COUP-TFII acts downstream of Wnt/�-cateninsignal to silence PPAR� gene expressionand repress adipogenesisMasashi Okamuraa,b, Hiromi Kudoa, Ken-ichi Wakabayashia,c, Toshiya Tanakaa, Aya Nonakaa,c, Aoi Uchidaa,Shuichi Tsutsumia,c, Iori Sakakibaraa, Makoto Naitod, Timothy F. Osbornee, Takao Hamakuboa, Sadayoshi Itob,Hiroyuki Aburatania,c, Masashi Yanagisawaf, Tatsuhiko Kodamaa,g, and Juro Sakaia,g,1

aLaboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba, Meguro-ku,Tokyo 153-8904, Japan; bDivision of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School ofMedicine, Sendai 980-8574, Japan; cGenome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1, Komaba,Meguro-ku, Tokyo 153-8904, Japan; dDepartment of Cellular Function, Division of Cellular and Molecular Pathology, Niigata University Graduate School ofMedical and Dental Sciences, Niigata 951-8510, Japan; eDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92717-3900;fHoward Hughes Medical Institute and Departments of Molecular Genetics University of Texas Southwestern Medical Center, Dallas, TX 75390-9050;and gTranslational Systems Biology and Medicine Initiative, University of Tokyo, Tokyo 153-8904, Japan

Contributed by Masashi Yanagisawa, February 18, 2009 (sent for review November 19, 2008)

Wnt signaling through �-catenin and TCF maintains preadipocytesin an un-differentiated proliferative state; however, the molecularpathway has not been completely defined. By integrating geneexpression microarray, chromatin immunoprecipitation-chip, andcell-based experimental approaches, we show that Wnt/�-cateninsignaling activates the expression of COUP-TFII which recruits theSMRT corepressor complex to the first introns located downstreamfrom the first exons of both PPAR�1 and �2 mRNAs. This maintainsthe local chromatin in a hypoacetylated state and represses PPAR�gene expression to inhibit adipogenesis. Our experiments definethe COUP-TFII/SMRT complex as a previously unappreciated com-ponent of the linear pathway that directly links Wnt/�-cateninsignaling to repression of PPAR� gene expression and the inhibi-tion of adipogenesis.

ChIP-chip � epigenome � histone modification � obesity � Wnt

The physiological differentiation program converting preadi-pocytes into adipocytes has been well characterized, pre-

dominantly in cultured mouse cell lines (1). This process, calledadipogenesis, is orchestrated by an elaborate cascade of sequen-tially acting transcription factors and chromatin modifying co-regulators that shape the differentiation through the actions ofhormones and other signaling pathways. During the very earlystages of adipogenesis, there is a rapid and transient inductionof CCAAT/enhancer-binding protein � (C/EBP�) and C/EBP�,followed by the activation of 2 critical pro-adipogenic transcrip-tion factors, C/EBP� and peroxisome proliferator-activatedreceptor � (PPAR�). These 2 factors are key activators for theglobal changes in gene expression that cause the loss of preadi-pocyte characteristics and the acquisition of the mature fat-ladenadipocyte phenotype.

The decision process used by preadipocytes to either remainproliferative or enter the differentiation pathway is influencedby both inhibitory and stimulatory factors (1), and one class ofendogenous factors proposed to repress adipogenesis includesthe Wnt proteins (2). There are several individual Wnt proteinsin the family and they influence a diverse array of developmentaland differentiation processes, including the fate of mesenchymalprogenitors. The endogenous Wnt isoform involved in main-taining preadipocytes in their quiescent state has been proposedto be Wnt10b (2). The canonical Wnt signaling pathway isinitiated by the binding of a specific Wnt as a ligand to the cellsurface LRP5/Frizzled receptor complex. Signaling throughFrizzled results in the stabilization and nuclear translocation of�-catenin where it associates with members of the T cellfactor/lymphoid enhancer factor (TCF/LEF) family, and the

resulting complex targets key genes to mediate the Wnt response(3, 4).

