Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) Binds to a Peroxisome Proliferator-responsive Element and Antagonizes Peroxisome Proliferator-mediated Signaling*

Peroxisome proliferators form a family of diverse xe- nobiotic compounds that includes hypolipidemic agents, herbicides, and plasticizers. These compounds activate transcription of a subset of nuclear genes in- cluding those encoding peroxisomal fatty acid P-oxida-tion enzymes, whose elevated activities can lead to hepatocarcinogenesis. Induction of the genes encoding fatty acyl-CoA oxidase and hydratase-dehydrogenase, the first and second enzymes of the pathway, is mediated by peroxisome proliferator-activated nuclear receptors (PPARs) that bind to upstream responsive elements (PPREs) through heterodimerization with retinoid X receptors. We demonstrate that the chicken ovalbumin upstream promoter transcription factor 1 (COUP-TFl), an- other member of the nuclear hormone receptor superfamily, binds to the hydratase-dehydrogenase PPRE in vitro and in vivo and antagonizes PPAR-depen- dent signaling. These data suggest that members of the COUP-TF family play a role in modulating receptor-me- diated activation of peroxisome proliferator-responsive genes.

cis-Acting peroxisome proliferator-responsive elements (PPREs) have been identified in the 5"flanking regions of the genes encoding fatty acyl-CoA oxidase and HD (9)(10)(11)(12). Both PPREs contain direct repeats of the sequence TGACCT, which is the consensus binding site for several members of the nuclear hormone receptor superfamily (13,141, including the vitamin DB, thyroid, and retinoic acid receptors. These receptors bind to their cognate sites through cooperativity with the retinoid X receptor, RXR (15)(16)(17). Signal transduction by peroxisome proliferators is mediated by a similar mechanism involving ligandactivated receptors called peroxisome proliferator-activated receptors (PPARs) that belong to this family of transcription factors (18)(19)(20)(21). PPARs bind cooperatively to PPREs through heterodimerization with the 9-cis-retinoic acid receptor, RXRa The transcriptional activation mediated by steroid receptors is subject to negative regulation. For example, the chicken ovalbumin upstream promoter-transcription factors (COUP-TFs), which are orphan members of the nuclear hormone receptor superfamily, have been shown to repress hormonal induction of vitamin D3, thyroid, and retinoic acid receptor target genes by competing with these receptors for their binding sites (27,28) and by heteromerization with RXR (28). COUP-TFs bind as homodimers to diverse response elements consisting of TGACCT repeats which exhibit wide variation in spacing and orientation (27); however, they have the greatest affinity for direct repeats separated by 1 base pair (DR1). The HD PPRE consists of three imperfect direct repeats of the TGACCT motif separated by 2 base pairs (DR2) and 1 base pair (DRl), respectively ( Fig. lA Cells-Rat hepatoma H4IIEC3 cells were cultured as monolayers in Dulbecco's modified Eagle's medium containing 10% horse serum and 5% fetal bovine serum. HeLa cells were maintained in suspension in Joklik's modified medium plus 5% fetal bovine serum. BSC4O cells were maintained in Dulbecco's modified Eagle's medium plus 10% calf serum. Plasmids-pCPSZuc is a luciferase expression vector containing the minimal promoter for the gene encoding rat liver carbamoyl phosphate synthetase (11). Plasmids for expression in BSC40 cells were constructed by cloning an oligonucleotide corresponding to the HD PPRE

( 5 ' -g a t C C T C T C C T T T G A C C T A ' I T G A A C T A C A and its complement 5 ' -g a t c T C A A A T G T A G C A G T A G G T C A A A G -
GAGAG or mutant forms of this PPRE, into the unique BamHI site of pCPSZuc located upstream of the carbamoyl phosphate synthetase promoter (25). pHD(X1)luc and pHD(X3)Zuc contain one copy and three direct tandem copies of the HD PPRE oligonucleotide, respectively, in the same 5' + 3' orientation found in the natural HD promoter (25). Rat (r) PPAR and RXRa vectors for in vivo and in vitro expression have hydroxyacyl-CoA dehydrogenase; COUP-TF, chicken ovalbumin up- The abbreviations used are: HD, enoyl-CoA hydratasd3stream promoter factor transcription factor; EMSA, electrophoretic mobility shift analysis; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-responsive element; RXR, retinoid X receptor; RXRa, 94s-retinoic acid receptor; r, rat; h, human.

