©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Novel Sequence Determinants in Peroxisome Proliferator Signaling (*)

Colin N. A. Palmer , Mei-Hui Hsu , Keith J. Griffin , Eric F. Johnson (§)

From the (1)Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gene encoding cytochrome P-450 4A6 (CYP4A6) is transcriptionally activated by peroxisome proliferators. This response is dependent on a strong enhancer element (Z) and weaker elements (X and -27). The peroxisome proliferator response is mediated by the binding of heterodimers containing the peroxisome proliferator-activated receptor (PPAR) and the retinoid X receptor (RXR) to these elements. These peroxisome proliferator response elements (PPREs) contain imperfect direct repeats of the nuclear receptor consensus recognition sequence with a spacing of one nucleotide (DR1) (AGGTCA N AGGTCA). This DR1 motif is seen in the binding sites for other nuclear receptor complexes, such as ARP-1, HNF-4, and RXR homodimers. Mutational analysis of the Z element reveals that the DR1 motif is required for the transcriptional activation of the CYP4A6 gene by peroxisome proliferators; however, deletion of sequences immediately upstream of this motif also abolishes this response. Oligonucleotides corresponding to truncated and mutated Z elements were assayed by gel retardation for binding to RXR, PPAR, and ARP-1. Deletions or mutations within six nucleotides 5` of the DR1 motif dramatically diminish PPARRXR binding without reducing the binding of either RXR or ARP-1 homodimers, whereas mutation or deletion of the core DR1 sequences abolishes the binding of PPARRXR heterodimers and of RXR or ARP-1 homodimers. Thus, the DR1 motif in the Z element is not sufficient to constitute a PPRE. Moreover, the binding of PPARRXR to the Z element requires sequences immediately 5` of the DR1. These sequences are conserved in natural PPREs and promote binding of PPARRXR heterodimers in preference to potential competitors such as ARP-1 and RXR.


INTRODUCTION

Peroxisome proliferators are non-genotoxic carcinogens that cause profound changes in the subcellular structure and lipid metabolism of hepatocytes in rodents(1, 2) . They are a structurally diverse group of chemicals that include hypolipidemic drugs, plasticizers, and herbicides. A well characterized biochemical marker for the action of peroxisome proliferators is the induction of the enzymes that catalyze the peroxisomal -oxidation and microsomal -hydroxylation of fatty acids. Recent studies have shown that peroxisome proliferators induce transcription of the rat fatty acid acyl-CoA oxidase gene (3, 4) and the rabbit CYP4A6 fatty acid -hydroxylase gene (5) via an orphan member of the nuclear receptor superfamily, the peroxisome proliferator-activated receptor (PPAR).()PPAR has been shown to act by binding to specific sequences in the 5`-flanking sequence of the acyl-CoA oxidase and CYP4A6 genes, and this binding requires the presence of accessory proteins such as the retinoid X receptor (RXR)(4, 6) .

We have previously identified three peroxisome proliferator response elements (PPRE) in the CYP4A6 gene(5, 6) . The most distal element (X) provides a weak response in the presence of cotransfected PPAR, and deletion of this element from CYP4A6 reporter constructs results in a 2-fold drop in response to peroxisome proliferators(5) . Deletion of the Z element from CYP4A6 reporter constructs results in the abolition of significant peroxisome proliferator responses in the presence of cotransfected PPAR. The proximal element(-27) is a cryptic element in that it is only activated in cotransfection studies when RXR is overexpressed together with PPAR. The cryptic nature of the -27 element has been shown to be due to the fact that it binds PPARRXR very inefficiently(6) . These and other PPRE sequences contain imperfect direct repeats of the consensus binding site for the nuclear receptor superfamily (AGGTCA)(7) , with a spacing of one nucleotide (DR1)(4, 5, 8, 9, 10) . These direct repeats are known to bind potential competitors such as homodimers of other nuclear receptors including RXR, ARP-1, chicken ovalbumin upstream promoter transcription factor-1, and hepatic nuclear factor-4(11, 12, 13) . This would suggest a level of promiscuity in the regulation of the genes controlled by these elements, and, in fact, ARP-1 and chicken ovalbumin upstream promoter transcription factor-1 have been shown to antagonize peroxisome proliferator signaling(6, 14) .

