p300 Interacts with the N- and C-terminal Part of PPARgamma 2 in a Ligand-independent and -dependent Manner, Respectively*

Laurent GelmanDagger §, Gaochao Zhou, Lluis FajasDagger , Eric RaspéDagger , Jean-Charles FruchartDagger , and Johan AuwerxDagger parallel

From the Dagger  Unité 325 INSERM, Département d'Athérosclérose, Institut Pasteur de Lille, 1, rue du Prof. Calmette, 59019 Lille Cédex, France and the  Merck Research Laboratories, Rahway, New Jersey 07065

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

The nuclear peroxisome proliferator-activated receptor gamma  (PPARgamma ) 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 PPARgamma is complex and involves multiple domains in each protein. p300/CBP not only bind in a ligand-dependent manner to the DEF region of PPARgamma 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 PPARgamma located in its N terminus (between residues 1 and 113 for CBP) and in the middle of the protein (between residues 1099 and 1460).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The three peroxisome proliferator-activated receptors (PPARs)1 alpha , delta  (or beta ), and gamma , each encoded by a separate gene and displaying different tissue distributions and distinct ligand selectivities, belong to the nuclear hormone receptor superfamily (1). PPARgamma is an important transcription factor involved in adipocyte differentiation and glucose metabolism. The PPARgamma gene gives rise to two different PPARgamma proteins, i.e. PPARgamma 1 and PPARgamma 2. PPARgamma 2 differs from PPARgamma 1 by the presence at its N terminus of an additional 28-amino acid domain whose function is so far unknown (2). Expression of both PPARgamma types is enriched in white adipose tissue (2), which is consistent with the major function this receptor plays in adipogenesis (3). 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 (4). 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 (5-9). Furthermore, the transcriptional activity of nuclear receptors can be influenced by cofactors, such as co-activators or co-repressors, which modulate signaling and interaction with the basal transcription machinery (10).

Among the cofactors shown to modulate nuclear receptor transcriptional activities, p300 and the CREB-binding protein (CBP), two homologous co-activators, have recently attracted interest because of the pivotal role they play in the cross-talk between different signal transduction pathways (11-13). 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 (14-20).

So far only a few studies addressed the interaction between PPARs and cofactors. Dowell et al. (21) have demonstrated that p300 could co-activate PPARalpha ligand-dependent transcriptional activity and could interact with the PPARalpha DEF domain in a ligand-dependent way. Aside from p300, the only cofactors described so far for PPARgamma are members of the steroid receptor co-activator-1 (SRC-1) family (22-25), the PPAR binding protein PBP (26), the PPAR gamma co-activator (PGC)-1 (27), and the receptor interacting protein (RIP)-140 (28). Although Mizukami and Taniguchi (29), using a yeast two-hybrid system, have shown an interaction between the ligand binding domain of PPARgamma 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 PPARgamma -mediated gene expression. A detailed analysis of the interaction domains between PPARgamma and p300/CBP revealed for the first time that PPARgamma 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 PPARgamma 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 PPARgamma 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.

    EXPERIMENTAL PROCEDURES

Materials

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-glutathione S-transferase (GST) constructs were a gift of Dr. R. Janknecht. The antibodies directed against the AB domain of PPARgamma were produced in our laboratory and were a kind gift of Dr. J. Najib (2). The antibodies directed against the ligand binding domain (LBD) of PPARgamma 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).

Cell Culture and Transient Transfection Assays

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 (30) 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 (30). 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 (31); pSG5-hPPARgamma 2, a construct containing the entire cDNA of the human PPARgamma 2 (hPPARgamma 2) (2); pcDNA3-BDGal4-hPPARgamma ABC, a construct where the A, B, and C regions of PPARgamma 2 (aa from 2 to 181) have been cloned downstream of the Gal4 DNA binding domain; pcDNA3-BDGal4-hPPARgamma DEF, a construct where the D, E, and F regions of PPARgamma 2 (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-beta Gal, a vector for the control of transfection efficiency.