COUP-TFl Antagonizes Signaling by Peroxisome Proliferators
been described (26). A plasmid expressing hCOUP-TF1 was constructed by ligating the HindIIYXbaI fragment from pCOUP-TFl (27) into the corresponding sites of pWCMV (Invitrogen). A plasmid encoding a truncated form of hCOUP-TF1 (tCOUP-TF1; Ref. 27) was constructed by cloning the SmYAatII fragment from pCOUP-TFl into the EcoRV and AatII sites of pGEM-5Zff +) (Promega). Transcription and translation of this plasmid in vitro generated a truncated hCOUP-TFl protein lacking 51 amino acids at its amino terminus. Dannfections and Measurement of Luciferase Activity-Transfections of H4IIEC3 cells were done by the calcium phosphate method followed by a dimethyl sulfoxide shock (11). BSC40 cells were transfected similarly except that cells were incubated for 24 h before and during transfection in medium without phenol red and containing 5% charcoalstripped fetal bovine serum (26). Transfections typically contained 5 pg of a reporter gene construct and, where indicated, 2 pg of rPPAR, 2 pg of RXRa, and 0-4 pg of hCOUP-TFl expression plasmids. Effector plasmid dosage was kept constant by the addition of appropriate amounts of the corresponding empty expression vector (pSG5 or pRC/CMV). The total amount of DNA was kept at 20 pg with sonicated salmon sperm DNA. Ciprofibrate ( x 100 stock in dimethyl sulfoxide) was added to fresh medium to a final concentration of 0.5 mM. Extracts were prepared 48-h post-transfection, and luciferase activity was measured.
ing rPPAFt, RXRa, HNF4, hCOUP-TFl, and tCOUP-TFl and subse-In Vitro Danscription /Dunslation-Transcription of cDNAs encodquent translation in rabbit reticulocyte lysate were performed using a commercially available kit (Promega). Translations of proteins for use in electrophoretic mobility shift assays (EMS&) were done with unlabeled methionine.
Electrophoretic Mobility Shift Analysis-Nuclear extracts from HeLa cells were prepared from cell suspensions (30). Nuclear extracts were prepared from monolayer cultures of H4IIEC3, BSC40, and BSC4O cells transfected with various expression plasmids as described (30,31). EMSA was performed as described (11.26). Double-stranded probes of the wild-type and mutant forms of the HD PPRE were end-labeled with [a-D2PldATP and the Klenow fragment of DNA polymerase I. Binding reactions were analyzed by electrophoresis a t 4 "C on prerun 3.5% polyacrylamide gels (30:l acrylamiddNJV"methylenebisacrylamide weight ratio) with 22 mM Tris base, 22 mM boric acid, 1 mM EDTA as running buffer. For binding reactions done with in vitro synthesized protein, 1-2 pl of translation mixture was incubated with labeled probe in a final volume of 15 pl. The total amount of reticulocyte lysate was kept constant in each reaction by the addition of unprogrammed lysate.

RESULTS AND DISCUSSION
The HD PPRE consists of three imperfect direct repeats of the consensus nuclear hormone binding motif TGACCT separated by 2 base pairs (DR2) and l base pair (DRl), respectively (Fig. L4). We have demonstrated that an oligonucleotide corresponding to the HD PPRE interacts with cellular factors present in H4IIEC3 cells, a rat hepatoma cell line responsive to peroxisome proliferators (11,25), and that the PPRE-binding proteins include rPPAR and RXRa (26). To explore whether members of the COUP-TF family are present in these complexes, EMSA was camed out with radiolabeled HD PPRE probe and nuclear extracts prepared from H4IIEC3 cells in the presence of antibody to hCOUP-TF1 (hereafter called COUP-TF1). This antibody recognizes both COUP-TF1 and a related receptor COUP-TF2 (27), which is also called ARP-1 (32). As shown in Fig. 1B, inclusion of anti-hCOUP-TF1 resulted in the formation of supershifted complexes (lane f ) , demonstrating that the PPRE-binding proteins include COUP-TF-related factors. The major protein-DNA complex formed between HD PPRE and nuclear extracts of HeLa cells was almost quantitatively supershifted with anti-hCOUP-TF1 (lane c). Therefore, COUP-TF-related factors in both rat hepatoma and HeLa nuclear extracts bind to the HD PPRE.