In this study, we demonstrate that the sequence requirements for the binding of PPARRXR to the CYP4A6 Z element are distinct from those seen for potential competitors such as homodimers of ARP-1 and RXR and that sequences 5` of the DR1 contribute to selective binding of PPARRXR. These additional sequences form an extended binding site that is conserved within natural PPREs.


EXPERIMENTAL PROCEDURES

Transient Transfection Experiments

The RK13 cell line was obtained from the American Type Culture Collection and maintained in minimal essential medium containing Earle's balanced salt solution (Life Technologies, Inc.) and 10% fetal calf serum (Gemini, Calabasas, CA). Luciferase reporter plasmids pLUCA6-880 and pLUCA6-155 (5) as well as the expression constructs pCMV-PPAR, pCMV-PPAR-G(5) , pRS-RXR(15) , pMT-ARP-1(16) , and pSVGal (Promega) have been previously described. The reporter and expression constructs were introduced into cultured cells by a modification of the calcium phosphate coprecipitation procedure(17) . After a 16-h exposure, the DNA containing culture medium was removed, and the cells were washed twice with minimal essential medium and then exposed to culture medium with either Wy-14,643 (50 µM) or the equivalent volume of solvent (MESO, 0.25% (v/v) final concentration). This medium was replaced after 24 h. After another 24 h, the cells were harvested, washed with phosphate-buffered saline (0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl), and then lysed by suspension in 0.1 M potassium phosphate buffer, pH 7.8, containing 1 mM dithiothreitol and 0.05% Triton X-100 followed by 3 cycles of freezing and thawing. Insoluble material was removed by centrifugation, and luciferase activity was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). -Galactosidase activities were determined as described (5). The luciferase activity obtained for individual cultures was expressed relative to the -galactosidase activity obtained for the same preparation of lysate.

Expression of PPAR/GST, RXR/MBP, and ARP-1/MBP Fusion Proteins and Non-fusion PPAR in Escherichia coli

The mPPAR, RXR, and ARP-1 receptors were expressed as fusion proteins and affinity purified as previously described(6) . The mPPAR cDNA was modified by PCR to contain an NdeI site at the initiator ATG. This allowed the cloning of the cDNA into the pCW vector (18) and subsequent expression of the PPAR protein. The PPAR cDNA was also modified at the second codon to encode an alanine residue (GCT), and a silent mutation was also incorporated into the fifth codon. These modifications have been previously shown to increase the expression of mammalian proteins in E. coli(18) . The upper primer corresponds to 5`-CCATCGATCATATGGCTGACACAGAAAGCCCCATCTGTCCT-CTCTCCCCACTGGAGGCAGATGAC-3` (bold type indicates changes from the published sequence). The lower primer was that used in the isolation of the mPPAR cDNA(5) . The growth conditions, induction, and lysis of E. coli containing this plasmid were as described for the fusion proteins(6) . The 100,000 g supernatants from the bacterial lysates were used directly in the EMSA.

EMSA

Oligonucleotides were end labeled with P using T4 polynucleotide kinase, and the complementary strands were annealed. Each double-stranded, labeled oligonucleotide was then purified by electrophoresis in a 20% polyacrylamide gel using 89 mM Tris borate buffer, pH 8.0, containing 2 mM EDTA (1 TBE). Following excision of the portion of the gel containing the labeled oligonucleotide, the probe was eluted from the gel with 0.5 M NHOAc, 1 mM EDTA, and 1% SDS and was collected following precipitation with ethanol. For EMSA, the partially purified fusion proteins were incubated with 1 µg of poly[d(I-C)] in 10 mM Tris-HCl, pH 8.0, containing 100 mM KCl, 10% glycerol, and 1 mM dithiothreitol in a total volume of 29 µl. The reaction was incubated for 10 min on ice, and after the addition of 1 µl of labeled probe, the incubation was continued on ice for another 10 min. Following the addition of 1 µl of loading buffer (30% glycerol, 5 mg/ml bovine serum albumin, 0.005% bromphenol blue), 4 µl of the reaction mixture was loaded onto a gel containing 4% acrylamide, 0.05% bisacrylamide, 0.5 TBE, 1.25% glycerol. Electrophoresis was performed at 160 V for 90 min at 4 °C. The gel was then dried and exposed to Kodak X-AR film at -70 °C. In some cases, the gel was analyzed using a Molecular Dynamics PhosphorImager, model SF (Sunnyvale, CA).

