From the 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
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ABSTRACT |
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The nuclear peroxisome proliferator-activated
receptor The three peroxisome proliferator-activated receptors
(PPARs)1 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 PPAR The aim of this work was to evaluate more precisely the role of p300
and CBP in PPAR 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 PPAR 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where the last 36 amino acids from the C terminus of p300 have been replaced by a hemagglutinin (HA) epitope (31); pSG5-hPPAR Production of Proteins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 PPAR Immunoprecipitation and Pull-down Experiments
Immunoprecipitations--
Polyclonal antibodies (5 µg)
directed against the AB domain of PPAR Pull-down Experiments--
The purified
PPAR Yeast Two-hybrid System
Different domains of hPPAR p300 Stimulates PPAR
To clarify the role of p300 toward each of the two PPAR p300 and PPAR p300 Interacts in Vitro with the AB and the DEF Domains of
PPAR
p300Nt-GST interacted with both the AB and the DEF domains of
PPAR
As each sub-region of PPAR CBP Interacts with the ABC Region of PPAR p300 and PPAR
Yeast co-transfected with bait vectors containing different regions of
the ABC domain of hPPAR
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 PPAR 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 PPAR In a series of transfection experiments, PPAR p300 or CBP have been shown to contact the DEF domains of the ER, RAR,
RXR, TR, and PPAR The existence of a ligand-independent interaction between p300 and the
AB domain of PPAR 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 PPAR In conclusion, this study has demonstrated that p300 is a bona
fide PPAR (PPAR
) 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 PPAR
is complex and involves multiple domains
in each protein. p300/CBP not only bind in a
ligand-dependent manner to the DEF region of PPAR
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 PPAR
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
,
(or
),
and
, each encoded by a separate gene and displaying different
tissue distributions and distinct ligand selectivities, belong to the
nuclear hormone receptor superfamily (1). PPAR
is an important
transcription factor involved in adipocyte differentiation and glucose
metabolism. The PPAR
gene gives rise to two different PPAR
proteins, i.e. PPAR
1 and PPAR
2. PPAR
2 differs from
PPAR
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
PPAR
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).
ligand-dependent transcriptional activity and could interact with the PPAR
DEF domain in a
ligand-dependent way. Aside from p300, the only cofactors
described so far for PPAR
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 PPAR
and CBP, they did not
provide any evidence for a co-activation function or a physiological
role for CBP in this interaction.
-mediated gene expression. A detailed analysis of the
interaction domains between PPAR
and p300/CBP revealed for the first
time that PPAR
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 PPAR
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 PPAR
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
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
PPAR
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).
2, a
construct containing the entire cDNA of the human PPAR
2
(hPPAR
2) (2); pcDNA3-BDGal4-hPPAR
ABC, a
construct where the A, B, and C regions of PPAR
2 (aa from 2 to 181)
have been cloned downstream of the Gal4 DNA binding domain;
pcDNA3-BDGal4-hPPAR
DEF, a construct where the D, E,
and F regions of PPAR
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-
Gal, a vector for the control of
transfection efficiency.
2AB1-146 (aa 1 to 146 of PPAR
), the
PPAR
2ABC1-181 (aa 1 to 181 of PPAR
), and the
PPAR
2DEF204-507 (aa 204 to 507 of PPAR
) proteins
were produced following the same procedure, and the GST domain was
removed by thrombin digestion.
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%
-mercaptoethanol), separated by 8% acrylamide SDS-PAGE, and transferred to nitrocellulose membranes. Blots were then
developed with anti-HA.11 antibodies.
2AB1-146, PPAR
2ABC1-181,
and PPAR
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 PPAR
2AB or PPAR
2DEF.
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
PPAR
2DEF181-507 were cloned by polymerase chain
reaction amplification on the pSG5-PPAR
2 construct of the
corresponding domains. The three PPAR
2DEF deletion
constructs PPAR
DEF181-501,
PPAR
DEF181-281, and PPAR
DEF181-224 were
generated by removing the 3'-ends of the
PPAR
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
-galactosidase assay was
performed as described before (30) but with yeast lysates from
saturated yeast cultures lyzed with acid-washed beads (Sigma).
RESULTS
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 PPAR
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 PPAR
2 (pSG5-hPPAR
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 PPAR
ligand (33) (Fig. 1). PPAR
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 PPAR 2
transcriptional activity in HeLa cells. HeLa cells were
co-transfected with an expression vector for hPPAR
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
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 hPPAR
2 fused to the
binding domain of the Gal4 yeast transcription factor (BDGal4-hPPAR
ABC and BDGal4-hPPAR
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-hPPAR
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-hPPAR
ABC and 1.5 times for
BDGal4-hPPAR
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-hPPAR
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
PPAR 2 AF-1 and AF-2 transcriptional activity
in RK13 cells. A,
pcDNA3-BDGal4-hPPAR
ABC is a construct where
the A, B, and C regions of PPAR
2 (aa from 2 to 181) have been cloned
downstream of the Gal4 DNA binding domain and
pcDNA3-BDGal4-hPPAR
DEF is a construct where the D, E,
and F regions of PPAR
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-hPPAR
AB (B), or
BDGal4-hPPAR
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-hPPAR
AB or
BDGal4-hPPAR
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.