Inhibition of endogenous Wnt signaling by a dominant neg-ative TCF7L2 (dnTCF7L2) results in spontaneous adipogenesis.Furthermore, an inhibitor of Wnt signaling in preadipocytes,harmine, has pro-adipogenic actions that target PPAR� (5).Expression of Wnts does not influence the rapid and transientinduction of C/EBP� and C/EBP� but completely prevent thedownstream induction of C/EBP� and PPAR� (2). However, themechanism by which Wnt/�-catenin blocks C/EBP� and PPAR�gene expression has not been defined.

Despite the fact that Wnts are clearly key negative regulatorsof adipocyte differentiation, no �-catenin target gene(s) thataccount for the inhibition of adipogenesis has been identified,and the downstream transcriptional cascade of �-catenin in theadipocyte lineage remains largely unexplored. To gain insightinto the molecular mechanisms that underlie the functions of�-catenin in regulating adipogenesis, we undertook a genome-wide identification of direct target genes regulated by �-cateninin 3T3-L1 preadipocytes using a combination of gene expressionprofiling, oligonucleotide microarray, and chromatin immuno-precipitation-chip (ChIP-chip) analyses. Our current resultsidentify �-catenin as a direct regulator of chicken ovalbuminupstream promoter-transcription factor II (COUP-TFII), a po-tent antiadipogenic factor (ref. 6 and the current study). Fur-thermore, we show that COUP-TFII recruits the silencingmediator for retinoic acid receptor and thyroid hormone recep-tor (SMRT) corepressor complex to binding sites close to bothpromoters for the PPAR� gene where the complex maintains thechromatin in a hypoacetylated and repressed state that is re-versed upon differentiation.

ResultsWnt/�-catenin Signaling Directly Regulates COUP-TFs Expression. Toaddress the regulatory program governing inhibition of adipo-genesis by canonical Wnt/�-catenin signaling; we first analyzedglobal gene expression profiles in 3T3-L1 preadipocytes com-pared with companion cultures that were induced to differen-tiate for 8 days with a hormonal cocktail containing 3-isobutyl-

Author contributions: M.O., M.Y., and J.S. designed research; M.O., H.K., K.W., T.T., A.N.,A.U., S.T., and I.S. performed research; T.T. and S.T. contributed new reagents/analytictools; M.N., T.H., S.I., H.A., T.K., and J.S. analyzed data; and M.O., T.F.O., and J.S. wrote thepaper.

The authors declare no conflict of interest.

1To whom correspondence should be addressed. E-mail: jmsakai-tky@umin.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901676106/DCSupplemental.

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1-methylxanthine, dexamethasone, and insulin (MDI) in theabsence or presence of exogenous Wnt3a protein as described inSI Materials and Methods. Total RNA was isolated and globalgene expression profiles were compared using Affymetrix mi-croarray mouse gene chip platform M430A2. There are 48nuclear receptors genes are encoded and 43 genes of them arerepresented on the chip. Of these, 23 were expressed at detect-able levels on either day 0 or day 8 of differentiation (here‘‘expressed’’ is defined as transcripts with a detection call of‘‘present’’ on the microarray for these time points). This chip-microarray analysis revealed that the mRNA for the orphanreceptor COUP-TFII was induced to the highest level (8-fold)relative to all of the expressed nuclear receptors in response toWnt3a addition during the 8 day differentiation program [sup-porting information (SI) Fig. S1 A]. Immunoblotting analysisdemonstrated that COUP-TFII protein declined and was almostundetectable at day 8 of MDI treatment in cells cultured withoutWnt and this was accompanied by a similar decline in nuclear�-catenin. However when recombinant Wnt3a was added duringthe differentiation process, the levels of both COUP-TFII andnuclear �-catenin remained high (Fig. S1B). Wnt3a also pre-vented the induction of C/EBP�, PPAR�, and liver X receptor� (LXR�) as expected for these genes that normally increasedduring adipogenesis (2).

