©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperative Formation of Higher Order Peroxisome Proliferator-activated Receptor and Retinoid X Receptor Complexes on the Peroxisome Proliferator Responsive Element of the Rat Hydratase-Dehydrogenase Gene (*)

(Received for publication, September 14, 1995; and in revised form, October 11, 1995)

Ruiyin Chu Yulian Lin M. Sambasiva Rao Janardan K. Reddy (§)

From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 (alpha, beta, ) 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 mPPAR2 isoform is adipose tissue-specific and plays a pivotal role in the adipogenic signaling cascade(6) . The rPPARalpha 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 beta-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.


EXPERIMENTAL PROCEDURES

Molecular Clones

The coding sequences for rPPARalpha and rRXRalpha were amplified by polymerase chain reaction from the respective cDNAs with two sets of primers: 5`AAT GCG GCC GCT ATG CAT CAC CAT CAC CAT CAC ATG GTG GAC ACA GAG AGC CCC3`/5`TGC TCT AGA GCA ACG GCC TAC CAT CTC AGG3`, and 5`AAT GCG GCC GCT ATG CAT CAC CAT CAC CAT CAC ATG GAC ACC AAA CAT TTC CTG3`/5`ATT GTC TAG AGC AGC TGT GTC CAG GCG GGG3`, respectively. After sequencing, the amplified cDNAs were introduced into a pVL1392 transfer vector as NotI/XbaI fragments. Baculovirus recombinants were recovered by using the BaculoGold in vivo recombination system (Pharmingen). For transient transfection studies, the rPPARalpha and rRXRalpha cDNAs were subcloned from these baculovirus transfer vectors into the EcoRI and BglII sites of the pSG5 vector (Invitrogen). Complementary oligonucleotides, corresponding to the HD PPRE and mutant derivatives (Fig. 1), were chemically synthesized, annealed, and inserted into the BglII site of a pGL2 (Promega)-based basal reporter construct (pTKLuc) that contains thymidine kinase promoter driving luciferase cDNA. The resulting plasmids were sequenced and recombinants that contain a single copy of the respective element in the same orientation as the native rat HD gene were selected for transfection assay.


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 [alpha-P]dCTP, dATP, dGTP, and dTTP by Klenow for gel mobility shift assay or cloned into the BglII site of pTKLuc after phosphorylation.



Electrophoretic Mobility Shift Assays

rPPARalpha and rRXRalpha proteins were obtained by infecting insect Sf9 cells with recombinant baculoviruses and purified by a Ni-NTA agarose column (Qiagen) as described elsewhere(15) . The purified proteins yielded a single band by SDS-polyacrylamide gel electrophoresis; they were diluted (10 ng/µl) and stored at -70 °C. In a standard protein/DNA binding reaction, 5 µl of P-labeled DNA probe (1 times 10^5 cpm) were incubated with 5 µl each of the purified rPPARalpha and rRXRalpha, and 10 µl (75 ng) of poly(dI-dC) in a total volume of 25 µl for 20 min at 20 °C. The protein-DNA complexes were analyzed by electrophoresis through a 4% polyacrylamide gel using 0.5 times TBE (45 mM Tris borate, 1 mM EDTA).

Transient Transfection Assays

Transfections were carried out by the calcium phosphate procedure using 10-cm dishes of CV1 cells as described(16) . Transfections typically contained 1 µg of a reporter gene construct, 0.2 µg of rPPARalpha expression plasmid, 0.2 µg of rRXRalpha expression plasmid, and 0.5 µg of a bacterial beta-galactosidase expression vector pCMVbeta (Invitrogen) as an internal control. Cells were incubated in the presence of 0.5 mM ciprofibrate or 0.5% dimethyl sulfoxide, as required. Following a 40-h incubation, cells were processed to assess luciferase activity; the activity obtained for individual transfections was expressed relative to the beta-galactosidase activity obtained for the same preparation of lysate(15) .


RESULTS AND DISCUSSION

PPAR/RXR Forms Two Different Complexes with Rat HD PPRE

Rat HD PPRE has been shown to form two types of cell-specific protein-DNA complexes (C1 and C2) when incubated with nuclear extracts from H4IIEC3 cells(13) . Incubation of a 36-bp probe (-2959 to -2924, Fig. 1b) with the purified rPPARalpha and rRXRalpha proteins revealed two bands corresponding to the C1 and C2 complexes (Fig. 2). When 9- and 7-bp sequences flanking the DR2 element were deleted from the 5` and 3` end, respectively, of the 36 bp probe, only one band was observed. In contrast, both the short (19 bp) and long (36 bp) probes of rat ACOX PPRE in the same region as the HD PPRE, exhibited a single band. It is of interest to note that the PPAR/RXR heterodimer (C2 complex) bound with the short and long probes of either HD PPRE or ACOX PPRE, respectively, displayed a different extent of migration; the reason for this difference is not clear at present. Nevertheless, the C2 complex formed on the HD PPRE was the same as the heterodimeric PPAR/RXR complex formed with the rat ACOX PPRE. The C1 complex formed only with the 36-bp HD PPRE probe thus appears to be sequence-specific.


Figure 2: Binding of PPAR and RXR to different PPREs. Purified rPPARalpha and rRXRalpha 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.



