(Received for publication, September 14, 1995; and in revised form, October 11, 1995)
From the
Peroxisome proliferator-activated receptor (PPAR) forms a heterodimer with retinoid X receptor (RXR) that binds to the peroxisome proliferator responsive element (PPRE) to regulate the expression of target genes. PPRE of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) gene has previously been shown to consist of three imperfect TGACCT half-sites and form two distinct complexes (C1 and C2) with the nuclear extracts from H4IIEC3 cells. The present study identifies another imperfect TGACCT motif involved in the PPAR/RXR-mediated trans-activation process and demonstrates that these four imperfect TGACCT motifs constitute an unique binding site consisting of two DR1 elements overlapping a DR2 element. PPAR and RXR cooperatively bind the two DR1 elements to form C1 complex or bind DR2 element to form C2 complex with a 1:1 ratio. Saturation of the HD PPRE probes with receptor proteins cannot convert the heterodimeric C2 complex to the higher order C1 complex, suggesting that they are formed independently. Transfection analyses indicate that mutation of any one of these TGACCT motifs or truncation of the entire HD PPRE into a separate DR1 and DR2 element significantly reduced the transcriptional response of HD PPRE to peroxisome proliferators. The rat HD PPRE differentially binds with one or two PPAR/RXR heterodimers providing the peroxisome proliferator signaling pathway with two levels of response.
Nuclear hormone receptors regulate gene expression by binding to
specific response elements in the promoter region of target gene(s).
The peroxisome proliferator-activated receptor (PPAR) ()is a
member of the nuclear hormone receptor superfamily which is activated
by a variety of fibrate hypolipidemic drugs and related nongenotoxic
rodent hepatocarcinogens termed peroxisome proliferators(1) .
Three PPAR isoforms (
,
,
) displaying divergent patterns
of expression have been identified; they may play different roles
during development and differentiation, as well as xenobiotic-induced
tissue-specific expression of
genes(2, 3, 4, 5) . For example, the
mPPAR
2 isoform is adipose tissue-specific and plays a pivotal role
in the adipogenic signaling cascade(6) . The rPPAR
isoform
is widely expressed, with highest levels in liver, kidney, heart, and
adrenal, and is required for the tissue-specific pleiotropic responses
induced by peroxisome proliferators(3, 7) .
PPARs
direct the transcriptional activation of peroxisome
proliferator-responsive genes by forming a heterodimer with the
retinoid X receptor (RXR), another member of the nuclear hormone
receptor superfamily(8, 9) . The PPAR/RXR heterodimer
binds to a peroxisome proliferator response element (PPRE) which is
identified as direct repeat motif of hexamer half-sites, TGACCT, spaced
by one nucleotide (DR1)(10) . PPREs have been identified in
various genes, including peroxisomal -oxidation enzymes, and
others that are transcriptionally activated by peroxisome
proliferators(1, 10) . PPRE of the rat enoyl-CoA
hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) gene, which closely
resembles the rat peroxisomal fatty acyl-CoA oxidase (ACOX)
PPRE(11, 12) , is located in a region about 3
kilobases upstream of the transcription start site(13) . Both
the HD and ACOX PPREs can mediate the transactivation of chimeric genes
by peroxisome proliferators(14) . Unlike the ACOX PPRE, HD PPRE
consists of three imperfect direct repeats of the consensus TGACCT
motifs separated by 2 bp (DR2) and 1 bp (DR1),
respectively(13, 14) . We demonstrate here that a
TCTCCT hexamer located 1 bp upstream of the DR2 element is another
imperfect TGACCT motif involved in the trans-activation
process. This imperfect TGACCT motif, together with three previously
identified TGACCT motifs, constitute a unified regulatory site (PPRE
binding unit). Thus, the rat HD PPRE consists of two DR1 elements
overlapping a DR2 element. Our results indicate that in this rat HD
PPRE binding unit, one PPAR/RXR heterodimer recognizes a DR2 motif
yielding C2 complex or two PPAR/RXR heterodimers recognize two
individual DR1 elements giving rise to C1 complex, providing the
peroxisome proliferator signaling pathway with two distinct responses.
Figure 1:
The
structure and oligonucleotide sequences of the wild type and mutant
derivatives of rat HD PPRE and ACOX PPRE. The consensus TGACCT motifs
are boxed and shaded. The mutant derivatives (g-j) created by transversion ((G/C) (A/T),
lowercase nucleotide) from the wild type in each of the TGACCT motifs
is boxed, but not shaded. Each of the probes with a BglII extension (in lowercase) was filled with
[
-
P]dCTP, dATP, dGTP, and dTTP by Klenow
for gel mobility shift assay or cloned into the BglII site of
pTKLuc after phosphorylation.
Figure 2:
Binding of PPAR and RXR to different
PPREs. Purified rPPAR and rRXR
proteins were incubated with
P-labeled complementary PPRE probes as indicated at the top. a-d, probes corresponding to the sequences
designated by the same letter in Fig. 1. Arrows indicate the C1 and C2 complexes.
Figure 3: Cooperative formation of two distinct complexes on HD PPRE by PPAR and RXR. A, gel shift assay using the full-length HD PPRE (see Fig. 1, sequence b) probe and different concentrations of PPAR and RXR (1, 2.5, and 5 µl) as schematically indicated at the top. B, saturation of the probe by receptor proteins. Each 5 µl of PPAR and RXR were incubated with 4, 3, 2, and 1 µl of probe, respectively, in each lane.
