(Received for publication, April 16, 1997, and in revised form, May 23, 1997)
From the Institut de Biologie Animale, Université de Lausanne, Bâtiment de Biologie, CH-1015 Lausanne, Switzerland
The malic enzyme (ME) gene is a target for both
thyroid hormone receptors and peroxisome proliferator-activated
receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to
bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome
proliferator signaling. Taking advantage of the close sequence
resemblance of MEp and MEd, we have identified crucial determinants of
a PPRE. Using reciprocal mutation analyses of these two elements, we
show the preference for adenine as the spacing nucleotide between the
two half-sites of the PPRE and demonstrate the importance of the two
first bases flanking the core DR1 in 5. This latter feature of the
PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound
to its cognate element. We demonstrate that, in contrast to the
polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements
respectively, PPAR binds to the 5
extended half-site of the response
element, while RXR occupies the 3
half-site. Consistent with this
polarity is our finding that formation and binding of the PPAR/RXR
heterodimer requires an intact hinge T region in RXR while its
integrity is not required for binding of the RXR/TR heterodimer to a
DR4.
The peroxisome proliferator-activated receptors
(PPAR)1 form a group of
lipid-activated transcription factors that belong to the nuclear
receptor superfamily. Members of this superfamily are characterized by
a structural organization in functional modules, comprising a
N-terminal domain, a DNA binding domain, a hinge region, and a ligand
binding domain that also contains a potent ligand-dependent
transactivation domain and offers several interfaces for dimerization
and protein-protein interaction. Although some of these receptors may
bind to DNA as monomers, the majority binds as dimers to specific DNA
sequences formed of two consensus half-sites. PPAR, thyroid hormone
receptor (TR), vitamin D receptor (VDR), and
all-trans-retinoic acid receptor (RAR) form a subgroup
within the superfamily which heterodimerize with the
9-cis-retinoic acid receptor (RXR) and bind to response
elements composed of two AGGTCA half-sites predominantly organized in a
direct repeat. All the natural PPAR response elements (PPREs) described
so far indeed consist of a direct repeat of two more or less conserved
AGGTCA hexamers separated by a single base pair and are thus referred to as DR1 elements (1, 2). This organization as direct repeat imposes a
head to tail polarity to the bound heterodimer complex. Recent work has
shown that in the case of TR/RXR and RAR/RXR bound to a DR4 and DR5,
respectively, RXR occupies the 5 half-site (3-5). However, the
polarity of RAR/RXR bound to a DR1 is opposite and results in a
silencing of the ligand-dependent transactivation properties of RAR (6-8). So far, the polarity of the PPAR/RXR heterodimer has not been determined.
Three different PPAR subtypes have been identified (,
/
, and
). Each of them displays a distinct expression pattern in adult
amphibians and rodents. PPAR
is predominantly expressed in
hepatocytes, cardiomyocytes, proximal tubule cells of the kidney, and
enterocytes. PPAR
(also called PPAR
or FAAR in rodents and NUCI
in man) is more widely and often more abundantly expressed than PPAR
and
, whereas PPAR
is mainly restricted to the adipose tissue
with some expression in spleen, retina, and hematopoietic cells
(reviewed in Refs. 9 and 10). PPARs were named by virtue of their
ability to be activated by peroxisome proliferators. However, new
specific activators and ligands for the different PPAR subtypes are now
emerging. Recent studies have shown that various fatty acids,
eicosanoids, and hypolipidemic compounds, such as fibrates, directly
bind to PPAR
,
, and
. In addition, PPAR
binds antidiabetic
thiazolidinediones, which lower the plasma levels of glucose,
triglycerides, and insulin (reviewed in Ref. 11). Interestingly, not
only can PPAR
direct adipocyte differentiation but all the PPAR
target genes identified so far are involved in major steps of fatty
acid metabolism, including fatty acid transport, intracellular binding,
- and
-oxidation, as well as fatty acid synthesis and storage as
triglycerides (reviewed in Ref. 10). Thus, the nature of the PPAR
ligands together with the observation that all PPAR
and
target
genes discovered so far are directly involved in lipid metabolism
reinforce the novel concept of fatty acids and their derivatives acting
as hormones and controlling their own fate through these specific
nuclear receptors (reviewed in Refs. 12-14).
