protein expression was also observed, and again treatment with GW7845 increased its levels (Figure 1C). To determine which component of the differentiation cocktail was primarily responsible for TLE3 induction, 10T1/2 cells were stimulated for 2 days with insulin (I), dexamethasone (D), methylisobutyl xanthine (M), and/or GW7845. TLE3 mRNA and protein expression were found to be responsive to both dexamethasone and PPARγ ligand (Figures 1D and 1E). We also employed confocal immunofluorescence microscopy to visualize TLE3 expression. In 10T1/2 cells, TLE3 expression colocalized with DAPI staining, consistent with nuclear localization (Figure 1F). Furthermore, the level of TLE3 protein increased robustly in cells induced to differentiate (DMI + GW), and lipid-laden mature adipocytes were consistently TLE3 positive (Figure 1F). In vivo, TLE3 protein expression was readily detected in WAT and BAT, but not in adjacent skeletal muscle (Figure S1E). Interestingly, immunoblot analysis revealed more prominent expression of TLE3 in WAT compared to BAT (Figure S2A). In fractionated mouse WAT, TLE3 was more abundant in adipocytes compared to the stromal-vascular fraction (Figure S2B). We also investigated whether TLE3 levels were altered in murine models of obesity. We found that ob/ob and db/db mice expressed more TLE3 mRNA in WAT, compared to WT controls (Figures 1G and S2C). Examination of the tissue distribution of the mammalian TLE family of proteins revealed that, although TLE3 was expressed in a number of tissues, its expression was particularly prominent in WAT (Figure 1H). The observation that TLE3 expression increased during differentiation and was responsive to PPARγ agonist administration led us to explore whether TLE3 might be a direct target of PPARγ. We confirmed that short-term treatment of PPARγ-expressing 10T1/2 cells with PPARγ agonist induced TLE3 mRNA (Figure 2A ). A similar induction by GW7845 was seen in 3T3-L1 cells. TLE3 expression was also responsive to PPARγ administration in vivo. Treatment of mice with PPARγ (rosiglitazone), but not PPARα (GW7647) or PPARδ (GW742) agonists, induced TLE3 mRNA in WAT and BAT (Figure 2B). To address whether PPARγ bound directly to the TLE3 promoter, we employed chromatin immunoprecipitation (ChIP) assays combined with deep sequencing (Nielsen et al., 2008Nielsen R. Pedersen T.A. Hagenbeek D. Moulos P. Siersbaek R. Megens E. Denissov S. Børgesen M. Francoijs K.J. Mandrup S. Stunnenberg H.G. Genome-wide profiling of PPARgamma:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis.Genes Dev. 2008; 22: 2953-2967Crossref PubMed Scopus (410) Google Scholar). Through analysis of the global PPARγ DNA binding data of Nielsen et al., we identified several putative PPARγ binding regions in the mouse TLE3 locus (Figure 2C). Four of these regions were located more than 50 kb upstream of the transcriptional start site (peaks 1–4), while one was located in an intronic region (peak 5). Sequence analysis revealed that DR-1 sequences were associated with each of these peaks, increasing our confidence that these were likely to be bona fide PPARγ binding sites. To confirm this, we performed ChIP-PCR analysis over the time course of 3T3-L1 adipocyte differentiation. Both RXR and PPARγ bound to the five putative binding sites in the TLE3 genomic region in a differentiation-dependent manner (Figure 2D). No binding was observed with a control region from the myoglobin promoter. These results indicated that TLE3 is a direct PPARγ target gene and suggested that induction of TLE3 by PPARγ might contribute to a positive feedback loop to promote adipogenesis. To further validate the adipogenic action of TLE3, we generated stable cell lines. Importantly, these lines expressed TLE3 at only moderately elevated levels, consistent with the degree of TLE3 regulation during adipogenesis. Retroviral TLE3 expression strongly promoted adipocyte differentiation in both 10T1/2 and 3T3-L1 cells, as assessed by oil red O staining (Figure 3A ). We also assayed the activity of TLE5, which lacks a WD40 domain and is postulated to act as a dominant negative (Chen and Courey, 2000Chen G. Courey A.J. Groucho/TLE family proteins and transcriptional repression.Gene. 2000; 249: 1-16Crossref PubMed Scopus (314) Google Scholar). TLE5 expression did not affect differentiation, suggesting that a functional WD40 domain