Wnt3a also induced COUP-TFII and nuclear �-catenin whenadded directly to preadipocytes (Fig. 1A, Left). Because Wntsignals through �-catenin, we hypothesized that COUP-TFIImight be a direct Wnt/�-catenin target gene. To test thispossibility, we knocked down �-catenin by the application ofsiRNA and analyzed whether Wnt dependent COUP-TFII ex-pression was altered. Two independent siRNA oligonucleotidesequences that target �-catenin were transfected into Wnt3atreated 3T3-L1 cells and both decreased �-catenin and COUP-TFII expression (Fig. 1 A, Middle). Similar results were obtainedwhen the Wnt pathway was inhibited by a separate method usinga retrovirus expressing a dnTCF7L2 (7). This dominant negativeTCF proteins, binds normally to TCF/LEF consensus bindingsites but cannot be activated by �-catenin (8) (Fig. 1 A, Right).Similar results were obtained in mouse mesenchymal stem ST2cells [Fig. S1C].

Identification of COUP-TFII as a Direct Target of Wnt/�-catenin. Theresults presented above suggest that COUP-TFII might be adirect target of the canonical Wnt signaling pathway through�-catenin. To determine if the �-catenin containing TCF com-plex directly controls COUP-TFII gene expression; we per-formed ChIP-chip analysis using a genome wide promoter tilingarray platform. In this experiment, chromatin was prepared from2 day postconfluent 3T3-L1 cells that were cultured for 16 h inthe presence of the MDI mixture and recombinant Wnt3afollowed by a standard procedure for ChIP analyses. Duplicatechromatin immunoprecipitations were performed with 3T3-L1preadipocyte chromatin that was enriched after incubation with

Fig. 1. �-catenin associates with COUP-TFII promoter and regulates COUP-TFII mRNA and protein expression. (A) Immunoblot analysis showing up-regulation of COUP-TFII upon Wnt3a treatment (Left) in 3T3-L1 preadipocytesand down-regulation of COUP-TFII in 3T3-L1 preadipocytes transfected withsiRNA specific to �-catenin (Middle) or transduced with retrovirus carryingdnTCF7L2 (Right). 3T3-L1 cells were cultured in the absence or presence 20ng/ml of Wnt3a for 16 h (Left). 3T3-L1 cells were transfected with siRNA foreither �-catenin (si-�-catenin#1 and #2) or control siRNA (Middle) or infectedwith retrovirus carrying dnTCF7L2 (Right). Cells were harvested and the ex-pression of COUP-TFII and �-catenin were assessed by immunoblot analysis.Equal loading of the proteins were confirmed by the detection of TBP. (B andC) Two-day post confluent 3T3-L1 cells were cultured for 16 h in the presenceof MDI mixture and recombinant Wnt3a (20 ng/ml) and were harvested forChIP. (B) ChIP-chip analysis identified COUP-TFII as �-catenin target gene. Themost enriched binding sites are shown by arrows. Individual data tracksrepresenting the enrichment ratio of ChIP versus input DNA hybridizationintensity is shown as P value (-log10), vertical axes. ChIP-chip analysis onTCF/LEF consensus binding sequences is highly conserved between species. (C)qPCR analysis of COUP-TFII promoter (Top) or CCND1 promoter (Bottom)sequences normalized to cyclophilin levels in control DNA and ChIP performed

with COUP-TFII antibody as described previously (20, 21). The data are theaverage of 3 replicates and error bars represent � SD. (D) 3T3-L1 cells weretransfected with 0.3 �g of COUP-TFII promoter luciferase reporter plasmid and0.05 �g of pCMV� together with the indicated amounts of the followingplasmids: 0.1 �g of pCMV-�-catenin (S33Y), 0.1 �g of pCMV-TCF7L2, 0.1 �g ofpCMV-ICAT as indicated. Three days after transfection, cells were harvestedand firefly luciferase activity was measured and normalized to �-galactosidaseactivity. Each bar represents mean � SE. of triplicate experiments. (E and F)3T3-L1 cells were transfected with siRNA specific for COUP-TFII (si-TFII#1 andsi-TFII#2) or control siRNA (si-ctrl). (E) Cells were induced for differentiationwith Dex and ORO was performed at day 8. (F) Cells were induced fordifferentiation with MDI in the presence of 20 ng/ml of Wnt3a and ORO wasperformed at day 8. Plate of cells and micrographs are shown (E and F). Theyellow rectangle designates the area in micrographs (E and F).