Figure 4:
Binding of PPAR and RXR heterodimer to
each of the individual and mutant elements on HD PPRE. A, gel
mobility shift assay using the HD DR1L, DR2, DR1R, and ACOX DR1
elements as indicated by e, a, f, and c corresponding to the sequences designated by the same letter in Fig. 1. 1, 2.5, and 5 µl of each of the purified rPPAR
and rRXR
proteins, respectively, as schematically indicated at the top were used in the assays. B, mutant probes as
indicated at the top (see Fig. 1, probes
g-j) were incubated with the same amount of purified
rPPAR
and rRXR
(5 µl of
each).
Figure 5:
Ciprofibrate responsiveness is efficiently
mediated by the Wt HD PPRE composed of all four TGACCT direct repeats.
CV1 cells were transfected with reporter plasmids and together with
rPPAR and rRXR
expression vectors in the presence or absence
of 0.5 mM ciprofibrate. Luciferase assays were performed after
a 40-h incubation with the inducer. pTKLuc, a basal reporter
plasmid, contains a 242-bp thymidine kinase promoter driving luciferase
cDNA. Reporter plasmids, pACOXLuc, pHDLuc,
pHDM1Luc, pHDM2Luc, pHDM3Luc,
pHDM4Luc, pHDDR1LLuc, pHDDR2Luc, and
pHDDR1RLuc harbor respective elements as described in Fig. 1. All the elements are single copy and in the same
orientation as the native rat HD gene as judged by sequencing. The
values represent average induction ratios (± S.E.) of luciferase
activity measured in the presence/absence of ciprofibrate. Values are
normalized to the ratio obtained with the pTKLuc control
plasmid (given the nominal value of 1; values lower than 1 are
attributed to toxicity of the inducer). Values reported are from three
independent transfections carried out in
duplicate.
PPREs have been identified in various genes with
variations in the binding site and spacer
sequence(1, 10) . We now show that the rat HD PPRE is
the first PPRE identified so far with four naturally occurring TGACCT
half-sites. Four half-sites have also been found in the cellular
retinol-binding protein element (CRBP-II element) which serves as an
efficient RXR element and facilitates the formation of a homotetrameric
complex on the CRBP-II element(18) . The homotetrameric complex
on the CRBP-II element was formed only with RXR and
, but
not with
, isoform(18) . In our study, only the rPPAR
and the rRXR
isoforms were tested to examine their ability to form
heterooligomers with HD PPRE. Additional studies are needed to
ascertain whether the C1 complex formed on the HD PPRE is also
isoform-specific. In fact, PPAR
plays a major role in the response
to peroxisome proliferators(7) . The binding of four RXR
molecules to the CRBP-II element is highly cooperative and interactive
and occurs without the detectable formation of dimeric
complexes(18) . It is not clear whether the C1 complex formed
on the HD PPRE simply represents either two noninteractive or two
highly interactive PPAR/RXR heterodimers. Unlike the RXR homotetramer
formed on the CRBP-II element, PPAR and RXR exhibit near equal amounts
of C1 and C2 complexes on the HD PPRE. Furthermore, saturation of the
probe cannot convert the heterodimeric C2 complex to the tetrameric C1
complex, suggesting that these two complexes are possibly formed by
different mechanisms as proposed in Fig. 6. The C1 complex,
composed of two PPAR/RXR heterodimers with minimal or no interaction,
occupies the two DR1 motifs, conferring a highly efficient
responsiveness to peroxisome proliferators. In contrast, the
heterodimeric C2 complex forms only on the DR2 element conferring a low
or non-responsiveness to peroxisome proliferators. Studies on the
binding of RXR heterodimers with thyroid hormone receptor (on DR4
element) and RAR (on DR2 and DR5 elements) have shown that RXR occupies
the 5`-half-site of AGGTCA direct repeats(19) . The binding of
RAR to the upstream half-site of DR1 elements results in switching of
the RAR from an activator to an inhibitor(20) . In cytochrome
P-450 4A6 PPRE, an extended 5` sequence of two imperfect AGGTCA direct
repeats is required for PPAR/RXR binding implying that PPAR
occupies the 5`-half-site of the two AGGTCA motifs(17) . The
AGGTCA motifs are presented on the lower strand of the HD PPRE relative
to the promoter of HD gene. Thus, in rat HD PPRE, we propose that PPAR
also occupies 5` AGGTCA motif and RXR occupies 3` AGGTCA motif, i.e. that RXR is in upstream and PPAR is in downstream
relative to the promoter. The polarity of RXR and PPAR on the central
DR2 is reversed in C1 complex. The failure of switching C2 to C1
complex is possibly due to the fact that the binding of a single
heterodimer to the DR2 element to form C2 complex is in the same
orientation as for the binding of the heterodimers to each of the DR1
elements in C1 complex and that it does not permit the binding of
additional receptors to the outer sites in the favorable orientation.
Thus, unlike the RXR homotetramer, RXR and PPAR form two distinct
complexes on HD PPRE.
Figure 6: Schematic diagram of the interaction of PPAR and RXR with HD PPRE. Rat HD PPRE consisting of four TGACCT direct repeats efficiently binds with PPAR and RXR heterodimer and forms C1 and C2 complexes. The C1 complex is composed of two PPAR/RXR heterodimers occupying the two DR1 elements, whereas the C2 complex contains only one PPAR/RXR heterodimer occupying the DR2 element. The orientations of C1 and C2 are the same.