The malic enzyme (ME) gene is one of the PPAR target genes whose
product is involved in lipogenesis. ME catalyzes the oxidative decarboxylation of malate to pyruvate, which results in the production of NADPH for fatty acid synthesis. Thyroid hormone (T3)
leads to a pronounced stimulation of ME gene expression in the liver (15) and is involved in adipose differentiation for which ME is a late
marker (16, 17). Interestingly, thyromimetic effects of hypolipidemic
fibrates on ME expression in rat liver have been observed (18, 19).
Since these compounds are PPAR ligands, these thyromimetic effects may
be mediated by PPAR/RXR heterodimers binding to the ME promoter.
We2 and others (20, 21) have
identified two DR1-like elements in the ME promoter, hereafter referred
to as MEp (proximal, located at bp 340/
328 with respect to the
transcription initiation site) and MEd (distal, located at bp
463/
451). However, conflicting results have been reported on the
respective role of these two elements in PPAR-mediated regulation of ME
gene expression; Castelein et al. (20) have identified MEp
and Hertz et al. (21) MEd as the functional PPRE. Our
interest in the hormonal cross-talk in adipocytes between the
T3 and fatty acids signaling pathways prompted us to pursue
the characterization of the ME promoter with respect to PPAR-mediated
gene regulation. In particular, we studied its regulation by PPAR
,
the subtype abundantly expressed in adipose tissue.
In this work, the demonstration that only MEp and not MEd is a functional PPRE gave us a tool to identify the determinants of a functional PPRE, which go beyond the characteristics of the core DR1 element. Furthermore, we unveiled the polarity of the heterodimer PPAR/RXR bound to a DR1, which explains the nature of the PPRE-specific requirements.
The Xenopus PPAR (1), human RXR
(22) and rat TR
1 were subcloned into a modified pSG5, pSG5PL (gift
of Dr. Hélène Richard-Foy). To optimize in vitro
translation of the PPAR
and RXR
receptors, a Kozak (23) consensus
sequence was introduced by recombinant polymerase chain reaction at the
translational start site of the receptor.
The P box mutants of PPAR and RXR
were obtained in two sequential
mutagenic steps using the Kunkel et al. (24) method. The
primers 5
-GCGTCCATGCATGTGGATCTTGCAAGGGGTTCTT-3
and
5
-GTGGATCTTGCAAGGTGTTCTTTAGAAGAAC-3
were used to create
PPARpgr; the primers 5
-GAGTGTACAGCTGCGGGTCGTGCAAGGGCTTCTT-3
and
5
-GCGGGTCGTGCAAGGTCTTCTTCAAGCGGAC-3
were used to create RXRpgr.
The primer 5
-GGCATGAAGCGGGAATTCGAGGGGGAGGAGCGGCAGCG-3
was used to
create the RXR(T) mutant, using the same approach.
pME775 was created by cloning the 809-bp
XbaI-BamHI fragment (bp 775/+34) of the pME882
plasmid (25) into pBLCAT3 (26). The mutant reporter plasmids pME775pko
and pME775dko, in which, respectively, the proximal or the distal
putative PPRE has been mutated, were generated by recombinant
polymerase chain reaction using the ExpandTM High Fidelity
kit (Boehringer Mannheim). The mutated sequences are as follow: within
pME775dko from bp
469 to
445: TGCACTAGATCTGTCCGGTCTAACA; within
pME775pko from bp
346 to
322: CATTCTAAGCTTGAGTTGATCCCCT.
The reporter constructs containing the wild-type or mutated PPREs upstream of the heterologous thymidine kinase promoter were created by cloning a single copy of the response element into the BamHI/HindIII sites of the pBL-CAT8+ plasmid. All the constructs were verified by DNA sequencing.