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an antibody against C-terminal domain of �-catenin. The en-riched DNA was then hybridized to an Affymetrix array platformcontaining tiled oligonucleotide probes that span between �6.0to � 2.5 kb relative to the transcription initiation site of allannotated 28,000 mouse proximal promoter sequences. Thisanalysis identified highly significant �-catenin binding (P �10�5) to the promoter region of COUP-TFII 4.7 kb upstreamrelative to translation initiation site (Fig. 1B). In agreement withthis result, a perfect TCF binding motif (CTTTGAA) that isconserved among species was found at this position (Fig. 1B). Aquantitative gene specific ChIP analysis confirmed that theCOUP-TFII promoter fragment from �4 kb region was enrichedby the �-catenin antibody relative to the signal derived from anirrelevant genomic region used for normalization (Fig. 1C, Top).As a positive control, we also showed that �-catenin wasrecruited to the well-known �-catenin target gene cyclin D1(CCND1) by the �-catenin antibody (Fig. 1C, Bottom).

Up-Regulation of COUP-TFII Promoter Activity by a Complex of �-catenin and TCF7L2. To determine whether the �-catenin/TCFcomplex directly activates the COUP-TFII promoter, we sub-cloned a DNA fragment containing the 5�-franking region (FR)of the mouse COUP-TFII gene, which contained one TCF/LEFbinding motif into a luciferase reporter construct, and trans-fected it into 3T3-L1 preadipocytes with or without expressionvectors for an activated form of �-catenin and/or wild-typeTCF7L2 (Fig. 1D). The reporter activity of the COUP-TFIIpromoter reporter construct was significantly enhanced (3.7-fold) by the combination of an activated form of �-catenin and

wild-type TCF7L2, which was abrogated by the cotransfection ofan expression vector for the Wnt signaling inhibitory protein,ICAT, (inhibitor of �-catenin and TCF4) (9) that interacts with�-catenin and prevents it from translocating into the nucleus(10). Taken together, these results all demonstrate that COUP-TFII is a bona fide target of �-catenin and Wnt signaling in3T3-L1 preadipocytes.

COUP-TFII Inhibits Adipocyte Differentiation. To specifically evalu-ate a potential role for COUP-TFII in adipogenesis, we exam-ined the effects of overexpression of COUP-TFII by retroviraltransduction during adipocyte differentiation. In this experi-ment, COUP-TFII overexpression blocked the morphologicalchanges associated with adipogenesis, most notably through amarked decrease in accumulation of Oil Red O (ORO) (Fig.S2A). These morphological effects were accompanied by adecrease in the induction of the key adipogenic transcriptionfactors C/EBP� and PPAR�. However, C/EBP� and C/EBP�were induced normally (Fig. S2B), suggesting that COUP-TFIIacts upstream of PPAR� and C/EBP� but independent ofC/EBP� and C/EBP� during adipogenesis.

Ectopic Expression of PPAR� or C/EBP� Overcomes COUP-TFII Inhibi-tion of Adipogenesis. To further assess the epistatic relationshipbetween COUP-TFII and PPAR� or C/EBP�; we infected3T3-L1 cells with retroviruses carrying cDNAs for each one andtested whether they could rescue differentiation in COUP-TFIIexpressing preadipocyte cells that fail to differentiate upon MDItreatment. After stable selection, cells were induced to differ-

Fig. 2. COUP-TFII/RXR� heterodimer binds to PPAR�1 and PPAR�2 loci and induces changes in the pattern histone H3-K4 tri-methylation (H3K4me3) withineach region. A schematic diagram shows the mouse Ppar�1 and �2 genes and their exons is displayed. Introns occupied by COUP-TFII/RXR� heterodimer orH3K4me3 are indicated by arrows. (A) The occupancy of COUP-TFII and RXR� in 3T3-L1 preadipocyte as examined by ChIP-chip. The most enriched binding sitesare indicated by arrows. Conserved imperfect direct or inverted repeats of the consensus sequence AGGTCA with various spacing in the first introns of bothPPAR�1 and �2 located downstream from the first exons of both PPAR�1 and �2 mRNAs are shown. COUP-TFII and RXR� binding was validated by ChIP-qPCR.(B) The occupancy of H3K4me3 as examined by ChIP-chip in preadipocytes versus cells allowed to differentiate for 8 days. (A and B) Individual data tracksrepresenting the enrichment ratio of ChIP versus input DNA hybridization intensity is shown as P value (�log10).