Oligonucleotide-directed Mutagenesis

The two complementary mutant Z element oligonucleotides were each used as PCR primers to introduce the nucleotide substitutions shown in Fig. 4into the CYP4A6 promoter. This procedure employed the pLUCA6-880 plasmid as template in two separate PCR reactions to produce fragments corresponding to nucleotides -880 to -642 of CYP4A6 and -671 of CYP4A6 to nucleotide 3122 of p19dLUC that each carried the mutations. Following the purification of the two fragments, they were denatured, mixed, annealed, and amplified in a PCR reaction employing the outer primers. This produced a fragment spanning from -880 of CYP4A6 to the nucleotide 3122 of p19dLUC. The fragment was digested with HindIII and KpnI, and the resulting fragment was inserted with the HindIII-SacI fragment of pLUCA6-155M or pLUCA6-155 into SacI-KpnI-digested p19dLUC. The PCR reactions employed Taq polymerase (Promega) and reaction buffers supplied by the manufacturer. The identity of the mutated fragments was confirmed by sequencing.


Figure 4: Effect of mutations on the binding of PPARRXR and ARP-1 to the CYP4A6 Z element. A, three point mutations, indicated by lowercase letters, were introduced into each half site on separate oligonucleotide pairs. This arrangement of mutations has proven effective in abolishing ARP-1 binding to the APOCIIIB (13) and the CYP4A6-27 elements (6). Mutants Zm1c and Zm5 are mutations designed to disrupt the consensus extended binding site derived from the known PPREs (Fig. 3). B, EMSA was performed with labeled mutant oligonucleotide pairs. These experiments employed 10 fmol of each double-stranded oligonucleotide, together with 1.5 µg of PPAR/GST and 0.2 µg of RXR/MBP fusion proteins. In the ARP-1 assays, 0.5 µg of affinity-purified ARP-1/MBP fusion protein was used.



PPRE Oligonucleotides Used in EMSA

The double-stranded oligonucleotides corresponding to the CYP4A6 elements, Z and -27, were as previously described(6) . The CYP4A6 X element oligonucleotide corresponds to -746 to -724 of the CYP4A6 gene as follows: 5`-GACAAGTAGGACAAAGGCCAGGG-3`. The acyl-CoA oxidase oligonucleotide (ACOXA) include nucleotides -553 to -575 of the rat peroxisomal acyl-CoA oxidase gene (19) as follows: 5`-acgtGGACCAGGACAAAGGTCACGTTCtcga-3`. The bifunctional enzyme PPRE corresponds to -2919 to -2942 of the rat peroxisomal bifunctional enzyme gene (8) and does not contain the overlapping DR2 structure that is not required for PPARRXR binding(14, 20) (5`-TCAAATGTAGGTAATAGTTCAATA-3`). The fatty acid binding protein PPRE (FABP) corresponds to -48 to -71 of the rat liver fatty acid binding protein gene (9) as follows: 5`-TCAAATATAGGCCATAGGTCAGTG-3`. The 3-hydroxy-3-methylglutaryl-CoA synthase PPRE corresponds to nucleotides -82 to -107 of the rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene (10) as follows: 5`-CAGAAAAACTGGGCCAAAGGTCTCAG-3`. The APOCIIIB element corresponds to nucleotides -63 to -84 of the human apolipoprotein CIII gene (16) as follows: 5`-AGGGCGCTGGGCAAAGGTCACC-3`. DR1P = 5`-ACAAAACTAGGTCAAAGGTCAGGG-3`, and DR1M = 5`-CGCGCGCCAGGTCAAAGGTCAGGG-3`.