Cell Culture and TransfectionsNIH3T3 cells were maintained
in culture and transfected as described previously (27). Briefly, each
cuvette for electroporation contained 4 × 106 cells
at a density of 12.5 × 106 cells/ml and a total of 70 µg of plasmid DNA (20 µg of chloramphenicol acetyl transferase
(CAT)-reporter plasmid, 12 µg of each expression vector as indicated
in the figure legends, 1 µg of pCMVgal as internal control for
transfection efficiency, and pUC19 or salmon sperm DNA to complete to
70 µg). After electroporation, cells were resuspended and equally
distributed in four 60-mm dishes containing the transfection medium
supplemented with the appropriate activator. After 48 h, cell
extracts were prepared and
-galactosidase and CAT activities were
determined as described previously (27).
Proteins for
electrophoretic mobility shift assays (EMSA) were obtained by in
vitro transcription and translation using the TNT®
coupled reticulocyte lysate system (Promega). Parallel translations using [35S]methionine (Amersham Corp.) followed by
SDS-PAGE analysis and exposure in the phosphor-analyst (Bio-Rad)
allowed standardization of the different protein preparations.
Alternatively, nuclear extracts from Sf9 cells infected with a
recombinant baculovirus overexpressing the mouse RXR were used.
The probes and competitors corresponded to the double-stranded
oligonucleotides indicated in the figures flanked on their 5 side and
3
side by the BamHI and HindIII overhang
sequence, respectively. The ACO(A) and METRE
oligonucleotides are (5
-GATCCCGAACGTGACCTTTGTCCTGGTCCCGATC-3
) and
(5
-GATCAGGACGTTGGGGTTAGGGGAGGACAGATC-3
), respectively.
In vitro translated proteins were preincubated for 15 min at
room temperature, in a buffer containing 25 mM HEPES, pH
7.5, 5 mM MgCl2, 1 mM EDTA, 10%
glycerol, 40 mM KCl, 1 mM dithiothreitol, and 8 µg poly(dI-dC). After a further 20-min incubation period at room
temperature in the presence of 20,000 cpm of labeled probe, the
complexes were separated on a 6% native polyacrylamide gel with
0.25 × TBE running buffer at 500 V, 25 mA, 4 °C. For DNA binding competition experiments, a 10-100-fold molar excess (as indicated) of the unlabeled double-stranded competitor oligonucleotide was added to the preincubation reaction. Gels were dried and exposed at
80 °C to a Kodak X-Omat AR film with an intensifying screen. Scanning and treatment of the images were performed using the Cirrus
1.2 software. When appropriate, gels were analyzed by phosphor-analyst or densitometry.
The
possibility of cross-talk between PPAR and TR signaling pathways at the
level of ME gene expression might be important in cells, such as
adipocytes, in which all three proteins, PPAR, TR, and ME, are
expressed. We thus tested if the ME promoter is responsive to PPAR,
the PPAR subtype which is crucial for adipogenesis and is present at
high levels in mature adipocytes (28, 29). As seen in the schematic
representation of the ME promoter in Fig.
1A, two DR1 elements, MEp
(proximal, at bp
340/
328) and MEd (distal, at bp
463/
451), are
located upstream of the thyroid hormone response element
METRE (27). Conflicting reports from Castelein et
al. (20) and Hertz et al. (21) indicated mediation of
the ME peroxisome proliferator response through either of these elements.
To clarify this point, we first tested whether the ME promoter, from
775 to +34 base pairs relative to the initiation site was responsive
to PPAR
and, as a positive control, to TR. Transfection analyses
using a CAT reporter gene in NIH3T3 cells confirm that TR
1 can
control the ME promoter, as a T3-dependent
7-fold stimulation of the reporter gene expression was observed.
Cotransfection of the reporter gene with a vector expressing PPAR
in
the absence of PPAR activator moderately but reproducibly induced
expression of the reporter gene (2.5-fold). Whether this induction was
due to a constitutive activity of PPAR
or to endogenous PPAR
activators was not further analyzed. Addition of the PPAR activator Wy
14,643 to the culture medium resulted in a 5.5-fold stimulation over the basal expression level of the reporter construct. A similar induction was obtained using the thiazolidinedione BRL49653, a PPAR
specific ligand (data not shown). These effects are receptor-specific, since T3 and Wy 14,643 stimulated the ME promoter activity
only in the presence of TR and PPAR, respectively (Fig.