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entiate with the treatment of the standard MDI adipogenicmixture and 8 days after the induction, cells expressing COUP-TFII alone remained fibroblastic and failed to accumulatesignificant amounts of lipid as expected; however, when eitherPPAR� or C/EBP� was coexpressed, almost all of the cellsdifferentiated into adipocytes (Fig. S2C). Increased expressionof LXR�, a known PPAR� target gene and another marker foradipocyte differentiation (11), was also observed and this wasalso decreased by COUP-TFII overexpression (Figs. S1B andS2D). These results suggest that COUP-TFII represses differ-entiation by inhibiting the expression of C/EBP� and PPAR�transcription factors, but not C/EBP� nor C/EBP�, which is alsoconsistent with a key role downstream of Wnt-mediated inhibi-tion of adipogenesis (2).

To complement the sufficiency results, we undertook necessitytests by predicting that a decrease in COUP-TFII expressionwould enhance differentiation of 3T3-L1 preadipocytes (Fig.1E). Because the MDI mixture is very efficient in converting3T3-L1 preadipocytes into mature adipocytes, we reasoned thatit would be difficult to assess an increase in adipogenesis usingMDI. Therefore, we only used dexamethasone treatment toinduce differentiation, which is much more inefficient by itself atinducing differentiation than the complete mixture (compareFig. S2 A and Fig. 1E). A control siRNA or 2 independent siRNAoligonucleotide sequences that target COUP-TFII were sepa-rately transfected into 3T3-L1 cells which were then treated withdexamethasone and cultured for 8 days. Treatment with eitherCOUP-TFII siRNA decreased COUP-TFII protein levels andincreased lipid accumulation (Figs. 1E and Fig. S2E) consistentwith the hypothesis that COUP-TFII is a negative regulator ofadipogenesis. Knockdown of COUP-TFII in ST2 cells alsoresulted in an increase in lipid accumulation (Fig. S2F).

If COUP-TFII acts as a downstream mediator of Wnt signal-ing then theoretically, reduction of COUP-TFII should preventWnt from inhibiting differentiation. We tested this prediction byusing siRNA. In control cells, 20 ng/well of recombinant Wnt3acompletely inhibited adipogenesis as measured by ORO accu-mulation (Fig. 1F). However, in cells treated with the COUP-TFII siRNA, Wnt exhibited a diminished ability to repressadipocyte differentiation, indicating that COUP-TFII contrib-utes at least in part to Wnt-induced suppression of adipogenesis(Figs. 1F and S2G).

Tri-methylation of Histone H3-K4 Within the PPAR�1 and PPAR�2 LociIs Altered by the Binding of the COUP-TFII/RXR� Heterodimer. Theabove results showed that the down-regulation of COUP-TFII isessential for adipogenesis. To identify COUP-TFII candidategenes with the potential to modulate adipogenesis, ChIP-chipanalysis was conducted with chromatin from 3T3-L1 preadipo-cytes using an antibody directed against COUP-TFII. BecauseCOUP-TFII is a nuclear receptor that forms a heterodimer withretinoid X receptor � (RXR�) to bind to response elements (12),we also used a RXR� antibody in a separate ChIP-chip exper-iment. This screen led to the identification of highly significantCOUP-TFII binding (P � 10�4) to sites downstream of thetranscription start sites for both PPAR�1 and PPAR�2 mRNAs(Fig. 2A). The separate analysis for RXR� binding revealedsignificant overlap between COUP-TFII and RXR� in bothregions. Validation of COUP-TFII and RXR� sites obtained bydirect gene specific ChIP analysis that showed that the 2 nuclearreceptors were indeed binding to the respective sites identifiedby ChIP-chip (Fig. 2 A). Inspection of the DNA sequence withineach region shows there are conserved nuclear receptor half-sites (AGGTCA) in both. These results suggest that COUP-TFIIbinds to sites in the first intron of both PPAR�1 and �2 mRNAisoforms to suppress their expression and thereby prevent adi-pogenesis. Intriguingly, independent ChIP-chip analysis with anantibody that detects H3-K4 tri-methylation (H3K4me3) re-