Preparation of Liver Lysates and Supershift Analysis

Frozen mouse liver (100 mg) was homogenized in 500 µl of phosphate-buffered saline solution containing 5 mM EDTA, 1 mM dithiothreitol, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 10% glycerol. The homogenates were then sonicated 10 s, diluted to 1 ml with the above buffer, and centrifuged at 200,000 g for 30 min. The protein content of the supernatant was determined by the Bio-Rad protein assay (Bradford Assay). For supershift analysis, 100 µg of lysate (20 µl) was combined with 1 µg (1 µl) of poly(dI-dC) and 1 µg (1 µl) of sheared salmon sperm DNA and incubated on ice for 10 min. Following the addition of 25 fmol of radiolabeled double-stranded oligonucleotide, the incubation was continued on ice for 10 min, after which 0.5 µl of either rabbit pre-immune or rabbit anti-PPAR serum was added, and the incubation on ice was continued for an additional 30 min. Gel loading and electrophoresis was performed as described for the EMSA protocol; however, the entire binding reaction was loaded onto the gel.


RESULTS

As previously shown(5) , cotransfection of CYP4A6 reporter constructs pLUCA6-880 and pLUCA6-663 (Fig. 1) with an expression vector for PPAR-G (a mutant form of mPPAR that displays an enhanced response to peroxisome proliferators) revealed that sequences 5` of the direct repeat (DR1) were required to confer responsiveness to peroxisome proliferators. pLUCA6-880 is activated 13.3-fold by the inclusion of a peroxisome proliferator (Wy-14,643) in the culture medium, whereas pLUCA6-663 does not respond to Wy-14,643. An oligonucleotide that spanned the deletion site (Z) was found to confer a peroxisome proliferator response to the non-responsive pLUCA6-155 construct. This element contains four imperfect repeats of the nuclear receptor binding site (AGGTCA) (half sites). We would anticipate that the binding of a PPARRXR heterodimer would require two such half sites, with a zinc finger from each receptor interacting with one of these repeats. The portion of the Z element found in pLUCA6-663 contains an imperfect direct repeated sequence (DR1) that is formed with half sites 2 and 3. DR1 motifs have been found in the PPREs of other genes, including the acyl-CoA oxidase gene, and are known to bind RXR homodimers. However, half sites 2 and 3 are retained in the inactive pLUCA6-663 construct. It appears that it is the disruption of half site 1 that leads to a loss of response in the -663 construct. Half site 1 (3`-AGTTCA-5`) is known to bind retinoic acid receptors with a high efficiency(21, 22) . This sequence forms imperfect inverted palindromes with the directly repeated half sites 2, 3, and 4. Nuclear receptors are known to bind inverted palindromic sequences of different spacings(22, 23) .


Figure 1: CYP4A6 reporter constructs and their activation by peroxisome proliferators. The relative positions of the three CYP4A6 PPREs (X, Z, and -27) are indicated schematically. The peroxisome proliferator-dependent stimulation of transcription is also shown (Fold Induction). Deletion of X has previously been shown to reduce the response to Wy 14,643 by 2-fold; however, deletion into the Z element at position -663 abolishes the response to peroxisome proliferators (-663 construct). The Z element acts as an enhancer element and confers peroxisome proliferator responsiveness to the pLUCA6-155 promoter construct (-155 versus -155Z). Cotransfection of expression vectors for PPAR and RXR with reporter constructs that do not contain the X and Z elements still respond to peroxisome proliferators. This is due to the utilization of the cryptic -27 element at higher concentrations of RXR (6). The position of the direct repeats of imperfect nuclear receptor binding sites (numbered 1-4) in the Z element are shown.