1B).
Second, we determined the relative contribution of the two putative
PPREs to PPAR responsiveness. For that purpose, we first mutated
either the MEd or the MEp sequence within the homologous promoter
creating pME775dko and pME775pko, respectively. The mutation of the MEd
sequence did not alter the response of the reporter gene to Wy 14,643, while mutation of MEp suppressed responsiveness, indicating that MEp is
the responsive element (Fig. 1B; see also Castelein et
al. (20)). To test if this result was independent of the position
or relative orientation of each of these elements within the ME
promoter (see Fig. 1A), we inserted, in the same orientation, the two elements encompassing the DR1 motif plus their 6 bp flanking either side, upstream of the herpes simplex virus thymidine
kinase heterologous promoter in a CAT reporter gene. pMEpfl, containing
the proximal element, and pMEdfl, containing the distal element, were
then transfected into NIH3T3 cells. As shown in Fig. 1B,
PPAR
increased the basal level of pMEpfl expression 7-fold in the
absence of an exogenous PPAR activator, and 28-fold in the presence of
Wy 14,643. In contrast, pMEdfl was not responsive to PPAR
, neither
in the absence nor in the presence of Wy 14,643 (Fig. 1B).
No response of either reporter construct was observed in presence of TR
and T3 (data not shown).
To test if the above reported difference in PPAR responsiveness of MEp
and MEd reflects their ability to bind PPAR/RXR heterodimers, we
performed EMSAs. We used MEpfl as a probe, in vitro
translated PPAR, and cellular extracts from Sf9 cells infected by a
recombinant baculovirus expressing RXR
. Fig. 1C shows
that PPAR
/RXR binding complex could form on MEpfl, whereas no PPAR
binding was observed in absence of RXR (data not shown). Unlabeled
double-stranded oligonucleotides encompassing either the PPRE of the
acyl-CoA oxidase gene (ACO(A)) or MEpfl itself efficiently competed
PPAR/RXR complex formation on the probe MEpfl, whereas MEdfl, which was unresponsive in the functional test, was a very inefficient competitor. As expected, METRE did not compete for the PPAR/RXR complex
binding.
These data show that of the two DR1 elements present in the ME
promoter, only the MEp is able to bind PPAR/RXR heterodimers in a
sequence-specific manner and can mediate peroxisome proliferator signaling.
The inability of MEd to
act as PPRE was puzzling since its sequence is closer to the consensus
DR1 than that of the functional element MEp. Analysis of the
compilation of the natural PPREs characterized so far (Fig.
2) and recent reports suggested that two
regions of the PPRE may be given particular attention: the spacing
nucleotide between the two half-sites which is predominantly an A and
the sequence immediately 5 upstream of the DR1 core element (Fig. 2)
(13, 31, 32).3 In contrast to
MEp, MEd strikingly diverges from the consensus in these two regions
suggesting that these differences might be responsible for the lack of
responsiveness of MEd to PPAR
and Wy 14,643. The role of the spacing
nucleotide was first analyzed by changing the spacing nucleotide A of
MEpfl to either G, C, or T, resulting in the MEp(G), MEp(C), and MEp(T)
elements, respectively (see Fig.
3A). The presence of an A as
in MEpfl reproducibly resulted in the strongest binding, while a C at
this position always resulted in the weakest interaction (Fig.
3A). Notably, the nonfunctional sequence MEd has a C at the
corresponding spacing position. Thus, we introduced the converse
mutation in MEd, changing its spacing nucleotide from a C to an A, and
observed a partial restoration of PPAR
/RXR binding (MEd(DR-A); Fig.