Production of Proteins

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 glutathione S-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 PPARgamma 2AB1-146 (aa 1 to 146 of PPARgamma ), the PPARgamma 2ABC1-181 (aa 1 to 181 of PPARgamma ), and the PPARgamma 2DEF204-507 (aa 204 to 507 of PPARgamma ) proteins were produced following the same procedure, and the GST domain was removed by thrombin digestion.

Immunoprecipitation and Pull-down Experiments

Immunoprecipitations-- Polyclonal antibodies (5 µg) directed against the AB domain of PPARgamma were added to nuclear extracts (150 µg at 0.5 mg/ml) prepared as described previously (32). The samples were incubated for 1 h at 4 °C in the presence or absence of 10-6 M 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% beta -mercaptoethanol), separated by 8% acrylamide SDS-PAGE, and transferred to nitrocellulose membranes. Blots were then developed with anti-HA.11 antibodies.

Pull-down Experiments-- The purified PPARgamma 2AB1-146, PPARgamma 2ABC1-181, and PPARgamma 2DEF204-507 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 PPARgamma 2AB or PPARgamma 2DEF.

Yeast Two-hybrid System

Different domains of hPPARgamma 2 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 PPARgamma 2DEF181-507 were cloned by polymerase chain reaction amplification on the pSG5-PPARgamma 2 construct of the corresponding domains. The three PPARgamma 2DEF deletion constructs PPARgamma DEF181-501, PPARgamma DEF181-281, and PPARgamma DEF181-224 were generated by removing the 3'-ends of the PPARgamma 2DEF181-507 insert located downstream of the SalI, 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 the HIS3 and the LacZ genes occur, and the yeasts can grow on histidine-deficient media. The beta -galactosidase assay was performed as described before (30) but with yeast lysates from saturated yeast cultures lyzed with acid-washed beads (Sigma).

    RESULTS

p300 Stimulates PPARgamma Transcriptional Activity in HeLa Cells-- 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 PPARgamma 2-mediated gene expression. HeLa cells were therefore co-transfected with the proliferator-responsive element-driven reporter construct pGL3-(Jwt)3TKCAT (30), together with an expression vector for human PPARgamma 2 (pSG5-hPPARgamma 2) (2) and increasing amounts of an expression vector for p300-CHA (CMV p300-CHA) (31), in the presence or absence of BRL 49,653, a synthetic PPARgamma ligand (33) (Fig. 1). PPARgamma 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.


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Fig. 1.   p300 enhances PPARgamma 2 transcriptional activity in HeLa cells. HeLa cells were co-transfected with an expression vector for hPPARgamma 2 (0.1 µg/well), increasing amounts of an expression vector for p300, and with either the pGL3-(Jwt)3TKCAT or the pGL3-TKCAT reporter constructs (1 µg/well). They were then grown during 24 h in the presence or absence of 10-7 M BRL 49,653. The numbers above the shaded bars indicate the -fold increase of the normalized CAT activity compared with control (ctrl). Each point was performed in triplicate, and this figure is representative of four independent experiments. DMSO, dimethylsulfoxide

To clarify the role of p300 toward each of the two PPARgamma 2 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 hPPARgamma 2 fused to the binding domain of the Gal4 yeast transcription factor (BDGal4-hPPARgamma ABC and BDGal4-hPPARgamma DEF, respectively, Fig. 2A). 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-hPPARgamma ABC protein by co-transfected p300, the stimulation of the DEF chimera was extremely weak (Fig. 2, B and C). These activities are maximally increased 2.4 times for BDGal4-hPPARgamma ABC and 1.5 times for BDGal4-hPPARgamma DEF 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-hPPARgamma DEF 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 10-7 M, respectively).


