Title: p300 Interacts with the N- and C-terminal Part of PPARγ2 in a Ligand-independent and -dependent Manner, Respectively
Abstract: The nuclear peroxisome proliferator-activated receptor γ (PPARγ) activates the transcription of multiple genes involved in intra- and extracellular lipid metabolism. Several cofactors are crucial for the stimulation or the silencing of nuclear receptor transcriptional activities. The two homologous cofactors p300 and CREB-binding protein (CBP) have been shown to co-activate the ligand-dependent transcriptional activities of several nuclear receptors as well as the ligand-independent transcriptional activity of the androgen receptor. We show here that the interaction between p300/CBP and PPARγ is complex and involves multiple domains in each protein. p300/CBP not only bind in a ligand-dependent manner to the DEF region of PPARγ but also bind directly in a ligand-independent manner to a region in the AB domain localized between residue 31 to 99. In transfection experiments, p300/CBP could thereby enhance the transcriptional activities of both the activating function (AF)-1 and AF-2 domains. p300/CBP displays itself at least two docking sites for PPARγ located in its N terminus (between residues 1 and 113 for CBP) and in the middle of the protein (between residues 1099 and 1460). The nuclear peroxisome proliferator-activated receptor γ (PPARγ) activates the transcription of multiple genes involved in intra- and extracellular lipid metabolism. Several cofactors are crucial for the stimulation or the silencing of nuclear receptor transcriptional activities. The two homologous cofactors p300 and CREB-binding protein (CBP) have been shown to co-activate the ligand-dependent transcriptional activities of several nuclear receptors as well as the ligand-independent transcriptional activity of the androgen receptor. We show here that the interaction between p300/CBP and PPARγ is complex and involves multiple domains in each protein. p300/CBP not only bind in a ligand-dependent manner to the DEF region of PPARγ but also bind directly in a ligand-independent manner to a region in the AB domain localized between residue 31 to 99. In transfection experiments, p300/CBP could thereby enhance the transcriptional activities of both the activating function (AF)-1 and AF-2 domains. p300/CBP displays itself at least two docking sites for PPARγ located in its N terminus (between residues 1 and 113 for CBP) and in the middle of the protein (between residues 1099 and 1460). peroxisome proliferator-activated receptors CREB-binding protein activating function ligand binding domain retinoid X receptor steroid receptor coactivator chloramphenicol acetyltransferase thymidine kinase hemagglutinin glutathione S-transferase amino acid(s) polyacrylamide gel electrophoresis cytomegalovirus The three peroxisome proliferator-activated receptors (PPARs)1 α, δ (or β), and γ, each encoded by a separate gene and displaying different tissue distributions and distinct ligand selectivities, belong to the nuclear hormone receptor superfamily (1Schoonjans K. Martin G. Staels B. Auwerx J. Curr. Opin. Lipidol. 1997; 8: 159-166Crossref PubMed Scopus (470) Google Scholar). PPARγ is an important transcription factor involved in adipocyte differentiation and glucose metabolism. The PPARγ gene gives rise to two different PPARγ proteins, i.e. PPARγ1 and PPARγ2. PPARγ2 differs from PPARγ1 by the presence at its N terminus of an additional 28-amino acid domain whose function is so far unknown (2Fajas L. Auboeuf D. Raspé E. Schoonjans K. Lefebvre A.-M. Saladin R. Najib J. Laville M. Fruchart J.-C. Deeb S. Vidal-Ping A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar). Expression of both PPARγ types is enriched in white adipose tissue (2Fajas L. Auboeuf D. Raspé E. Schoonjans K. Lefebvre A.-M. Saladin R. Najib J. Laville M. Fruchart J.-C. Deeb S. Vidal-Ping A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar), which is consistent with the major function this receptor plays in adipogenesis (3Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3166) Google Scholar). To date, we have only a limited insight into the molecular basis by which PPARs control gene expression. Like other nuclear receptors, PPARs are suggested to have a modular structure consisting of six functional domains, A/B, C, D, and E/F (4Schoonjans K. Staels B. Auwerx J. J. Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). The A/B and E/F regions are each endowed with transcriptional activities: the activating functions (AF)-1 and -2, respectively. The E/F region also the ligand binding domain (LBD) and the AF-2 is ligand-dependent. Classically it is suggested that ligand binding facilitates the heterodimerization of PPAR with the retinoid X receptor (RXR) and the binding of the PPAR/RXR heterodimers to peroxisome proliferator-responsive elements. Consecutively the ligand-activated heterodimer stimulates transcription of the target gene. In addition to this ligand-dependent regulation, it was recently demonstrated that the transcriptional activity of PPARs could be also altered by covalent modifications such as phosphorylation (5Hu E. Kim J.B. Sarraf P. Spiegelman B.M. Science. 1996; 274: 2100-2103Crossref PubMed Scopus (949) Google Scholar, 6Zhang B. Berger J. Zhou G. Elbrecht A. Biswas S. White-Carrington S. Szalkowski D. Moller D.E. J. Biol. Chem. 1996; 271: 31771-31774Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 7Adams M. Reginato M.J. Shao D. Lazar M.A. Chatterjee V.K. J. Biol. Chem. 1997; 272: 5128-5132Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 8Camp H.S. 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Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (852) Google Scholar, 12Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-C. Heyman R.A. Rose D.W. Glass C.K. et al.Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1931) Google Scholar, 13Hanstein B. Eckner R. DiRenzo J. Halachmi S. Liu H. Searcy B. Kurokawa R. Brown M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11540-11545Crossref PubMed Scopus (344) Google Scholar). Acting as factors capable of both influencing chromatin structure and establishing contacts between the nuclear receptors and the basal transcription machinery, p300 and CBP provide a model to explain how nuclear receptors exert their effect on gene expression (14Abraham S.E. Lobo S. Yaciuk P. Heidi H.-G. Moran E. Oncogene. 1993; 8: 1639-1647PubMed Google Scholar, 15Smith C.L. Oñate S.A. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. 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Chem. 1997; 272: 33435-33443Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) have demonstrated that p300 could co-activate PPARα ligand-dependent transcriptional activity and could interact with the PPARα DEF domain in a ligand-dependent way. Aside from p300, the only cofactors described so far for PPARγ are members of the steroid receptor co-activator-1 (SRC-1) family (22DiRenzo J. Sàderstràm M. Kurokawa R. Ogliastro M.-H. Ricote M. Ingrey S. Hàrlein A. Rosenfeld M.G. Glass C.K. Mol. Cell. Biol. 1997; 17: 2166-2176Crossref PubMed Scopus (255) Google Scholar, 23Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar, 24Zhu Y. Qi C. Calandra C. Sambasiva R. Janardan K.R. Gene Expression. 1996; 6: 185-195PubMed Google Scholar, 25Li H. Gomes P.J. Don Chen J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (507) Google Scholar), the PPAR binding protein PBP (26Zhu Y. Qi C. Rao M.S. Reddy J.K. J. Biol. Chem. 1997; 272: 25500-25506Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), the PPAR gamma co-activator (PGC)-1 (27Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3177) Google Scholar), and the receptor interacting protein (RIP)-140 (28Treuter E. Albrektsen T. Johansson L. Leers J. Gustafsson J.A. Mol. Endocrinol. 1998; 12: 864-881Crossref PubMed Scopus (0) Google Scholar). Although Mizukami and Taniguchi (29Mizukami J. Taniguchi T. Biochem. Biophys. Res. Commun. 1997; 240: 61-64Crossref PubMed Scopus (88) Google Scholar), using a yeast two-hybrid system, have shown an interaction between the ligand binding domain of PPARγ and CBP, they did not provide any evidence for a co-activation function or a physiological role for CBP in this interaction. The aim of this work was to evaluate more precisely the role of p300 and CBP in PPARγ-mediated gene expression. A detailed analysis of the interaction domains between PPARγ and p300/CBP revealed for the first time that PPARγ contacts p300/CBP not only through its DEF domain in a ligand-dependent manner but also through its AB domain in a ligand-independent manner. CBP itself contacts PPARγ through several domains located in its N terminus and in a region located in the middle of the protein. As a consequence, in transfection experiments, p300 was able to co-activate independently the AF-1- and AF-2-mediated transcriptional activities of PPARγ when its ABC domain, on the one