3B). The inability of MEdfl to function as a PPRE might also
be determined by the 5
-flanking nucleotides: TTCT in MEp
versus TTAG in MEd. Indeed, transversion of AG
to CT, creating MEd(CT), enhances the formation of PPAR
·RXR complexes. Finally, combination of the mutations in the two regions, the spacing and the 5
-flanking nucleotides, in MEd(CTA) had a synergistic effect on PPAR
/RXR binding (Fig. 3B), which
was comparable to that observed on MEpfl.
To correlate PPAR binding affinity and transcriptional activity, MEp(C)
and the MEd mutants were inserted upstream of the herpes simplex
thymidine kinase promoter and used as reporter constructs in
cotransfection experiments (Fig. 3C). Compared with MEpfl,
MEp(C) lost most of its capacity to mediate PPAR induction. While
the mutants MEd(DR-A) and MEd(CT) did not confer a PPAR responsiveness
to the reporter gene, the double mutant MEd(CTA) mediated a
PPAR-dependent increase of the basal level of expression, which was further induced by Wy 14,643. This transactivation pattern of
MEd(CTA) correlates well with its capacity to bind PPAR/RXR and closely
resembled that observed with MEpfl, confirming the importance of both
the central adenylate and the 5
flank in defining a PPRE.
Previous work on PPREs stressed their DR1-type
structure, particularly since a synthetic perfect DR1 core sequence
exhibits a high PPAR/RXR binding affinity. The newly discovered role of the 5 flank in native elements raises the question as to whether it
mainly compensates for a weak interaction of the heterodimer with
imperfect DR1 core sequences as found in native elements (see Fig. 2).
In that respect, the poorly conserved 3
half-site (AGTTGA) of the MEp
PPRE is an excellent example. To answer the above question, we assessed
the importance of the flanking sequences for PPAR/RXR binding both in
the context of the native MEp and that of a perfect synthetic DR1
element. EMSAs, as the one shown in Fig.
4, demonstrate that PPAR/RXR binding to
MEp is 80% less efficient (mean of two independent experiments) when
its specific 5
flank is mutated. In contrast, binding to the perfect
DR1 is affected to a lesser extent by the sequence of the 5
flank,
exhibiting a 26% loss of binding efficiency in presence of the mutated
versus the wild-type 5
MEp flank (mean of three independent
experiments). This result reinforces the hypothesis that the
5
-flanking sequence does play a role in the specific DNA recognition
by PPAR, as PPAR/RXR heterodimer; together with an imperfect core DR1,
it contributes to selective binding of PPAR (see "Discussion").
Polarity of the PPAR
Based on the
results described above, we hypothesized that PPAR may bind to the
extended 5 half-site of a PPRE, while RXR binds to the 3
hexamer. To
determine the PPAR/RXR binding polarity, we converted the P box of PPAR
and of RXR, into that of the glucocorticoid receptor (GR), creating
PPARpgr and RXRpgr, respectively (Fig. 5A). Such hybrid receptors
will recognize the consensus hexamer -AGAACA- of the GR response
element (GRE) (3, 4). Consequently, we tested in EMSA two hybrid MEp
elements in which either the 5
hexamer or the 3
hexamer was replaced
by a GRE half-site, giving MEpfl:GRE5
and MEpfl:GRE3
, respectively
(Fig. 5A). The formation of PPAR/RXR complex was barely
detectable either on MEpfl:GRE5
or on MEpfl:GRE3
(Fig. 5A,
compare lane 1 to lanes 5 and 9). In
contrast, the combination of PPAR and RXRpgr led to the formation and
binding of a complex to MEpfl:GRE3
but neither to MEpfl nor to
MEpfl:GRE5
(Fig. 5A, compare lane 6 to
lanes 2 and 10). This result suggested that RXR
indeed binds to the 3
half-site of the PPRE. As expected, the
heterodimer PPARpgr/RXR did not bind to MEpfl:GRE3
(Fig.
5A, lane 7); however, it was not able to bind to
MEpfl:GRE5
either (Fig. 5A, lane 11). As shown
in the top panel of Fig. 5, that latter probe associates a
GRE half-site -AGAACA- in 5
and the poorly conserved MEp 3
half-site -AGTTGA-. Thus, it is likely that the divergence of the
overall sequence of MEpfl:GRE5
from a DR1-like element is too
important to accommodate nuclear receptor binding.