To determine the minimal sequences required for the binding of PPARRXR to the Z element, EMSAs of double-stranded oligonucleotides containing various combinations of these repeated sequences were performed. For this purpose, the murine PPAR was expressed as a fusion protein with GST, and the human RXR was expressed as a fusion protein with the MBP. As previously shown(6) , a complex is observed for PPARRXR with the Z element (Fig. 2B, leftpanel, Z1234). Truncation of the Z element by six nucleotides on the 5`-end and three nucleotides on the 3`-end does not reduce the binding of PPARRXR (Fig. 2B, left panel, Z123). This result demonstrates that half site 4 is not involved in the binding of PPARRXR. Truncations of the 5`-end by a further six nucleotides, which disrupts half site 1, results in a loss of binding of PPARRXR (Fig. 2B, left panel, Z234, Z23). Deletion of a further nine nucleotides from the 3`-end, which removes half site 3, results in a loss of binding of PPARRXR (Fig. 2B, left panel, Z12). EMSA employing finer truncations of the Z123 oligonucleotide revealed that progressive truncations that disrupt half site 1 result in a progressive loss of PPARRXR binding (Fig. 2, center panel, Z123-Z23). Truncations 3` of half site 3 revealed the requirement of one additional nucleotide (Fig. 2B, centerpanel, ZD6-ZVVS) for the binding of PPARRXR. Therefore, it appears that the minimal PPARRXR binding sequence spans the regions corresponding to half sites 1, 2, and 3. Mutagenesis of half site 2 (Fig. 2B, leftpanel, Z13) results in the loss of PPARRXR binding. The requirement for three half sites (1, 2, and 3) was unexpected as the binding of a heterodimer should require only two repeats. As such, we decided to compare the binding requirements of PPARRXR with those for ARP-1. EMSA was performed with the partially purified ARP-1/MBP fusion protein and double stranded oligonucleotides corresponding to various truncations of the Z element (Fig. 2B, rightpanel). ARP-1 forms a single complex with the Z element in contrast to that seen with the -27 element of CYP4A6 and other characterized ARP-1 binding sites where both monomeric and dimeric binding is observed(6) . The mobility of the complex formed with the Z element approximates that of the lower mobility complex seen with the -27 element. The lower mobility complex is presumed to consist of a dimeric rather than monomeric complex as the slower mobility of the complex is likely to reflect a larger molecular weight. The appearance of a single, dimeric complex with the Z element and ARP-1 suggests that the monomer binds to the Z element with a very low efficiency. Truncations that disrupt half site 1 and abolish PPARRXR binding do not affect ARP-1 binding (Fig. 2B, rightpanel, Z123-Z23). ARP-1 binds readily to the Z23 oligonucleotide, indicating that only half sites 2 and 3 are required for binding; however, ARP-1 binding is abolished by the deletion of the 3`-nucleotide adjacent to the DR1 (Fig. 2B, rightpanel, ZD5 versus ZVVS). Therefore, the 3`-sequence requirements for the binding of ARP-1 are identical to those seen for RXRPPAR.


Figure 2: Analysis of PPARRXR binding to oligonucleotides containing different repeated half sites. A, the Z element contains four imperfect repeats of the nuclear receptor binding consensus sequence (AGGTCA) or ``half sites'' that are indicated by the arrows. Half sites 2 and 3 constitute a direct repeat with a spacing of one nucleotide. The sequences of truncated oligonucleotides used in panelB are shown. Changes from the wild type sequence are denoted in bold and lower case. B, EMSA analysis was performed using double-stranded oligonucleotides that were truncated to assess the contribution of flanking nucleotides to PPARRXR and ARP-1 binding of the Z element. Affinity-purified PPAR/GST (1000 ng), RXR/MBP (200 ng), and ARP-1/MBP (200 ng) fusion proteins were used in this assay.



The half site 1 region is required for the function of the Z element and the binding of PPARRXR but is not required for the binding of ARP-1 (Fig. 2B). This region, along with half site 2, constitutes an imperfect palindrome that is not conserved between PPREs. Comparison with other known PPRE sequences reveals that the region 5` of the DR1, which constitutes half site 1 of the Z element, exhibits a loose consensus (C(A/G)(A/G)A(A/T)CT) (Fig. 3). This consensus does not appear to be similar to the core nuclear receptor binding sequence (AGGTCA) but does resemble the extended consensus binding site for the monomeric nuclear receptors (Rev-ErbA, NGFI-B, and ROR) that contains an A-T-rich region 5` of a single AGGTCA motif (24, 25, 26) (Fig. 3).