To determine whether COUP-TF1 could bind directly to the HD PPRE, EMSA was camed out with in vitro translated receptor. As shown in Fig. 2A, in oitro translated COUP-TF1 bound to the HD PPRE (lane e; the identity of COUP-TF1 in these complexes was established using anti-hCOUP-TF1 serum; data not presented). COUP-TF1 also bound to the PPRE of the gene encoding fatty acyl-CoA oxidase (data not shown). The arrows designate the TCACCT direct repeat motifs. E . electrophoretic mobility shift assays. Nuclear extracts prepared from H e h cells or rat hepatoma H41IEC3 cells were incubated with radiolabeled HD PPRE probe alone (lanes a and d ) or with addition of preimmune serum (lanes b and e ) or of polyclonal anti-hCOUP-TF1 serum (lane8 c and f 1, as indicated. The brackets indicate novel complexes of reduced mobility generated in the presence of anti-hCOUP-TF1 serum. Where indicated, 0.25 pl of preimmune serum ( P I ) or of anti-hCOUP-TF1 serum was added to the binding reactions, which were then preincubated for 5 min prior to addition of probe.
Several distinct PPARa bind to the HD PPRE cooperatively through heteromerization with RXRa (26). To determine the DNA sequence requirements for binding COUP-TF1 versus the cooperative binding of PPAR and RXRa on the HD PPRE. we used oligonucleotides in which each of the core TGACCT motifs was individually mutated. Fig. 2B shows that mutation of the first repeat (M4) had no effect on binding of in vitro translated rPPAR/RXRa  (lane h). Therefore, COUP-TF1 and rPPAR/RXRa recognize distinct, yet overlapping, core elements within the HD PPRE, with the first two repeata being the important determinant for COUP-TF1 binding and the second and third repeats being necessary for rPPAR/RXRa binding. The DR2 spacing of the first two repeats is not necessary for COUP-TF1 binding, as an oligonucleotide (M6) in which the DR2 was converted to a DR1 bound COUP-TF1 strongly (lane j). Therefore, the primary sequence of the first two repeats, and not their relative spacing, are important for binding of COUP-TF1.
The mutated PPREs were assessed for their ability to confer peroxisome proliferator responsiveness onto a luciferase reporter gene in transient transfection assays of rat H4IIEC3 cells. As shown in Fig. 3, the activities of the wild-type HD PPRE reporter constructs pHD(X1)luc and pHMX3)luc were induced approximately 3and l&fold, respectively, in the presence of the peroxisome proliferator ciprofibrate, similar to what has been shown previously (25). Reporter constructs containing either one copy or three tandem copies of the different mutant  (lanes a , c, e, g. and i) or COUP -TF1 (lanes b, d , f, h, a n d j ) , a s indicated.
PPREs were unresponsive to ciprofibrate. These results extend our previous observation (25) that multimerization of the wildtype HD PPRE serves only to amplify the behavior of the response element to ciprofibrate and has little effect on basal level transcription with either the wild-type or mutant HD PPREs. Importantly, the results of Fig. 3 demonstrate that the sequences required for COUP-TF1 binding in uitm are also necessary for peroxisome proliferator responsiveness in uiuo. Moreover, they confirm our finding that PPAR/RXRa binding is necessary but not sufficient for peroxisome proliferator mediated induction via the HD PPRE (26) and demonstrate that all three repeats of the HD PPRE and the particular spacing of the repeats are involved in the responsiveness to proliferators, sug- were individually cloned as single copies (X1 ) or as three direct tandem copies (X3) upstream of reporter plasmid pCPSluc and transfected into H4IIEC3 cells, which were subsequently treated with dimethyl sulfoxide alone or with the peroxisome proliferator ciprofibrate. The values were normalized to the activity of control transfections done with pCPSluc in the absence of ciprofibrate. which was taken as 1. The values are from two independent transfections done in duplicate and did not vary by more than 15%.
gesting the involvement of additional transcription factors in regulating the response to ciprofibrate.