To circumvent this experimental limitation, we repeated the same
experiment using the synthetic element DR1fl and chimeric response
elements in which we introduced a GRE half-site in place of the 5
hexamer or of the 3
hexamer giving DR1fl:GRE5
and DR1fl:GRE3
,
respectively (Fig. 5B). Very clearly, the PPAR/RXRpgr heterodimer bound to DR1fl:GRE3
but not to DR1fl:GRE5
(Fig. 5B, lanes 11 and 7, respectively),
whereas PPARpgr/RXR heterodimer bound to DR1fl:GRE5
but not to
DR1fl:GRE3
(Fig. 5B, lanes 8 and 12,
respectively). In other words, the complex that contained the mutant
PPARpgr could form only on the response element that bears the GRE
sequence in the 5
position while the complex containing the mutant
RXRpgr could form only on the element with the GRE sequence in the 3
position. Together these results provide evidence for a defined binding
polarity of the PPAR/RXR heterodimer onto its response element, with
PPAR anchored to the 5
extended half-site and RXR to the 3
half-site.
Interestingly, the role of the 5
-flanking sequences clearly appeared
in the context of the mutant receptors and mutant PPREs, as exemplified
by PPAR/RXR and PPARpgr/RXR which bound to DR1fl:GRE5
with a greater
efficiency than to DR1:GRE5
(Fig. 5B, compare lane
6 to lane 18 and lane 8 to lane
20; see "Discussion").
In agreement with these binding studies, PPARpgr was unable to activate
the expression of the MEpfl reporter construct while it could
efficiently activate the reporter DR1fl:GRE5 (Fig. 5C). Cotransfection of RXRpgr with either of these reporter genes strongly inhibited the PPAR
-induced expression, suggesting a dominant negative effect of this mutant receptor. In contrast, the reporter construct that contains the GRE at the 3
half-site was not activated by PPARpgr, was poorly activated by PPAR in presence of the endogenous RXR, but was significantly stimulated if RXRpgr is cotransfected with
PPAR (Fig. 5C). Thus, these functional studies further
confirmed the binding polarity of PPAR/RXR to PPRE.
The binding
polarity of PPAR/RXR onto a PPRE should be reflected in the
dimerization surface used by each partner. Structural and biochemical
analyses of nuclear receptor heterodimers bound to direct repeat
elements demonstrate that the second zinc finger of the receptor which
binds to the 5 half-site contacts the T box and the first zinc finger
of the receptor which binds to the 3
half-site (3-5, 32-37). Hence
we mutated 3 amino acids in the T box of RXR, creating RXR(T) (Fig.
6). These mutations should affect the
function of RXR as the 3
-binding receptor and thus the formation and
binding of the heterodimer PPAR/RXR to a PPRE such as MEp. In contrast,
they should not alter the function of RXR as the 5
-binding receptor,
and consequently still allow the formation and binding of the complex
RXR/TR on a TRE such as the METRE. Indeed, as seen in Fig.
6, PPAR is not able to bind MEp when using RXR(T) as partner while a
complex corresponding to the heterodimer TR/RXR(T) can form on
METRE albeit with a weaker affinity than TR/RXR (Fig. 6,
lanes 3 and 6, respectively). These results are
clearly consistent with the polarity that we demonstrate for the
PPAR/RXR complex bound to PPRE and further suggest that the T region of
RXR is involved in the dimerization surface between RXR and PPAR.
The analysis of the regulation by peroxisome proliferators of the
ME promoter led us to a better understanding of the molecular mechanisms governing PPAR-mediated gene regulation. First, the observation that in our experimental model, only the proximal DR1
sequence located at 340/
328 (MEp) can function as a PPRE, while the
element
451/
463 (MEd) does not, prompted us to further analyze the
structural definition of a PPRE. Our results add the following three
main properties of native PPREs to the initial definition as DR1: (i)
an extended 5
half-site, (ii) an imperfect core DR1, and (iii) an
adenine as a spacing nucleotide between the two hexamers. Second, we
provide evidence that this PPRE structure reflects the polarity with
which PPAR/RXR binds to the element: PPAR recognizes the 5
extended
half-site and RXR the 3
half-site.