Figure 3: Conservation between known PPREs. The DR1 motif is highly conserved between the CYP4A6 Z element and the PPREs found in the genes encoding the rat CYP4A1 (CYP4A1) (37), fatty acyl CoA oxidase (ACOXA) (3, 19), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (BIF) (8, 38), FABP (9), and the 3-hydroxy-3-methylglutaryl-CoA synthase (HMG) (10). A region of limited conservation is present immediately 5` of the DR1 motif. This region is similar to that seen in the binding site for the monomeric nuclear receptor, Rev-ErbA (24).



To test the nature of the sequence requirements for the binding of PPARRXR within the Z element, double-stranded oligonucleotides containing grouped point mutations in each of the four half sites were synthesized (Fig. 4A) and assayed by EMSA for binding to PPARRXR and ARP-1 (Fig. 4B). Equivalent grouped mutations have been shown to abolish binding of ARP-1 to the apolipoprotein CIIIB element and the CYP4A6 -27 element (6, 13) and have also been shown to abolish PPARRXR binding to the CYP4A6 -27 element(6) . Mutation of half sites 2 and 3 greatly diminished the binding of PPARRXR and ARP-1, confirming the importance of the DR1 motif for binding of these receptors. Mutations that disrupt the nuclear receptor binding motif in half site 1 but retain an A-T-rich motif similar to the monomeric nuclear receptor binding site (Zm1b) did not affect the binding of PPARRXR to the Z element. In contrast, mutations (Zm1c and Zm5) that disrupt the loose consensus in the extended binding site region display diminished binding to PPARRXR (Fig. 4B) but have no significant effect on ARP-1 binding. These results confirm the specific requirement for sequences 5` of the DR1 in the binding of PPARRXR and demonstrate the requirement for an extended binding site such as that seen for the monomeric receptors.

To test the effects of these mutations on transcriptional responses to peroxisome proliferators, oligonucleotide-directed mutagenesis was used to generate the above mutations in the 5`-flanking regions of the CYP4A6 gene. Cotransfection of mutant CYP4A6 reporter constructs and the expression vector for PPAR revealed that the mutations in half site 3 that severely reduced PPARRXR binding to the Z element also reduced the transcriptional response to peroxisome proliferators (Fig. 5, 880 versus Zm3). This reduction is not as complete as may have been expected from the DNA binding studies, suggesting that the other elements (X and -27) may partially compensate for the loss of function of the Z element. Mutation of the -27 element in the CYP4A6 reporter construct (880m) has little effect on its own (Fig. 5, 880 versus 880m), although previous studies have shown that this mutation abolishes the binding of PPARRXR to this element(6) . The -880m construct was used to analyze the other mutations of the Z element to minimize the effects of the -27 element in this analysis. Using these double mutant CYP4A6 reporter constructs, it is clear that mutations that diminish binding of PPARRXR to the Z element also abolish the response to peroxisome proliferators (Fig. 5, Zm1c-d, Zm2-d, Zm3-d). Mutations that do not affect DNA binding of PPARRXR do not diminish the response to peroxisome proliferators (Fig. 5, Zm1b-d, Zm4-d). Mutation of half sites 2 and 3 (Fig. 5, Zm2-d, Zm3-d) results in a loss of responsiveness to peroxisome proliferators, demonstrating the requirement for the DR1 in the function of the Z element. The Zm1c-d construct, which contains mutations in the extended binding site, also shows no response to peroxisome proliferators. This result, along with the deletion analysis of the CYP4A6 reporter constructs (Fig. 1), demonstrates the requirement of the sequences 5` of the DR1 for the function of the Z element.