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Fig. 2.   p300 co-activates the PPARgamma 2 AF-1 and AF-2 transcriptional activity in RK13 cells. A, pcDNA3-BDGal4-hPPARgamma ABC is a construct where the A, B, and C regions of PPARgamma 2 (aa from 2 to 181) have been cloned downstream of the Gal4 DNA binding domain and pcDNA3-BDGal4-hPPARgamma DEF is a construct where the D, E, and F regions of PPARgamma 2 (aa from 181 to 507) have been cloned downstream of the Gal4 DNA binding domain. HeLa cells were co-transfected with expression vectors for the BDGal4, BDGal4-hPPARgamma AB (B), or BDGal4-hPPARgamma DEF (C) chimeric proteins (5 ng/well), increasing amounts of an expression vector for p300, and the pGL3-(Gal4)5TKLuc reporter construct (1.5 µg/well). Cells were then grown 24 h in the presence or absence of 10-6 M BRL 49,653. The histograms present the transcriptional activity of the BDGal4-hPPARgamma AB or BDGal4-hPPARgamma DEF chimeric proteins compared with the activity of the BDGal4 protein in the same conditions. The numbers above the shaded bars indicate the relative increase when p300 was added compared with control (ctrl). Each point was performed in triplicate, and the means and standard deviations where calculated with data from three independent experiments. Comparisons between groups were made by nonparametric Mann-Whitney tests. *, indicates a statistically significant difference (p < 0.05) with control. DMSO, dimethylsulfoxide.

p300 and PPARgamma Interact in a Cellular Context-- The enhancement of PPARgamma 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 pSG5hPPARgamma 2 and CMV p300-CHA expression constructs and of the corresponding empty expression vectors. PPARgamma 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 PPARgamma and p300-CHA. For the immunoprecipitates from cells which had been transfected either by PPARgamma or p300-CHA alone, no clear band was visible in the immunoblot. This specific co-immunoprecipitation of p300-CHA with PPARgamma suggests that PPARgamma 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.


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Fig. 3.   p300 and PPARgamma interact in vivo. HeLa cells were transfected either with an empty expression construct or with expression vectors coding for hPPARgamma 2 or p300-CHA. hPPARgamma 2 was immunoprecipitated from the nuclear extracts with an antibody directed against the AB domain of PPARgamma after incubation of the samples in absence or presence 10-6 M BRL 49,653. The immunoprecipitates were separated on an 8% SDS-PAGE gel and immunoblotted with anti-HA.11 antibodies.

p300 Interacts in Vitro with the AB and the DEF Domains of PPARgamma 2-- The association of p300 with PPARgamma 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 (11, 12). To verify that the same domain is involved in the interaction of p300 with PPARgamma , 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 PPARgamma 2AB1-146 (aa from 1 to 146 of PPARgamma ), the PPARgamma 2ABC1-181 (aa from 1 to 181 of PPARgamma ), and the PPARgamma 2DEF204-507 (aa from 204 to 507 of PPARgamma ) 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 PPARgamma 2 but following two different modes: in a ligand-independent way with the AB domain (Fig. 4A) and in a ligand-dependent way with the DEF domain of PPARgamma 2 (Fig. 4B). The ligand-dependent interaction between the PPARgamma 2DEF and p300Nt-GST was enhanced by increasing amounts of BRL 49,653. Similar data were obtained when another synthetic PPARgamma ligand, troglitazone, was used (data not shown). No interaction was detected with the GST protein alone.


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Fig. 4.   p300 and CBP interact directly with the ABC and the DEF domains of PPARgamma in vitro. A, the purified PPARgamma 2AB1-146 and PPARgamma 2ABC1-181 proteins were incubated with purified GST or p300Nt-GST protein and glutathione-Q-Sepharose beads in presence or absence of BRL 49,653. The beads were then washed and the samples separated on a 12% SDS-PAGE gel. Blots were developed with antibodies directed against the AB domain of PPARgamma 2. B, the purified PPARgamma 2DEF protein was incubated with purified p300Nt-GST protein, glutathione-Q-Sepharose beads, and different concentrations of BRL 49,653. The beads were then washed and the samples separated on a 12% SDS-PAGE gel. Blots were developed with antibodies directed against the DEF domain of PPARgamma 2. C, full-length PPARgamma 2 synthetized with rabbit reticulocyte lysates or purified PPARgamma 2DEF protein mixed with nonprogrammed lysate were incubated with the purified p300Nt-GST or GST proteins and glutathione-Q-Sepharose beads in presence or absence of BRL 49,653. The beads were then washed and the samples separated on a 4-20% SDS-PAGE gel. Blots were developed with antibodies directed against the DEF domain of PPARgamma 2.