Binding of PPAR/RXR to DR1-like elements raises the
question of the selectivity of this interaction since these elements
are binding sites for several members of the nuclear receptor
superfamily. DR1 elements were shown to mediate
9-cis-retinoic acid responsiveness through binding of RXR
homodimers, as first demonstrated on the cellular retinol binding
protein II element (CRBP-II) (38). Moreover, an unbiased search for
RXRE as well as analyses of synthetic elements revealed the importance
of an A or a G as the base immediately 5 of perfect core
hexanucleotides in a DR1 configuration (39-42). Interestingly,
however, no or very weak binding of RXR homodimer was observed in
previous work describing naturally occurring PPREs, indicating that
discriminating parameters must account for RXR homodimer and PPAR/RXR
heterodimer selectivity. In addition, DR1 elements are also binding
sites for at least three orphan members of the nuclear receptor
superfamily: HNF-4, ARP-1, and ear-3 (or COUP-TF), the most promiscuous
response element being a synthetic DR1-G element, in which the two
consensus hexamer are flanked by a G in 5
(see Ref. 41 and references
therein). Competition for binding and functional interference can
indeed occur between PPAR/RXR and HNF4 (43, 44) as well as between
PPAR/RXR and COUP-TF (45, 46). However, the native HNF4 binding site
characterized in the
1-antitrypsine gene only poorly
fits the DR1 consensus and binds neither COUP-TF nor ARP1 (47). Again
this underscores that subtle differences in the natural DR1-type
response elements must be important for their selectivity. The
alignment in Fig. 2 of the sequence of 19 native PPREs previously
characterized, with the number of occurrences for each nucleotide at
each position (from 1 to 17) presented in the bottom panel,
reveals specific characteristics of native PPREs. DR1 motifs clearly
appear between position 5 and 17, with an obvious lack of nucleotide
preference only at a single position (nucleotide 8), while the spacing
nucleotide between the half-sites (position 11) is remarkably
conserved. Our mutation analyses show that indeed an adenine as the
spacing base results in the strongest heterodimer binding, whereas
cytidine is the least desirable of the four possibilities at that
position. A certain degree of conservation is also present in the
5
-flanking nucleotides (AACT, position 1-4), suggesting a potential
role for this region. Herein, we demonstrate that the nucleotides in positions 3 and 4 extend the 5
half-site and are an integral part of
the PPRE, in agreement with recent work done with the PPRE of the
CYP4A6 gene (31). Importantly, the role of the 5
-flanking sequence is
especially apparent when the DR1 sequence is poorly conserved with
respect to a perfect DR1, as for MEp and MEpfl (Fig. 4) but also as in
the chimeric elements DR1:GRE5
and DR1fl:GRE5
(Fig. 5B).
The same applies when RXR itself binds poorly because it has been
altered, as in RXRpgr, leading to a PPAR/RXRpgr complex that binds to
DR1fl but not to DR1. Thus, it appears that a weakened interaction of
PPAR/RXR with the core DR1 is tolerated as long as specific contacts in
the 5
flank can stabilize it. These results clearly plead for the
importance of the association of a specific 5
-flanking sequence, an
imperfect core DR1, and a central adenine as structural characteristics
allowing the discrimination of PPRE from other DR1 response elements by
the PPAR/RXR heterodimer.