vealed that patterns of H3-K4 tri-methylation close to theCOUP-TFII sites increases during differentiation (Fig. 2B).Since H3-K4 tri-methylation is a mark of histone chromatinmodification associated with gene activation, these results alsosuggest that PPAR� gene expression is regulated throughchanges in histone modification close to both COUP-TFIIbinding sites.

COUP-TFII Mediates Inactivation of PPAR� Gene Expression by Re-pressing Histone Modification. To further analyze the mechanismsby which COUP-TFII suppresses PPAR�1 and �2, we performeda ChIP of endogenous histone modifications within their firstintrons of both PPAR�1 and �2 mRNAs close to the COUP-TFII binding regions we identified above (Fig. 3). We usedantibodies that detect acetylated forms of both histone H3 andH4 that are known to correlate positively with high levels of geneexpression (13, 14). Before differentiation, we observed lowlevels of mono-acetylation of histones H3 and H4 at the COUP-TFII binding regions from the PPAR�2 locus in both controlempty vector transduced and COUP-TFII retrovirus transducedcells and very low level of tri-methylation of H3-K4 at PPAR�2locus (Fig. 3 D-F, lanes 3 and 6). In contrast, we found hyper-acetylation of histones H3 and H4 together with a strikingincrease in tri-methylation of histone H3-K4 in the first intron ofPPAR�2 at day 8 of differentiation in control empty vector

Fig. 3. COUP-TFII regulates the histone modifications of PPAR�1 and PPAR�2genes. 3T3-L1 cells transduced with retrovirus carrying empty or COUP-TFIIvector were subjected to ChIP with anti-AcH3 (A and D), anti-AcH4 (B and E),and anti-H3K4me3 (C and F) antibodies and IgG as a control. qPCR analysis onPPAR�1 and �2 first intron regions were normalized to hoxc4a levels in controlDNA and ChIP as described previously (20, 21). The data are the average of 3replicates and error bars represent � SD. Schematic diagrams of the PPAR�1and PPAR�2 genes above the panels indicate the DNA fragments that wereamplified by qPCR following ChIP.

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transduced cells (Fig. 3 D-F, lane 9). These changes werecompletely abrogated in COUP-TFII overexpressing cells (Fig.3 D-F, compare lane 9 and lane 12). A very similar pattern wasalso observed for the PPAR�1 COUP-TFII binding region withthe exception that a significant level of tri-methylation of H3-K4was already observed together with increased acetylation ofhistone H3 before the induction of differentiation (Fig. 3 A, lane3, and C, lane 3). These results suggest that changes in COUP-TFII binding to the first introns located downstream from thefirst exons of both PPAR�1 and �2 mRNAs is required foralterations in histone acetylation that accompany gene inductionduring 3T3-L1 differentiation.

siRNA-mediated Knockdown of SMRT Reverses COUP-TFII InducedSuppression of Adipogenesis. A recent report indicates that DNAbound COUP-TF represses gene expression by using a domainwithin the ligand-binding region that recruits a corepressor tothe DNA (i.e., nuclear receptor corepressor (NCoR) and SMRT;ref. 15). This results in a repressed chromatin structure that hasbeen proposed to block transcriptional activation of target genes(12). Taken together with our results, this suggests that COUP-TFII might recruit an inhibitory complex to the first intron ineach PPAR� locus to inhibit transcription. To test this hypoth-esis, we performed ChIP analysis on both PPAR� loci using