COUP-TFs repress induction by hormone receptors through the formation of inactive DNA-binding heterodimers (28). Since COUP-TF1.DNA complexes had the same electrophoretic mobility vis a uis those formed with rPPAR/RXRa (see Fig. 2A,  lanes d and e ) , we constructed a truncated form of COUP-TF1 (tCOUP-TF1) that permitted better resolution of potential heteromeric complexes. As seen in Fig. 4 Mixing tCOUP-TF1 with RXRa (fane f), rPPAR (fane i ) . or HNF4 (lane g), another member of the nuclear hormone recep tor family (33). did not generate any novel complexes, indicating that COUP-TF1 does not form PPRE-binding heteromera with these receptors. In addition, an excess of RXRa itself was unable to inhibit COUP-TF1 binding (data not presented), s u g gesting that COUP-TF1 homodimer binding to the PPRE is not appreciably affected by the formation of COUP-TF1.RXRu heterodimers in solution.
We wished to determine whether COUP-TFl could downregulate PPAR/RXRa-mediated transactivation in uiuo. The pHD(X3)Zuc reporter plasmid was cotransfected with expression vectors for rPPAR, RXRa, and COUP-TF1 into BSC4O cells. BSC40 cells are unresponsive to peroxisome proliferatora in the absence of co-transfected PPAR and RXRa (Fig. 5 ) and do not contain factors that bind to the HD PPRE (11). A 10-fold induction in luciferase activity was observed in the presence of both rPPAR and RxRa, which was further increased by the addition of the ciprofibrate. Addition of increasing amounta of COUP-TF1 expression vector resulted in the progressive repression of rPPAR/RXRa-mediated induction with pHD(X3)luc. Repression of induction was specific, as the transfection of COUP-TF1 expression vector alone had no effect on the basal Fro. 5. COUP-TF1 antagoniee~~ peroxieome proliferator-mediated dgnaling. pHWX3)luc was transfected into BSC4O cells along with effector plasmids expressing rPPAR, RxRa, and COUP-TFl, as indicated. The values shown are relative activities from repeat transfections done in duplicate and were normalized to the value obtained for ciprofibrate-treated cells cotransfected with rPPAR and RXRa expression plasmids, which was taken as 100%. The numbers for COUP-TFl refer to the amount (in pg) of cotransfected plasmid expressing COUP-TF1.

COUP-TF1 Antagonizes Signaling by Peroxisome Proliferators
activity of pHD(X3)Zuc. Expression of reporter constructs containing mutant HD PPREs was not stimulated by cotransfection with plasmids expressing rPPAR and RxRa in either the presence or absence of ciprofibrate (data not shown), in agreement with the transfection results presented in Fig. 3. Therefore, COUP-TF1 antagonizes PPAR-mediated peroxisome proliferator signaling in vivo. This finding, combined with the results of Fig. 4, suggests that COUP-TF1-mediated antagonism occurs via competition with PPAFURXRa for occupancy of the DNA target site on the HD PPRE.
The promiscuous binding of members of the COUP-TF recep tor family to diverse response elements composed of TGACCT repeats, with its consequences on transcriptional induction mediated by cognate receptors, has positioned COUP-TFs as central regulators of hormone-responsive networks. COUP-TFs have been shown to play an important role in the modulation of expression of apolipoprotein genes, whose products are involved in lipid homeostasis (34). Our demonstration that COUP-TF1 binds to a PPRE and antagonizes PPAR-mediated signaling implicates members of the COUP-TF family an playing a role in lipid homeostasis also through the modulation of peroxisome-mediated metabolism of fatty acids and in the cellular response to peroxisome proliferators and other xenobiotic compounds.