is required for the adipogenic effect. Prior studies have identified loss-of-function groucho point mutations (Jennings et al., 2006Jennings B.H. Pickles L.M. Wainwright S.M. Roe S.M. Pearl L.H. Ish-Horowicz D. Molecular recognition of transcriptional repressor motifs by the WD domain of the Groucho/TLE corepressor.Mol. Cell. 2006; 22: 645-655Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Introduction of a loss-of-function point mutation in the WD40 domain (V708D) blocked the ability of TLE3 to stimulate adipogenesis (Figure S3B). Gene expression analysis confirmed increased expression of PPARγ and many of its downstream target genes in cells stably expressing TLE3 (Figures 3B, S3A, and S3B). We turned to the NIH 3T3 fibroblast system to investigate potential combinatorial effects of TLE3 and PPARγ. NIH 3T3 cells are unable to differentiate into adipocytes because they lack PPARγ. Expression of TLE3 in this context had little if any effect on the expression of PPARγ target genes (Figures 3C and S3C). Consistent with prior work (Tontonoz et al., 1994cTontonoz P. Hu E. Spiegelman B.M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor.Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3007) Google Scholar), introduction of PPARγ conferred the ability to accumulate lipid and express adipogenic genes (Figure 3C) (data not shown). The combination of PPARγ and TLE3 was highly synergistic, both in terms of target gene expression and morphological differentiation (Figure 3C) (data not shown). These data demonstrate that the effects of TLE3 on adipogenic gene expression are highly dependent on PPARγ expression. In order to address whether endogenous TLE3 activity contributes to adipogenesis, we used retroviruses encoding inhibitory shRNAs to knock down TLE3 expression. TLE3 knockdown was confirmed by immunoblotting (Figure S3D). Stable 3T3-L1 cell lines expressing two different shRNA sequences targeting TLE3 exhibited reduced differentiation capacity, compared to those expressing a control shRNA (Figure 3D). In agreement with the morphological differentiation, the expression of adipocyte-selective genes was also impaired in TLE3 knockdown cells (Figures 3E and S3E). As a complement to the knockdown studies, we generated mice lacking TLE3 using a Gene Trap embryonic stem cell line obtained from The Sanger Centre (XP0165), in which the insertion of the trapping vector resulted in a null allele. Unfortunately, homozygous deletion of TLE3 results in embryonic lethality due to multiple developmental defects (data not shown). However, we succeeded in deriving primary MEFs from TLE3−/− mice. Multiple preparations of primary embryonic fibroblasts derived from TLE3−/− mice showed reduced capacity for adipogenesis, compared to WT controls (Figure 3F). Adipogenic gene expression was correspondingly reduced in MEFs lacking TLE3 (Figure S3F). Together, these data demonstrate that TLE3 is a physiologic component of the adipogenic program that works cooperatively with PPARγ to promote differentiation. Next we addressed the mechanism of TLE3 action in adipogenesis. As chronic (stable) overexpression of TLE3 was able to induce expression of PPARγ, aP2, and CD36 in undifferentiated 10T1/2 cells, it was possible that TLE3 was functioning primarily by inducing PPARγ expression, leading to secondary effects on downstream target genes. To address whether acute expression of TLE3 could also induce adipogenic gene expression, we transduced 10T1/2 cells expressing the coxsackie adenovirus receptor (CAR) with an adenoviral vector encoding TLE3. Surprisingly, TLE3-tranduced cells showed increased expression of aP2 and CD36 and enhanced response to PPARγ agonist, despite expressing similar endogenous levels of both PPARγ1 and PPARγ2 (Figure 4A ) (data not shown). These effects occurred in the absence of morphological differentiation. Many PPARγ target genes are also regulated by C/EBPs; however, the expression of C/EBPs was also not altered by TLE3 (Figure S4A). The ability of TLE3 to regulate target genes without affecting PPARγ expression suggested that TLE3 might be enhancing PPARγ activity. We further explored this possibility by acutely expressing TLE3 in the presence of stably expressed PPARγ. As shown in Figure 4B, TLE3 strongly promoted the ability of PPARγ to induce its target genes in response to agonist. These data suggest