As each sub-region of PPARgamma 2 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 PPARgamma 2 produced with rabbit reticulocyte lysates or purified PPARgamma 2DEF were incubated with p300Nt-GST in presence or absence of BRL 49,653. Nonprogrammed reticulocyte lysate was added to the samples with purified PPARgamma 2DEF to rule out any artifact because of the potential presence of a PPARgamma ligand in this crude lysate (used for the full-length PPARgamma ). In presence of ligand, both full-length PPARgamma 2 and PPARgamma 2DEF interacted with p300Nt (Fig. 4C), but in absence of ligand, only the interaction between full-length PPARgamma 2 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 PPARgamma 2 observed in the co-immunoprecipitation experiments.

CBP Interacts with the ABC Region of PPARgamma 2 through Multiple Domains-- 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 PPARgamma 2ABC1-181 or PPARgamma 2AB1-146 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 PPARgamma 2 mainly through their N-terminal part, i.e. aa from 2 to 516 for p300 (Fig. 5A, lane 2), and aa from 1 to 113 for CBP (Fig. 5B, 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 PPARgamma 2 (Fig. 5A, lanes 9 and 10). It appears therefore that p300/CBP and PPARgamma 2 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 PPARgamma ligand-binding domain (24) did not interact with the PPARgamma N-terminal domain (Fig. 5B, lane 6), suggesting that the interaction observed with p300/CBP is specific.


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Fig. 5.   PPARgamma 2ABC interacts with different domains of CBP. A, the purified PPARgamma 2ABC1-181 protein was incubated with the purified p300Nt-GST protein or CBP-GST constructs and glutathione-Q-Sepharose beads. The beads were then washed and the samples separated on a 12% SDS-PAGE gel. Blots were developed with antibodies directed against the AB domain of PPARgamma 2. B, the purified PPARgamma 2ABC1-146 protein was incubated with the purified p300Nt-GST, CBP1-113-GST, or SRC568-780-GST proteins and glutathione-Q-Sepharose beads. The beads were then washed and the samples separated on a 12% SDS-PAGE gel. Blots were developed with antibodies directed against the AB domain of PPARgamma 2.

p300 and PPARgamma Interact in the Yeast Two-hybrid System-- The yeast two-hybrid system provides a very sensitive and functional test to study interactions between p300 and PPARgamma . 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 hPPARgamma were cloned downstream of the DNA binding domain of the Gal4 protein (Figs. 6A and 7A. In yeast, the BDGal4-PPARgamma 2DEF181-507 and the ADGal4-p300Nt fusion proteins interact without addition of any PPARgamma ligand. It is unclear whether this interaction is because of the presence of potential PPARgamma ligands in the yeast cells or whether a constitutive interaction between the DEF domain of PPARgamma and the N-terminal part of p300 can actually occur in absence of any ligand in vivo (Fig. 6A). This interaction is disrupted when the AF-2 domain of PPARgamma is deleted, pointing to an important role for this domain in the interaction between the two molecules.


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Fig. 6.   The N-terminal part of p300 interacts with the DEF domain of PPARgamma in the yeast two-hybrid system. A, yeasts were co-transformed with bait vectors containing different parts of the DEF domain of hPPARgamma 2 (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-PPARgamma 2DEF181-507 bait vector and the pADGal4-p300Nt or pADGal4-SV40 constructs and grown in absence or presence of BRL 49,653 (10-6 M). The beta -galactosidase activity was then measured in each yeast culture lysate. Data are presented as means of triplicates ± standard deviations. 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


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Fig. 7.   The N-terminal part of p300 interacts with the ABC domain of PPARgamma in the yeast two-hybrid system. A, yeasts were co-transformed with bait vectors containing different parts of the ABC domain of hPPARgamma 2 (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-PPARgamma 2ABC1-182 bait vector and the pADGal4-p300Nt or pADGal4-SV40 constructs and grown in absence or presence of BRL 49,653 (10-6 M). The beta -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.