anti-NCoR and anti-SMRT antibodies. Before differentiation,we observed the association of both proteins at the COUP-TFIIbinding regions from the PPAR�2 locus in COUP-TFII retro-virus transduced cells (Fig. 4A) indicating that COUP-TFIIrecruits inhibitory complex containing these repressors to thePPAR�2 locus. To test the relevance of NCoR and SMRTbinding here, we predicted that knockdown of NCoR or SMRTwould rescue COUP-TFII-induced suppression of PPAR� geneexpression and adipogenesis. Accordingly, we transfected asiRNA designed to inactivate SMRT into COUP-TFII trans-duced cells and examined adipocyte differentiation induced byMDI. Similar to the results in Fig. S2 A, transduction with aretroviral expression vector for COUP-TFII significantly inhib-ited the ability of the MDI mixture to induce differentiation and,consistent with our hypothesis, specific knockdown of SMRTreversed the inhibition of COUP-TFII on adipogenesis (Fig. 4 Band C).

Expression of COUP-TFI and -TFII Are Down-Regulated in Diet andGenetic Mouse Models of Obesity. COUP-TFI is related to COUP-TFII and both are expressed in adipose tissues of rats as shownby immunostaining (Fig. 5A). Because COUP-TFII expression isdown-regulated during adipocyte differentiation, it is possiblethat a low level of COUP-TFII expression is associated withobesity. To investigate this possibility, we examined potentialchanges in expression of both COUP-TF family members inadipose tissue of 2 different mouse models of obesity; one thatoccurs as a result of high fat feeding in C57BL/6J mouse (DIO)and the other is the genetically predisposed obese ob/ob mouse.For DIO, we randomized 6-week old C57BL/6J littermates andfed them either normal or high fat chow for 8 weeks, extractedRNA from epididymal fat depots, and examined expression ofCOUP-TFs by quantitative PCR (qPCR). We found that DIOsignificantly decreased mRNA levels for both COUP-TFI andTFII in white adipose tissue (Fig. 5B). A similar down-regulationof COUP-TFI and TFII expression was observed in adiposetissues from 10-week old ob/ob genetically obese mice comparedto matched controls (Fig. 5B). The concordant expression

Fig. 4. Knockdown of SMRT restores the COUP-TFII mediated suppression ofadipogenesis. (A) Cells were subjected to ChIP with anti-NCoR or anti-SMRTantibodies. qPCR analysis of PPAR�2 promoter sequences normalized to cy-clophilin levels in control DNA and ChIP performed with NCoR or SMRTantibody as described in SI Text (20, 21). The data are the average of 3replicates and error bars represent � SD. Schematic diagrams of the PPAR�2genes above the panels indicate the DNA fragments that were amplified byqPCR following ChIP. (B) Cells transduced COUP-TFII were transfected withsiRNA specific to SMRT or control siRNA. Cells were induced to differentiatewith MDI and were stained with ORO at day 8 of differentiation. (C) SMRTmRNA levels were quantified by qPCR in control and SMRT siRNA transfectedcells. To correct for variations in input RNA, data were normalized to thequantity of cyclophilin.

Fig. 5. COUP-TFI and -TFII are expressed in white adipose tissue of rats anddown regulated in diet-induced and genetically predisposed obese ob/obmice. (A) Immunohistochemistry of paraffin-embedded section from rat usinganti-COUP-TFI and -TFII antibodies. (B) Relative amounts of COUP-TFI and TFIImRNA in epididymal white adipose tissues from C57BL/6J male mice on eitherhigh fat diet (HFD) or normal chow diet (NCD) or those from male ob/ob miceon NCD (n � 6 for each group). Male C57BL/6J mice (6 weeks of age) and ob/obmice were fed with either HFD or NCD for 8 weeks as described under SI Text.Total RNA was extracted from 6 independent mice from each group fromepididymal adipose tissues were prepared independently from each groupand subjected to qRT-PCR. Each bar represents mean � SD. of 3 independentexperiments performed in triplicate. *P � 0.01 compared with C57BL/6J micefed NCD. Detail methods for immunohistochemistry and animals and diet aredescribed in SI Text.

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patterns in these 2 different obesity models support the idea thatCOUP-TFI and TFII signals may be germane to fat differenti-ation and possibly pathophysiology.