The C1 and C2 Complexes Are Formed in a Highly Cooperative Manner

PPAR and RXR combine to form a heterodimer and bind to ACOX PPRE(8) . The C2 complex formed on HD PPRE displayed similar mobilities as the heterodimer bound on the ACOX PPRE implying that it is also a PPAR/RXR heterodimer. The C1 complex displayed a slower extent of migration suggesting that it may contains more than two receptor molecules. To examine whether PPAR and RXR are equally presented in the C1 complex, gel mobility shift assays were done using different concentrations of protein and probe. Increasing RXR concentration and keeping PPAR constant resulted in a cooperative increase in both the C1 and C2 complexes (Fig. 3A, lanes 1-3). Likewise, increasing PPAR concentration while keeping the amount of RXR constant also resulted in an increase of C1 and C2 complexes (Fig. 3A, lanes 4-6). These results indicate that, as with the C2 complex, the C1 complex also consists of an equal molar amount of PPAR and RXR. Saturation of the probes with receptor proteins showed almost no effect on the C1:C2 ratio (Fig. 3B), indicating that the C1 and C2 complexes are independently formed and the heterodimeric C2 complex cannot convert to the multimeric C1 complex.


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.



PPAR and RXR Heterodimers Can Bind to DR2 or Either One of the DR1 Elements

The C1 complex is sequence-specific and is composed of equal amounts of PPAR and RXR. The formation of C1 complex is due to the special structure of HD PPRE. By comparing the nucleotide sequence of rat HD and ACOX PPRE, we anticipated that a TCTCCT hexamer, which is similar to the consensus TGACCT motif and linked by a T nucleotide to the previously identified first TGACCT motif, can potentially serve as a binding site in HD PPRE (Fig. 1b). If it is the case, this hexamer, together with three other imperfect TGACCT motifs previously identified, can form two DR1 elements (Fig. 1, e and f; designated as DR1L and DR1R, respectively) overlapping the original DR2 element. Gel mobility shift assays indicated that the DR1L, DR1R, as well as, DR2 element, can individually bind with PPAR and RXR heterodimers (Fig. 4A). Mutation of any one of the TGACCT motif, as expected, formed no C1 complex when compared to the wild type probe (Fig. 4B). The second TGACCT motif seems to be the most critical one, as mutation of this motif also significantly reduced the formation of the heterodimeric C2 complex (Fig. 4B, lane 3). A very small amount (about 1/20 of the wild type) of C1 complexes remained when the last TGACCT motif was altered (Fig. 4B, lane 5) suggesting that these four TGACCT motifs are unified, and each of these TGACCT motifs contributes differently. These results suggest that with a special structure, the rat HD PPRE can alternatively bind with two PPAR/RXR heterodimers to form the C1 complex, or with one PPAR/RXR heterodimer to form the C2 complex.


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 rPPARalpha and rRXRalpha 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 rPPARalpha and rRXRalpha (5 µl of each).



Formation of Tetrameric C1 Complex on Rat HD PPRE in Vitro Correlates with Efficient Reporter Gene Activation in Vivo

It has been reported that a single copy of rat HD PPRE is more efficient in activating the reporter gene than an equivalent length of ACOX PPRE in the peroxisome proliferator-responsive H4IIEC3 cells(14) . Transfection of CV1 cells with the reporter constructs together with rPPARalpha and rRXRalpha expression plasmids confirmed the results obtained from H4IIEC3 cells (Fig. 5). In correlating with the gel shift results, mutation of any one of the four TGACCT motifs significantly reduced the transcriptional response to ciprofibrate. The M2 and M3 mutants which altered the second and third TGACCT motifs, respectively, abolished the response to ciprofibrate almost totally. Whereas the M1 and M4 mutants which can still bind with one PPAR/RXR heterodimer displayed marginal activities. Although, the individual DR1 and DR2 elements can bind efficiently with the PPAR/RXR heterodimer, they are not sufficient to activate the reporter gene in response to a peroxisome proliferator. The lack of responses of those individual DR1 and DR2 elements to the inducer may be due to the requirement of additional flanking sequences. Palmer et al.(17) reported that in rabbit cytochrome P-450 4A6 PPRE (which exhibits two imperfect AGGTCA motifs), six nucleotides 5` of the DR1 element appear necessary for both receptor binding and reporter gene activation. The oligonucleotides used in our study disrupt this extended binding site as it is replaced with BglII overhangs used for cloning.


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 rPPARalpha and rRXRalpha 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 alpha and , but not with beta, isoform(18) . In our study, only the rPPARalpha and the rRXRalpha 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, PPARalpha 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 PPARalpha 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.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant R37 GM 23570 and by the Joseph L. Mayberry Sr. Endowment Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago IL 60611. Tel.: 312-503-8144; Fax: 312-503-8240.

(^1)
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; PPRE, peroxisome proliferator responsive element; HD, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; ACOX, peroxisomal fatty acyl-CoA oxidase; TR, thyroid hormone receptor; RAR, retinoic acid receptor; CRBP-II, cellular retinol-binding protein II; bp, base pair(s); m, mouse; r, rat.


ACKNOWLEDGEMENTS

We thank Dr. Frank Gonzalez for the gift of rat PPARalpha cDNA. We greatly appreciate the comments of Dr. J. Larry Jameson on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.