Yeast co-transfected with bait vectors containing different regions of the ABC domain of hPPARgamma 2 and the pADGal4-p300Nt vector can also grow on histidine-deficient plates (Fig. 7A), confirming that p300Nt interacts with the ABC domain of PPARgamma 2. The different constructs used suggest that the interaction domain in PPARgamma 2 is located between aa 31 and 99 and that the B exon of PPARgamma 2 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 PPARgamma ligand on the strength of the interaction between p300Nt and PPARgamma 2DEF181-507 or PPARgamma 2ABC1-182 (Figs. 6B and 7B). Similar to the pull-down experiments, two distinct mechanisms of interaction for the two domains of PPARgamma 2 and p300 were observed. PPARgamma 2ABC and p300Nt interact in absence of any ligand and the addition of BRL 49,653 has no effect on this interaction. In contrast, although hPPARgamma DEF and p300Nt can interact in absence of any ligand, a significant increase of the beta -galactosidase activity is observed in presence of BRL 49,653, suggesting an enhancement of the interaction.

    DISCUSSION

Among the cofactors shown to interact with several transcription factors, the homologous molecules p300 and CBP are two of the most studied co-activators (19, 20). Concerning nuclear receptors, there is now evidence for a direct interaction between p300/CBP and the estrogen receptor (ER), the retinoic acid receptor (RAR), RXR, and the thyroid hormone receptor (TR) (11, 12, 15, 16). It has been shown recently that p300 could interact with PPARgamma 2DEF (29) and that p300 could enhance PPARalpha ligand-dependent transcriptional activity by binding to its DEF domain (21). The scope of this work was to evaluate the role of p300 toward both the ligand-dependent and ligand-independent transcriptional activities of PPARgamma .

In a series of transfection experiments, PPARgamma 2-transcriptional activity was enhanced in the presence of p300. The observation that full-length PPARgamma is more activated than PPARgamma 2ABC or PPARgamma 2DEF alone sustains the hypothesis that nuclear receptors AF-1 and AF-2 are co-activated in a cooperative manner and confirms previous observations made with the progesterone receptor and SRC-1 (34). This result shows also for the first time that p300 is a co-activator of PPARgamma and raises the question of whether p300 is crucial in PPARgamma 2 physiology, most notably in fat tissue and colon (2) where PPARgamma is expressed to high levels. We can speculate that some mutations or altered levels of expression of p300 or PPARgamma could result in the disruption or the enhancement of their interaction and have important consequences in these processes.

p300 or CBP have been shown to contact the DEF domains of the ER, RAR, RXR, TR, and PPARalpha in a ligand-dependent manner (11, 12, 15, 16, 21). These interactions involve different domains of p300, all located in its N-terminal region (21). In this study, different parts of PPARgamma were tested for their interaction with the N terminus of p300. A ligand-dependent interaction between the N-terminal part of p300 and the DEF domain of PPARgamma 2 was demonstrated, and the importance of the AF-2 domain in this process has been highlighted (Fig. 7). Most interestingly, it was shown that the AB domain of PPARgamma 2 also displays a docking site for p300 and that this interaction is ligand-independent. This result is not surprising because p300 is already known to interact with transcription factors that do not have any ligand such as CREB (35) or the AP-1 complex (12, 36, 37). The AB region of nuclear receptors, which includes the AF-1 domain, hence could be considered as a ligand-independent interaction surface. Such a ligand-independent interaction through the AB region might not be restricted to PPARgamma and p300/CBP. Indeed, CBP as well as the F-SRC-1 and RIP140 co-activators have been shown to co-activate the androgen receptor (AR) AF-1 activity (38) even though the existence of a direct interaction between CBP and the N terminus of the AR has not been demonstrated. Along the same lines, it was recently shown that also the estrogen receptor AF-1 works by binding p160 co-activator proteins (39). Another argument for different docking domains in PPARgamma for cofactors comes from a paper by Puigserver et al. (27) who recently cloned a new cofactor that interacts only in a ligand-independent way with part of the DNA-binding and hinge domains of PPARgamma .