Interact in a Cellular Context--
The
enhancement of PPAR
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 pSG5hPPAR
2 and CMV p300-CHA expression
constructs and of the corresponding empty expression vectors. PPAR
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 PPAR
and p300-CHA. For the immunoprecipitates from cells which
had been transfected either by PPAR
or p300-CHA alone, no clear band
was visible in the immunoblot. This specific co-immunoprecipitation of
p300-CHA with PPAR
suggests that PPAR
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 PPAR
interact in vivo. HeLa cells were
transfected either with an empty expression construct or with
expression vectors coding for hPPAR
2 or p300-CHA. hPPAR
2 was
immunoprecipitated from the nuclear extracts with an antibody directed
against the AB domain of PPAR
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.
2--
The association of p300 with PPAR
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 PPAR
, 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
PPAR
2AB1-146 (aa from 1 to 146 of PPAR
), the
PPAR
2ABC1-181 (aa from 1 to 181 of PPAR
), and the
PPAR
2DEF204-507 (aa from 204 to 507 of PPAR
)
proteins were also produced and purified following the same procedure.
The GST part of these proteins was then removed by thrombin cleavage.
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
PPAR
2 (Fig. 4B). The ligand-dependent interaction between the PPAR
2DEF and p300Nt-GST
was enhanced by increasing amounts of BRL 49,653. Similar data were
obtained when another synthetic PPAR
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 PPAR in
vitro. A, the purified
PPAR
2AB1-146 and PPAR
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 PPAR
2. B, the purified PPAR
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 PPAR
2. C,
full-length PPAR
2 synthetized with rabbit reticulocyte
lysates or purified PPAR
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 PPAR
2.
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 PPAR
2 produced with rabbit reticulocyte lysates or purified PPAR
2DEF were incubated
with p300Nt-GST in presence or absence of BRL 49,653. Nonprogrammed reticulocyte lysate was added to the samples with purified
PPAR
2DEF to rule out any artifact because of the
potential presence of a PPAR
ligand in this crude lysate (used for
the full-length PPAR
). In presence of ligand, both full-length
PPAR
2 and PPAR
2DEF interacted with p300Nt (Fig.
4C), but in absence of ligand, only the interaction between
full-length PPAR
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 PPAR
2 observed in the co-immunoprecipitation experiments.
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 PPAR
2ABC1-181
or PPAR
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
PPAR
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 PPAR
2 (Fig. 5A, lanes 9 and 10).
It appears therefore that p300/CBP and PPAR
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 PPAR
ligand-binding domain (24) did not interact with the PPAR
N-terminal domain (Fig.
5B, lane 6), suggesting that the interaction
observed with p300/CBP is specific.
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Fig. 5.
PPAR 2ABC
interacts with different domains of CBP. A,
the purified PPAR
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 PPAR
2. B,
the purified PPAR
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 PPAR
2.
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 PPAR
. 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 hPPAR
were cloned downstream of the DNA
binding domain of the Gal4 protein (Figs.
6A and
7A. In yeast, the
BDGal4-PPAR
2DEF181-507 and the ADGal4-p300Nt fusion proteins interact without addition of any PPAR
ligand. It is unclear
whether this interaction is because of the presence of potential
PPAR
ligands in the yeast cells or whether a constitutive interaction between the DEF domain of PPAR
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 PPAR
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 PPAR in the yeast
two-hybrid system. A, yeasts were co-transformed with
bait vectors containing different parts of the DEF domain of hPPAR
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-PPAR
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
-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 PPAR in the yeast two-hybrid
system. A, yeasts were co-transformed with bait vectors
containing different parts of the ABC domain of hPPAR
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-PPAR
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
-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.
2 and the pADGal4-p300Nt vector can also grow
on histidine-deficient plates (Fig. 7A), confirming that
p300Nt interacts with the ABC domain of PPAR
2. The different constructs used suggest that the interaction domain in PPAR
2 is
located between aa 31 and 99 and that the B exon of PPAR
2 is not
required for this interaction.
ligand on
the strength of the interaction between p300Nt and
PPAR
2DEF181-507 or PPAR
2ABC1-182 (Figs. 6B and 7B). Similar to the pull-down
experiments, two distinct mechanisms of interaction for the two domains
of PPAR
2 and p300 were observed. PPAR
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
hPPAR
DEF and p300Nt can interact in absence of any
ligand, a significant increase of the
-galactosidase activity is
observed in presence of BRL 49,653, suggesting an enhancement of the interaction.
DISCUSSION
2DEF (29) and that p300
could enhance PPAR
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 PPAR
.
2-transcriptional
activity was enhanced in the presence of p300. The observation that
full-length PPAR
is more activated than PPAR
2ABC or
PPAR
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 PPAR
and raises the question of whether p300 is
crucial in PPAR
2 physiology, most notably in fat tissue and colon
(2) where PPAR
is expressed to high levels. We can speculate that some mutations or altered levels of expression of p300 or PPAR
could
result in the disruption or the enhancement of their interaction and
have important consequences in these processes.
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 PPAR
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 PPAR
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
PPAR
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 PPAR
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
PPAR
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 PPAR
.
2 might also indicate that beside the PPAR
ligands the interaction between PPAR
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).
PPAR
activity itself is also subject to regulation by phosphorylation (5-9). During adipocyte differentiation for example, p300 or PPAR
could each be differentially phosphorylated and/or could undergo other post-translational modifications that might drastically affect their ability to interact and control transcription.
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 PPAR
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.
co-activator and constitutes a node at which
the PPAR
-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 PPAR
conformation, it will be of interest to test whether it also interferes
with ligand- or responsive element-specificity.
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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
PPAR2DEF 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.
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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.
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.
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ABBREVIATIONS |
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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.
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REFERENCES |
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