that the primary effect of TLE3 is on PPARγ activity and that the elevated level of PPARγ observed in cells stably expressing TLE3 is secondary to increased adipocyte differentiation. We next employed transcriptional profiling to compare the PPARγ and TLE3 transcriptomes. We acutely expressed TLE3 and PPARγ individually and in combination in 10T1/2 cells by viral transduction. Gene expression was analyzed after 48 hr using Affymetrix Mouse Gene 1.0 ST Arrays. Despite previous characterization of TLEs as repressors, a greater number of transcripts were induced in response to TLE3 expression than suppressed (Figure S4B). Furthermore, the transcriptional programs engaged by TLE3 and PPARγ were highly overlapping, as indicated by the Venn diagram in Figure 4C. Approximately 25% of PPARγ-responsive genes overlapped with those regulated by TLE3, indicating a significant degree of specificity for the PPARγ transcriptional program. Cluster analysis of the entire set of overlapping genes is presented in Figure S4C and Tables S1 and S2. A more limited set of genes, whose expression was altered more than 1.4-fold, is presented in Figure 4D. Importantly, the common regulated genes identified in this analysis included many established PPARγ targets, including aP2, CD36, and adiponectin (Figure 4D). Furthermore, it is clear from the heat map of Figure 4D that PPARγ and TLE3 have additive or synergistic effects on the expression of a large battery of genes. In agreement with the additive effects of these factors in adipogenesis (Figure 3C), maximal expression of this gene set was achieved in the presence of TLE3, PPARγ, and GW7845 (Figure 4D). Our global transcriptional analysis did not reveal a significant effect of TLE3 expression on the programs of other nuclear receptors, including LXR, FXR, or RAR (data not shown). We validated our array results for a number of direct PPARγ targets by real-time PCR analysis (Figure 4E). The results confirmed that the vast majority of established direct PPARγ target genes in adipocytes were additively responsive to TLE3 and PPARγ. For some genes, such as aP2 and perilipin, the response was synergistic. A minority of putative PPARγ target genes was responsive to PPARγ but not TLE3 (e.g., Angptl4) or TLE3 but not PPARγ (e.g., HK2) (Figure 4E). The ability of TLE3 to selectively promote PPARγ-dependent target gene expression suggested that TLE might function as a positive transcriptional cofactor for PPARγ. To test this idea, we performed transfection assays with a −5.4 kb aP2-luciferease reporter. 10T1/2 cells transfected with PPARγ and RXR expression vectors showed robust luciferase activity in the presence of GW7845 (Figure 5A ). TLE3 had no effect in the absence of PPARγ and RXR, but enhanced reporter activity when these nuclear receptors were present. Introduction of the point mutation in the WD40 domain of TLE3 (V708D) that prevented TLE from inducing adipogenesis also prevented coactivation of PPARγ (Figure 5A). TLE3 showed similar ability to enhance PPARγ-dependent transcription when a reporter driven by isolated PPAR response elements (PPREs) was used (Figure 5B). This result strongly suggested that TLE3 was acting to increase aP2-promoter transcription by increasing PPARγ activity on its cognate response element, rather than by acting through other binding sites. Preliminary studies indicated that TLE3 also promoted the action of PPARα and PPARδ in transfection assays (Figure S5A). To provide additional evidence for the ability of TLE3 to enhance PPARγ activity on PPREs, we performed ChIP assays in 10T1/2 cells. We initially analyzed PPARγ and TLE3 occupancy along the 5′-flanking region of the aP2 gene extending from −5.4 kb to the transcriptional start site. Strong differentiation-dependent binding of PPARγ was detected in the region corresponding to the previously characterized PPREs in the aP2 enhancer (Tontonoz et al., 1994aTontonoz P. Graves R.A. Budavari A.I. Erdjument-Bromage H. Lui M. Hu E. Tempst P. Spiegelman B.M. Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR ga" @default.
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• W2010364183 date "2011-04-01" @default.
• W2010364183 modified "2023-10-18" @default.
• W2010364183 title "TLE3 Is a Dual-Function Transcriptional Coregulator of Adipogenesis" @default.
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