The existence of a ligand-independent interaction between p300 and the AB domain of PPARgamma 2 might also indicate that beside the PPARgamma ligands the interaction between PPARgamma and p300 is susceptible to modulation by other transduction pathways. Indeed, it has previously been demonstrated that p300 could undergo phosphorylation during cell differentiation (40) and that the state of phosphorylation of p300 could orientate the interactions with its different partners (41). PPARgamma activity itself is also subject to regulation by phosphorylation (5-9). During adipocyte differentiation for example, p300 or PPARgamma could each be differentially phosphorylated and/or could undergo other post-translational modifications that might drastically affect their ability to interact and control transcription.

Several studies demonstrated the crucial role played by the N-terminal domain of nuclear receptors for promoter- and cell-specificity determination, ligand-dependent transactivation, and recruitment of co-activator (34, 38, 42-44). Our results further sustain the idea that cofactor recruitment by the LBD of nuclear receptors is influenced by their N-terminal part and show that this could be because of the presence in this domain of one or several docking sites for these cofactors. More generally, the finding that domains other than the LBD of PPARgamma and the N terminus p300/CBP are involved in the interaction between the two molecules (Figs. 4 and 6) points out that their dimerization (and most likely the dimerization of p300/CBP with other nuclear receptors) is more complex than was previously thought to be and complement the growing body of evidence showing that associations between nuclear receptors, cofactors, and the basic transcription machinery involve multiple molecular interaction domains (or `boxes') in each partner (45-48). We recently obtained preliminary results showing a direct interaction in vitro between the N- and C-terminal regions of PPARgamma 2, an interaction strengthened by the presence of p300.2 It is tempting to speculate that, by contacting both the AB and the DEF regions of nuclear receptors, p300 could act as an adaptor and could favor the interaction between the AF-1 and the AF-2 regions, thereby allowing for full receptor activation. p300 could favor this interaction by simply bringing both domains next to another. It is also possible that p300 induces concomitantly a conformational change in either one or both domains thereby unmasking new interfaces for interaction.

In conclusion, this study has demonstrated that p300 is a bona fide PPARgamma co-activator and constitutes a node at which the PPARgamma -mediated transduction pathway is susceptible to be integrated in a more complex cellular signaling system. This work points also to a new ligand-independent mode of interaction between the two molecules. If these interactions are to modify PPARgamma conformation, it will be of interest to test whether it also interferes with ligand- or responsive element-specificity.

    ACKNOWLEDGEMENTS

We thank Dr. R. Eckner for the gift of the p300-CHA expression vector, Drs. L. Hamann and R. Heyman for the gift of BRL 49,653, Drs. M. Leibovitz and J. Berger for the gift of the PPARgamma 2DEF antibody, and Dr. R. Janknecht for the gift of the CBP-GST constructs. Helpful discussions with Drs. R. Heyman, Michael Briggs, and I. Schulman are kindly acknowledged.

    FOOTNOTES

* This work was supported by grants of the Institut Pasteur de Lille/Région Nord-Pas de Calais, the INSERM, Ligand Pharmaceuticals, Laboratoires Fournier, and the Association pour la Recherche contre le Cancer (ARC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a grant from Laboratoires Fournier.

parallel Research Director of CNRS. To whom correspondence should be addressed: L.B.R.E., Unité 325 INSERM, Département d'Athérosclérose, Institut Pasteur de Lille, 1, rue du Prof. Calmette, 59019 Lille Cédex, France. Fax: +33-320 87 73 60; E-mail: Johan.Auwerx{at}pasteur-lille.fr.