hand, and its DEF domain, on the other hand, were fused to the yeast Gal4 DNA-binding domain. The finding that the interaction between a cofactor such as p300/CBP and nuclear receptors involves numerous domains in both partners might help to understand how the N terminus region is able to regulate the whole activity of nuclear receptors. BRL 49,653 was a kind gift of Dr. L. Hamann and R. Heyman (Ligand Pharmaceuticals, San Diego, CA). The CMV p300-CHA expression vector was a gift of Dr. R. Eckner. The different CBP-glutathioneS-transferase (GST) constructs were a gift of Dr. R. Janknecht. The antibodies directed against the AB domain of PPARγ were produced in our laboratory and were a kind gift of Dr. J. Najib (2Fajas L. Auboeuf D. Raspé E. Schoonjans K. Lefebvre A.-M. Saladin R. Najib J. Laville M. Fruchart J.-C. Deeb S. Vidal-Ping A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar). The antibodies directed against the ligand binding domain (LBD) of PPARγ were a kind gift of Dr. J. Berger and Dr. M. Leibowitz (Merck Research Laboratories, Rahway, NJ). Anti-hemagglutinin antibodies (anti-HA.11) were purchased at BabCo (Richmond, CA). The protease inhibitor mixture was purchased at ICN (Orsay, France). The HeLa cell line was maintained in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% delipidated and charcoal-treated fetal calf serum, l-glutamine, and antibiotics. Transfections with chloramphenicol acetyltransferase (CAT) reporter constructs were carried out exactly as described previously (30Vu-Dac N. Schoonjans K. Kosykh V. Dallongeville J. Fruchart J.-C. Staels B. Auwerx J. J. Clin. Invest. 1995; 96: 741-750Crossref PubMed Scopus (368) Google Scholar) in 6-well plates. The pGL3-(Jwt)3TKCAT reporter construct contains three tandem repeats of the J site of the apolipoprotein A-II promoter cloned upstream of the herpes simplex virus thymidine kinase (TK) promoter and the CAT reporter gene (30Vu-Dac N. Schoonjans K. Kosykh V. Dallongeville J. Fruchart J.-C. Staels B. Auwerx J. J. Clin. Invest. 1995; 96: 741-750Crossref PubMed Scopus (368) Google Scholar). The following expression vectors were used: CMV p300-CHA, a construct where the last 36 amino acids from the C terminus of p300 have been replaced by a hemagglutinin (HA) epitope (31Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (937) Google Scholar); pSG5-hPPARγ2, a construct containing the entire cDNA of the human PPARγ2 (hPPARγ2) (2Fajas L. Auboeuf D. Raspé E. Schoonjans K. Lefebvre A.-M. Saladin R. Najib J. Laville M. Fruchart J.-C. Deeb S. Vidal-Ping A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar); pcDNA3-BDGal4-hPPARγABC, a construct where the A, B, and C regions of PPARγ2 (aa from 2 to 181) have been cloned downstream of the Gal4 DNA binding domain; pcDNA3-BDGal4-hPPARγDEF, a construct where the D, E, and F regions of PPARγ2 (aa from 181 to 507) have been cloned downstream of the Gal4 DNA binding domain; pGL3-(Gal4)5TKLuc, a reporter construct consisting of five tandem repeats of the Gal4 upstream activating sequence (UAS) cloned in front of the TK promoter and driving the expression of the luciferase reporter gene; and pCMV-βGal, a vector for the control of transfection efficiency. The p300Nt-GST, CBP-GST, and SRC1 fusion proteins were generated by cloning the N-terminal part of the p300 protein (aa 2 to 516), or different domains of CBP, or the domain comprised between amino acids 568 and 780 of SRC-1 downstream of the glutathioneS-transferase (GST) protein in the pGex-T1 vector (Amersham Pharmacia Biotech, Orsay, France). The p300Nt-GST and CBP-GST fusion proteins were then expressed in Escherichia coli and purified on a glutathione affinity matrix (Amersham Pharmacia Biotech). The PPARγ2AB1–146 (aa 1 to 146 of PPARγ), the PPARγ2ABC1–181 (aa 1 to 181 of PPARγ), and the PPARγ2DEF204–507 (aa 204 to 507 of PPARγ) proteins were produced following the same procedure, and the GST domain was removed by thrombin digestion. Polyclonal antibodies (5 μg) directed against the AB domain of PPARγ were added to nuclear extracts (150 μg at 0.5 mg/ml) prepared as described previously (32Dignam J.P. Lebowitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (10033) Google Scholar). The samples were incubated for 1 h at 4 °C in the presence or absence of 10−6m BRL 49,653. Hydrated protein A-Q-Sepharose beads (20 μl, Sigma, St. Quentin Fallavier, France), which had been first blocked with 3% bovine serum albumin in lysis buffer (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 40 mm NaCl, 1% Nonidet P-40, protease inhibitor mixture), were then added, and the samples were incubated under constant agitation for 20 min at 21 °C. The beads were then washed four times in lysis buffer. Complexes were recovered by boiling the beads in 2× sample buffer (12.5 mm Tris-HCl, 20% glycerol, 0.002% bromphenol blue, 5% β-mercaptoethanol), separated by 8% acrylamide SDS-PAGE, and transferred to nitrocellulose membranes. Blots were then developed with anti-HA.11 antibodies. The purified PPARγ2AB1–146, PPARγ2ABC1–181, and PPARγ2DEF204–507 proteins were incubated 1 h at 22 °C in pull-down buffer (1× phosphate-buffered saline, 10% glycerol, 0.5% Nonidet P-40) with either GST or the different GST fusion proteins, glutathione-Q-Sepharose beads, and different concentrations of BRL 49,653 when necessary. The beads were then washed four times in pull-down buffer and boiled in 2× sample buffer. The samples were separated by 12% acrylamide SDS-PAGE and transferred to nitrocellulose membranes. Blots were developed with antibodies directed against PPARγ2AB or PPARγ2DEF. Different domains of hPPARγ2 were cloned in the pBDGal4 vector for the construction of bait plasmids (Stratagene, La Jolla, CA); the different parts of the ABC region as well as PPARγ2DEF181–507 were cloned by polymerase chain reaction amplification on the pSG5-PPARγ2 construct of the corresponding domains. The three PPARγ2DEF deletion constructs PPARγDEF181–501, PPARγDEF181–281, and PPARγDEF181–224 were generated by removing the 3′-ends of the PPARγ2DEF181–507 insert located downstream of theSalI, EcoRI, and BglII sites, respectively. The N-terminal part of p300 (aa 2 to 516) was cloned in the pADGal4 vector (Stratagene). The pADGal4-SV40 construct was purchased from Stratagene. YRG-2 competent yeasts (Stratagene) were transformed with different combinations of expression vectors following the instructions of the manufacturer and grown at 30 °C on synthetic medium agar plates in the presence of the appropriate amino acids for selection. When the Gal4-chimera proteins interact, induction of theHIS3 and the LacZ genes occur, and the yeasts can grow on histidine-deficient media. The β-galactosidase assay was performed as described before (30Vu-Dac N. Schoonjans K. Kosykh V. Dallongeville J. Fruchart J.-C. Staels B. Auwerx J. J. Clin. Invest. 1995; 96: 741-750Crossref PubMed Scopus (368) Google Scholar) but with yeast lysates from saturated yeast cultures lyzed with acid-washed beads (Sigma). As p300 has been shown to co-activate the transcriptional activity of several nuclear receptors, we first addressed the question of whether the co-activator p300 could also enhance PPARγ2-mediated gene expression. HeLa cells were therefore co-transfected with the proliferator-responsive element-driven reporter construct pGL3-(Jwt)3TKCAT (30Vu-Dac N. Schoonjans K. Kosykh V. Dallongeville J. Fruchart J.-C. Staels B. Auwerx J. J. Clin. Invest. 1995; 96: 741-750Crossref PubMed Scopus (368) Google Scholar), together with an expression vector for human PPARγ2 (pSG5-hPPARγ2) (2Fajas L. Auboeuf D. Raspé E. Schoonjans K. Lefebvre A.-M. Saladin R. Najib J. Laville M. Fruchart J.-C. Deeb S. Vidal-Ping A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar) and increasing amounts of an expression vector for p300-CHA (CMV p300-CHA) (31Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (937) Google Scholar), in the presence or absence of BRL 49,653, a synthetic PPARγ ligand (33Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3496) Google Scholar) (Fig. 1). PPARγ transcriptional activity is stimulated in a dose-dependent way by co-transfection with the CMV p300-CHA expression vector. This effect is maximal in presence of 0.8 μg of CMV p300-CHA. To clarify the role of p300 toward each of the two PPARγ2 transcriptional activities (AF-1 and AF-2), we performed transfections with expression vectors coding for chimeric proteins composed of either the A, B, and C or the D, E, and F domains of hPPARγ2 fused to the binding domain of the Gal4 yeast transcription factor (BDGal4-hPPARγABC and BDGal4-hPPARγDEF, respectively, Fig. 2 A). These vectors were co-transfected in HeLa cells together with increasing amounts of CMV p300-CHA. Whereas we observed a significant stimulation of the transcriptional activity of the chimeric BDGal4-hPPARγABC protein by co-transfected p300, the stimulation of the DEF chimera was extremely weak (Fig. 2, Band C). These activities