Consistent
with the discriminatory role of the PPRE 5 flank, we demonstrated that
PPAR binds to the extended 5
half-site of the PPRE, while RXR binds to
the 3
hexamer. This is in contrast to the RXR/VDR, RXR/TR, and RXR/RAR
heterodimers which bind with the reverse polarity to DR3, DR4, and DR5,
respectively (3-5), but is in register with the RAR/RXR complex bound
to a DR1 (6-8). Asymmetric contacts operating between the two partners
of a heterodimer bound to a direct repeat occur between their
respective DNA binding domain (DBD) and carboxyl-terminal extension
(CTE) that comprises the T box and A box. The hinge region, which also
includes the CTE, would allow adequate rotation of the interacting
ligand binding domains with respect to the DBD (reviewed in Refs. 49
and 50). While the three-dimensional structure of the DBD and CTE
region of PPAR has not yet been solved, some of its properties can be inferred from the polarity to which PPAR binds to PPRE and from detailed biochemical studies and structural analyses of RAR/RXR and
TR/RXR heterodimers bound to direct repeat sequences (3-5, 32-37). In
the configuration of an RAR/RXR heterodimer bound to a DR1 element,
the crucial amino acids for heterodimerization of the 5
-positioned
receptor (RAR) are located in the second zinc finger outside its first
knuckle or D box while the 3
-positioned receptor (RXR) contributes to
the dimerization interface via its T box. Exclusion from the
dimerization interface of the D box of the 5
-positioned receptor could
explain why PPARs have a D box of 3 amino acids instead of 5 in other
members of the superfamily. Consistently, exchanging this D box with
that of RXR did not alter PPAR/RXR binding to a
PPRE.4
Because of the 5 location of the PPAR molecule on a PPRE, its T/A
region may be involved in the recognition of the 5
extension of the
PPRE half-site. This has been described for receptors which can bind
extended half-sites as monomers such as FTZ-F1, NGFI-B/Nurr1, ROR/RZR,
Rev-erb
, and TR
1. Their recognition of the base pairs extending
the consensus hexamer in 5
involves critical amino acids in their
respective CTE region (48, 50-55). In PPARs the corresponding region
is highly conserved between the different subtypes but differs from the
other above mentioned receptors. The closest similarity to PPAR within
this region is found in ROR/RZR and Rev-erbA
, which correlates with
the closest 5
-extended sequence similarity of their respective
response elements (see Fig. 7). However,
in contrast to these latter receptors, PPAR is unable to bind as a
monomer.
The polarity of PPAR/RXR onto the direct repeat may also explain the
spacing of 1 nucleotide between the two half-sites of the PPRE. Indeed,
VDR, TR, and RAR by occupying the 3 half-site of DR3, DR4, and DR5
respectively, dictate the spacing that provides the specificity of
their respective response element, since all of them have the same
partner RXR. Accordingly, RXR on the 3
half-site of RXRE and PPRE
elements dictates the common spacing of 1 nucleotide, likely through
physical constraints residing in the role of its T region as
dimerization surface. One consequence of this reasoning is that if RXR
binds to the 3
half-site, as it does in the context of the RAR/RXR,
PPAR/RXR and RXR/RXR dimers, selectivity cannot be conferred by
spacing. Instead, like PPAR, the heterodimerization partner might
select a specific 5
extended half-site, allowing discrimination
between DR1 elements.
Elucidating the polarity with which nuclear receptors bind to their cognate response elements is not only important for understanding the molecular mechanism of DNA-protein interaction and of dimerization properties, but it may give insight into some functional aspects of receptor activation by the ligand and of transactivation. Along this line of thought, Kurokawa et al. (7) recently demonstrated that in the context of the DR1-bound RAR/RXR complex, RAR fails to release NCoR in the presence of ligand, suggesting a molecular mechanism for the repression caused by RAR/RXR complexed to a DR1. How do co-activators and co-repressors interact with each receptor within a PPAR/RXR heterodimer and how do the respective ligands influence these interactions remain to be solved. Interestingly, cooperativity in PPAR and RXR signaling pathways is also mediated by PPRE, as demonstrated by the additive and synergistic transcriptional effect of their respective ligands in cell culture (56, 57) and in vivo (58). In that respect, our detailed characterization of the ME PPRE unveils some critical mechanisms by which PPAR can achieve functional specificity and as such is a first step toward the understanding of the molecular mechanisms underlying cooperativity and hormonal cross-talk via the ME promoter.
We thank H. Richard-Foy and A. Hihi for the gift of pSG5-PL and RXR-containing Sf9 cellular extracts, respectively. We also thank E. Beale for reading the manuscript.
Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors (64).