2 J. Auwerx, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptors; CBP, CREB-binding protein; AF, activating function; LBD, ligand binding domain; RXR, retinoid X receptor; SRC, steroid receptor coactivator; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; HA, hemagglutinin; GST, glutathione S-transferase; aa, amino acid(s); PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
REFERENCES
  1. Schoonjans, K., Martin, G., Staels, B., and Auwerx, J. (1997) Curr. Opin. Lipidol. 8, 159-166[Medline] [Order article via Infotrieve]
  2. Fajas, 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., and Auwerx, J. (1997) J. Biol. Chem. 272, 18779-18789[Abstract/Free Full Text]
  3. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve]
  4. Schoonjans, K., Staels, B., and Auwerx, J. (1996) J. Lipid Res. 37, 907-925[Abstract]
  5. Hu, E., Kim, J. B., Sarraf, P., and Spiegelman, B. M. (1996) Science 274, 2100-2103[Abstract/Free Full Text]
  6. Zhang, B., Berger, J., Zhou, G., Elbrecht, A., Biswas, S., White-Carrington, S., Szalkowski, D., and Moller, D. E. (1996) J. Biol. Chem. 271, 31771-31774[Abstract/Free Full Text]
  7. Adams, M., Reginato, M. J., Shao, D., Lazar, M. A., and Chatterjee, V. K. (1997) J. Biol. Chem. 272, 5128-5132[Abstract/Free Full Text]
  8. Camp, H. S., and Tafuri, S. R. (1997) J. Biol. Chem. 272, 10811-10816[Abstract/Free Full Text]
  9. Shalev, A., Siegrist-Kaiser, C. A., Yen, P. M., Wahli, W., Burger, A. G., Chin, W. C., and Meier, C. A. (1996) Endocrinology 137, 4499-4502[Abstract]
  10. Glass, C. K., Rose, D. W., and Rosenfeld, M. G. (1997) Curr. Opin. Cell Biol. 9, 222-232[CrossRef][Medline] [Order article via Infotrieve]
  11. Chakravarti, D., LaMorte, V. J., Nelson, M. C., Nakajima, T., Schulman, I. G., Juguilon, H., Montminy, M., and Evans, R. M. (1996) Nature 383, 99-103[CrossRef][Medline] [Order article via Infotrieve]
  12. Kamei, 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.. (1996) Cell 85, 403-414[Medline] [Order article via Infotrieve]
  13. Hanstein, B., Eckner, R., DiRenzo, J., Halachmi, S., Liu, H., Searcy, B., Kurokawa, R., and Brown, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11540-11545[Abstract/Free Full Text]
  14. Abraham, S. E., Lobo, S., Yaciuk, P., Heidi, H.-G., and Moran, E. (1993) Oncogene 8, 1639-1647[Medline] [Order article via Infotrieve]
  15. Smith, C. L., Oñate, S. A., Tsai, M.-J., and O'Malley, B. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8884-8888[Abstract/Free Full Text]
  16. Yao, T.-P., Ku, G., Zhou, N., Scully, R., and Livingston, D. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10626-10631[Abstract/Free Full Text]
  17. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[Medline] [Order article via Infotrieve]
  18. Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve]
  19. Eckner, R. (1996) Biol. Chem. 377, 685-688
  20. Janknecht, R., and Hunter, T. (1996) Curr. Biol. 6, 951-954[Medline] [Order article via Infotrieve]
  21. Dowell, P., Ishmael, J. E., Avram, D., Peterson, V. J., Nevrivy, D. J., and Leid, M. (1997) J. Biol. Chem. 272, 33435-33443[Abstract/Free Full Text]
  22. DiRenzo, J., Sàderstràm, M., Kurokawa, R., Ogliastro, M.-H., Ricote, M., Ingrey, S., Hàrlein, A., Rosenfeld, M. G., and Glass, C. K. (1997) Mol. Cell. Biol. 17, 2166-2176[Abstract]
  23. Krey, G., Braissant, O., L'Horset, F., Kalkhoven, E., Perroud, M., Parker, M. G., and Wahli, W. (1997) Mol. Endocrinol. 11, 779-791[Abstract/Free Full Text]