Because COUP-TFI is also a negative regulator of geneexpression, we analyzed its role in over/under expression andChIP-chip analyses similar to those described here for COUP-TFII. We were able to confirm that COUP-TFI has a similar roleas COUP-TFII in regulating PPAR� gene expression down-stream of Wnt (Fig. S3).

DiscussionPPAR� expression is necessary and over-expression is sufficientfor adipogenesis (16), and it was previously known that Wntsignaling inhibits adipogenesis by silencing PPAR� gene expres-sion (2). However, the molecular pathway for this regulation wasincompletely defined and a big challenge in the field of Wntsignaling and adipogenesis has been to integrate known com-ponents of the Wnt signaling pathway with the downstreameffectors controlling adipogenesis and PPAR� activity.

In the current study, by combining global gene expressionanalyses with a ChIP-chip microarray approach, we demonstratethat COUP-TFII provides a direct link between the canonicalWnt signaling pathway and PPAR� gene activation. Furtherstudies demonstrated that histone acetylation patterns predictiveof gene activation were increased in the proximity of theCOUP-TFII binding sites at the PPAR� locus during adipocytedifferentiation, and over-expression of COUP-TFII not onlyprevented adipocyte conversion but also blunted the increase inhistone acetylation as well.

Consistent with an important role for COUP-TFII in histonehypoacetylation and gene repression in preadipocytes, we alsoshowed that its binding resulted in the corecruitment of NCoRand SMRT deacetylase containing corepressor complexes andthat a knockdown of SMRT reversed the repressive effects ofCOUP-TFII on adipogenesis.

Since COUP-TFII is down-regulated during conditions asso-ciated with excess nutrition, it is tempting to speculate thatdown-regulation of COUP-TFII, in response to over nutrition,induces adipogenesis and hence links the development of obesity

and the metabolic syndrome. We and others have reported thatproteins involved in the Wnt signal cascade including the Wntcoreceptors LRP5 and LRP6 and its nuclear effector proteinTCF7L2 are linked to type 2 diabetes (3, 17). Thus, the Wntsignaling cascade acting through COUP-TF to modulate PPAR�expression may be a key pathway in the development of obesity,insulin resistance, and the metabolic syndrome.

It was recently reported that COUP-TFII has an importantrole downstream of Hedgehog signaling to inhibit adipogenesisacting at the C/EBP� promoter (6). When combined with ourstudy, the results define COUP-TFII as a molecular hub thatintegrates the input through 2 key developmental signalingpathways, Wnt and Hedgehog, with PPAR� gene expression andadipocyte differentiation.

Materials and MethodsThe complete methods are described in detail in SI Text. All the PCR primersused in this article are listed in Tables S1 and S2 .

ChIP-Chip. ChIP DNA samples were amplified by 2 cycles of in vitro translation(IVT) as described (18). ChIP and input DNA samples were hybridized on arraysaccording to the manufacture’s instruction. After scanning and data extrac-tion, enrichment values (ChIP/input DNA) were calculated by using the model-based analysis of tiling-array (MAT) algorithm (19). The details are describedin SI Text.

Statistics. For multiple comparison, one-way analysis of variance, followed byTurkey’s honestly significant difference test was used.

ACKNOWLEDGMENTS. We thank Drs. Takeshi Inagaki, Naoko Nishikawa,Naoko Kamimura, Shuying Jiang, and Sigeo Ihara for helpful discussions; Dr.Toshio Kitamura for a retroviral packaging cell line and pMX plasmids; Drs.Tetsu Akiyama, Bert Vogelstein, Ming-Jer Tsai, and Masahiro Takiguchi forplasmid constructs. This study was supported in part by Translational SystemsBiology and Medicine Initiative, Grants-in-Aid for Scientific Research, andTechnology, a grant of the Genome Network Project from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan, by the grants fromthe Program of Fundamental Studies in Health Sciences of the NationalInstitute of Biomedical Innovation, by NFAT project of New Energy andIndustrial Technology Development Organization. M.Y. is an investigator atthe Howard Hughes Medical Institute.

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