are maximally increased 2.4 times for BDGal4-hPPARγABC and 1.5 times for BDGal4-hPPARγDEF in presence of 60 and 100 ng of co-transfected pCMV p300-CHA, respectively. The AF-1 activity was stimulated in the absence of ligand, whereas BRL 49,653 was required for the stimulation of the AF-2 activity. Surprisingly, the BDGal4-hPPARγDEF chimera, lacking the ABC region, not only was weakly co-activated by p300 but also needed higher amounts of BRL 49,653 than the full-length receptor to be fully activated (10−6 versus 10−7m,respectively). The enhancement of PPARγ transcriptional activity by p300 suggested that the two molecules are part of the same protein complex driving gene expression. To verify this, co-immunoprecipitation experiments were carried out. HeLa cells were therefore transfected with different combinations of the pSG5hPPARγ2 and CMV p300-CHA expression constructs and of the corresponding empty expression vectors. PPARγ was then immunoprecipitated from the cell nuclear extracts with antibodies directed against its AB domain, either in presence or in absence of BRL 49,653. The immunoprecipitates were analyzed by immunoblotting using anti-HA antibodies (Fig.3). A clear band corresponding to the p300-CHA protein with an approximate molecular mass of 270 kDa was observed only for the immunoprecipitates from cells co-transfected with both PPARγ and p300-CHA. For the immunoprecipitates from cells which had been transfected either by PPARγ or p300-CHA alone, no clear band was visible in the immunoblot. This specific co-immunoprecipitation of p300-CHA with PPARγ suggests that PPARγ and p300 associate in the cell. A 2-fold increase in the amount of immunoprecipitated p300-CHA was observed when BRL 49,653 was added. The association of p300 with PPARγ in a cellular environment could be due either to a direct interaction between the two molecules or to the interaction of both of them with a third partner, either a cofactor such as SRC-1, or a nuclear receptor such as RXR. To test the hypothesis of a direct interaction, pull-down experiments with purified proteins were carried out. The domain by which p300 interacts with the LBDs of other nuclear receptors has been localized in the N-terminal part of the protein (11Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (852) Google Scholar, 12Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-C. Heyman R.A. Rose D.W. Glass C.K. et al.Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1931) Google Scholar). To verify that the same domain is involved in the interaction of p300 with PPARγ, the N-terminal part of p300 (amino acids from 2 to 516) was produced as a GST fusion protein in E. coli and purified. The PPARγ2AB1–146 (aa from 1 to 146 of PPARγ), the PPARγ2ABC1–181 (aa from 1 to 181 of PPARγ), and the PPARγ2DEF204–507 (aa from 204 to 507 of PPARγ) proteins were also produced and purified following the same procedure. The GST part of these proteins was then removed by thrombin cleavage. p300Nt-GST interacted with both the AB and the DEF domains of PPARγ2 but following two different modes: in a ligand-independent way with the AB domain (Fig. 4 A) and in a ligand-dependent way with the DEF domain of PPARγ2 (Fig. 4 B). The ligand-dependent interaction between the PPARγ2DEF and p300Nt-GST was enhanced by increasing amounts of BRL 49,653. Similar data were obtained when another synthetic PPARγ ligand, troglitazone, was used (data not shown). No interaction was detected with the GST protein alone. As each sub-region of PPARγ2 apparently displayed different properties for the binding to p300Nt, we studied the overall mode of interaction between the full-length receptor and its co-activator. In a pull-down experiment, full-length PPARγ2 produced with rabbit reticulocyte lysates or purified PPARγ2DEF were incubated with p300Nt-GST in presence or absence of BRL 49,653. Nonprogrammed reticulocyte lysate was added to the samples with purified PPARγ2DEF to rule out any artifact because of the potential presence of a PPARγ ligand in this crude lysate (used for the full-length PPARγ). In presence of ligand, both full-length PPARγ2 and PPARγ2DEF interacted with p300Nt (Fig.4 C), but in absence of ligand, only the interaction between full-length PPARγ2 and p300Nt was substantial, indicating that the ABC domain was also involved in the interaction of the full-length nuclear receptor with its co-activator and giving a potential explanation for the important ligand-independent association