  24. Zhu, Y., Qi, C., Calandra, C., Sambasiva, R., and Janardan, K. R. (1996) Gene Expression 6, 185-195[Medline] [Order article via Infotrieve]
  25. Li, H., Gomes, P. J., and Don Chen, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8479-8484[Abstract/Free Full Text]
  26. Zhu, Y., Qi, C., Rao, M. S., and Reddy, J. K. (1997) J. Biol. Chem. 272, 25500-25506[Abstract/Free Full Text]
  27. Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M. (1998) Cell 92, 829-839[Medline] [Order article via Infotrieve]
  28. Treuter, E., Albrektsen, T., Johansson, L., Leers, J., and Gustafsson, J. A. (1998) Mol. Endocrinol. 12, 864-881[Abstract/Free Full Text]
  29. Mizukami, J., and Taniguchi, T. (1997) Biochem. Biophys. Res. Commun. 240, 61-64[CrossRef][Medline] [Order article via Infotrieve]
  30. Vu-Dac, N., Schoonjans, K., Kosykh, V., Dallongeville, J., Fruchart, J.-C., Staels, B., and Auwerx, J. (1995) J. Clin. Invest. 96, 741-750[Medline] [Order article via Infotrieve]
  31. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 869-884[Abstract]
  32. Dignam, J. P., Lebowitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489[Abstract]
  33. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T., and Kliewer, S. A. (1995) J. Biol. Chem. 270, 12953-12956[Abstract/Free Full Text]
  34. Onate, S. A., Boonyaratanakornkit, V., Spencer, T. E., Tsai, S. Y., Tsai, M.-J., Edwards, D. P., and O'Malley, B. W. (1998) J. Biol. Chem. 273, 12101-12108[Abstract/Free Full Text]
  35. Lundblad, J. R., Kwok, R. P., Laurance, M. E., Harter, M. L., and Goodman, R. H. (1995) Nature 374, 85-88[CrossRef][Medline] [Order article via Infotrieve]
  36. Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-229[CrossRef][Medline] [Order article via Infotrieve]
  37. Bannister, A. J., Oehler, T., Wilhelm, D., Angel, P., and Kouzarides, T. (1995) Oncogene 11, 2509-2514[Medline] [Order article via Infotrieve]
  38. Ikonen, T., Palvimo, J. J., and Jänne, O. (1997) J. Biol. Chem. 272, 29821-29828[Abstract/Free Full Text]
  39. Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., Chen, D., Huang, S. M., Subramanian, S., McKinerney, E., et al.. (1998) Mol. Endocrinol. 12, 1605-1618[Abstract/Free Full Text]
  40. Kitabayashi, I., Eckner, R., Arany, Z., Chiu, R., Gachelin, G., Livingston, D. M., and Yokoyama, K. K. (1995) EMBO J. 14, 3496-3509[Abstract]
  41. Avantaggiati, M. L., Carbone, M., Graessmann, A., Nakatani, Y., Howard, B., and Levine, A. S. (1996) EMBO J. 15, 2236-2248[Abstract]
  42. McInerney, E. M., Tsai, M., O'Malley, B. W., and Katzenellenbogen, B. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10069-10073[Abstract/Free Full Text]
  43. Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B., and Hogness, D. S. (1997) Cell 91, 777-788[Medline] [Order article via Infotrieve]
  44. Norris, J. D., Fan, D., Stallcup, M. R., and McDonnell, D. P. (1998) J. Biol. Chem. 273, 6679-6688[Abstract/Free Full Text]
  45. Voegel, J. J., Heine, M. J., Tini, M., Vivat, V., Chambon, P., and Gronemeyer, H. (1998) EMBO J. 17, 507-519[Abstract/Free Full Text]
  46. Kalkhoven, E., Valentine, J. E., Heery, D. M., and Parker, M. G. (1998) EMBO J. 17, 232-243[Abstract/Free Full Text]
  47. Leong, G. M., Wang, K. S., Marton, M. J., Blanco, J. C. G., Wang, I.-M., Rolfes, R. J., Ozato, K., and Segars, J. H. (1998) J. Biol. Chem. 273, 2296-2305[Abstract/Free Full Text]
  48. Zamir, I., Zhang, J., and Lazar, M. A. (1997) Genes Dev. 11, 835-846[Abstract]


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