of p300 and PPARγ2 observed in the co-immunoprecipitation experiments. Because the direct interaction in vitro of p300/CBP with the ABC domain of a nuclear receptor had never been studied so far, we investigated more precisely the regions in CBP susceptible to contact this part of the receptor. We performed pull-down experiments using the purified PPARγ2ABC1–181or PPARγ2AB1–146 proteins and different sub-regions of CBP fused to the GST protein (Fig. 5,A and B). p300 and CBP contact the ABC domain of PPARγ2 mainly through their N-terminal part, i.e. aa from 2 to 516 for p300 (Fig. 5 A, lane 2), and aa from 1 to 113 for CBP (Fig. 5 B, lane 4). Surprisingly, another domain located between amino acids 1099 and 1460 of CBP displayed a weaker though unambiguous interaction with the ABC domain of PPARγ2 (Fig. 5 A, lanes 9 and 10). It appears therefore that p300/CBP and PPARγ2 can associate through multiple contact points. A constitutive interaction occurs in absence of any ligand because of the presence of the ABC domain. Upon ligand binding, the DEF domain also contacts the co-activator, thereby strengthening the association. It is noteworthy that the domain in the SRC-1 co-activator known to interact with the PPARγ ligand-binding domain (24Zhu Y. Qi C. Calandra C. Sambasiva R. Janardan K.R. Gene Expression. 1996; 6: 185-195PubMed Google Scholar) did not interact with the PPARγ N-terminal domain (Fig.5 B, lane 6), suggesting that the interaction observed with p300/CBP is specific. The yeast two-hybrid system provides a very sensitive and functional test to study interactions between p300 and PPARγ. Therefore, the N-terminal part of p300 (aa from 2 to 516) was cloned downstream of the activating domain of the Gal4 transcription factor (pADGal4-p300Nt), whereas different parts of hPPARγ were cloned downstream of the DNA binding domain of the Gal4 protein (Figs.6 A and7 A. In yeast, the BDGal4-PPARγ2DEF181–507 and the ADGal4-p300Nt fusion proteins interact without addition of any PPARγ ligand. It is unclear whether this interaction is because of the presence of potential PPARγ ligands in the yeast cells or whether a constitutive interaction between the DEF domain of PPARγ and the N-terminal part of p300 can actually occur in absence of any ligand in vivo(Fig. 6 A). This interaction is disrupted when the AF-2 domain of PPARγ is deleted, pointing to an important role for this domain in the interaction between the two molecules.Figure 7The N-terminal part of p300 interacts with the ABC domain of PPARγ in the yeast two-hybrid system. A, yeasts were co-transformed with bait vectors containing different parts of the ABC domain of hPPARγ2 (pBDGal4 constructs) and with the pADGal4-p300Nt or pADGal4-SV40 constructs. Growth on a histidine-deficient media is indicated by a "+."B, yeasts were co-transformed with the pBDGal4-PPARγ2ABC1–182 bait vector and the pADGal4-p300Nt or pADGal4-SV40 constructs and grown in absence or presence of BRL 49,653 (10−6m). The β-galactosidase activity was then measured in each yeast culture lysate. Data are presented as means of triplicates ± S.D. The mean activity for the lysates from yeasts transformed with the pADGal4 vector and grown without BRL 49,653 was set to be 1. Comparisons between groups were made by nonparametric Mann-Whitney tests. *, indicates a statistically significant difference (p < 0.05) with the points where the empty pADGal4 vector was used.DMSO, dimethylsulfoxide.View Large Image Figure ViewerDownload (PPT) Yeast co-transfected with bait vectors containing different regions of the ABC domain of hPPARγ2 and the pADGal4-p300Nt vector can also grow on histidine-deficient plates (Fig. 7 A), confirming that p300Nt interacts with the ABC domain of PPARγ2. The different constructs used suggest that the interaction domain in PPARγ2 is located between aa 31 and 99 and that the B exon of PPARγ2 is not required for this interaction. Beside the HIS3 reporter system, YRG-2 yeast cells also have a Gal4-dependent lacZ reporter system that can be quantified more easily. We used that quantitative system to further investigate the effect of the presence of a PPARγ ligand on the strength of the interaction between p300Nt and PPARγ2DEF181–507 or PPARγ2ABC1–182(Figs. 6 B and 7 B). Similar to the pull-down experiments, two distinct mechanisms of interaction for the two domains of PPARγ2 and p300 were observed. PPARγ2ABC and p300Nt interact in absence of any ligand and the addition of BRL 49,653 has no effe