Figure 5: Transactivation of mutant CYP4A6 promoter constructs by mPPAR-G. Oligonucleotide-directed mutagenesis was used to mutate the CYP4A6 Z element in the reporter construct pLUCA6-880 to generate pLUCA6-880Zm3 (Zm3). This and the other mutants were also cloned into pLUCA6-880m to generate the respective double mutant clones pLUCA6-880Zm1b-d (Zm1b-d), pLUCA6-880Zm1c-d (Zm1c-d), pLUCA6-880Zm2-d (Zm2-d), pLUCA6-880Zm3-d (Zm3-d), and pLUCA6-880Zm4-d (Zm4-d). These reporter constructs were cotransfected with pCMV-PPAR-G into RK13 cells. After incubation with DNA for 16 h, the cells were washed and then either solvent (MESO; no drug) or peroxisome proliferator (Wy-14,643) was added to fresh medium for an additional incubation of 48 h. The bargraphs represent the mean values plus standard deviation obtained from three independent transfections and show the activity normalized to that obtained with pLUCA6-880 in the presence of peroxisome proliferator.



The Z element binds RXR homodimers very weakly; this is in contrast to certain other DR1-containing elements, such as the rat cellular retinol binding protein II RXRE(12, 27) . The rat cellular retinol binding protein II RXRE contains a DR1 that is a perfect repeat of the AGGTCA motif, which binds RXR homodimers efficiently and confers a response to both all-trans and 9-cis retinoic acid(27) . The natural PPRE elements, such as the Z element, are divergent from the idealized DR1 sequence (Fig. 3). This divergence may reduce the binding of RXR homodimers, and the extended binding site may stabilize the binding of PPARRXR heterodimers to these divergent sequences.

Oligonucleotides containing a perfect consensus DR1 and either a consensus or divergent extended 5`-binding site (DR1P and DR1M, respectively) were assayed for their ability to bind RXR, PPAR, PPARRXR, and ARP-1 (Fig. 6). These results were compared to those obtained with natural elements that contain imperfect direct repeats. DR1P and DR1M display significant binding to RXR (Fig. 6A, complexesI and II). The 5`-extended binding site on the DR1P element appears to diminish RXR binding (Fig. 6B). The binding of RXR to DR1P and DR1M is about 20-fold greater than that seen with the APOCIIIB and ACOXA elements and over 100-fold greater than that seen with the other elements (Fig. 6B).


Figure 6: Analysis of PPAR, RXR, and ARP-1 binding to DR1 containing elements. A, EMSA analysis was performed using double-stranded oligonucleotides that correspond to DR1 containing elements that have been shown to be regulated by nuclear receptors. Binding assays used crude bacterial lysates containing expressed PPAR (6 µg) or RXR/MBP(0.5 µg) fusion protein or affinity-purified ARP-1/MBP fusion protein (70 ng). RXR homodimer complexes are shown (I and II) as are the putative PPARRXR heterodimeric complexes (III) and the ARP-1 monomer (M) and homodimer (D) complexes. B, the sequences of the PPREs used in the above binding assays are shown. Also shown is the number of matches the 5`-region has with the consensus sequence and a summary of the binding results obtained from the phosphorimager analysis. The values shown represent relative binding activities (% oligonucleotide bound) normalized to those obtained with the DR1P oligonucleotide pair. Perfect nuclear receptor binding sites are indicated in boldtype.



The addition of PPAR and RXR to DR1P and DR1M results in a complex of greater mobility than that seen with RXR alone (Fig. 6A, complexIII). The binding to DR1M demonstrates that the extended binding site is not required for the binding of PPARRXR to a perfect direct repeat of the consensus nuclear receptor binding motif. The DR1P oligonucleotide binds the PPARRXR more efficiently than DR1M (Fig. 6B), suggesting a modulatory role for the extended half site. A complex of similar mobility is formed with the addition of both PPAR and RXR to the natural elements. The degree of binding of PPARRXR to the natural elements is relatively similar to that observed for the consensus DR1, except for the APOCIIIB element and the CYP4A6-X and -27 elements (Fig. 6B). The APOCIIIB element has not been shown to function as a PPRE, and the CYP4A6-X and -27 elements are known to be weak PPREs.

The addition of ARP-1 to the DR1P and DR1M oligonucleotide pairs results in the formation of a single complex (Fig. 6A) that presumably consists of a homodimer (D) of ARP-1. A complex of similar mobility is seen for the other elements; however, complexes of higher mobility (M) are seen for the APOCIIIB, ACOXA, FABP, and CYP4A6-27 elements. These complexes may consist of monomeric ARP-1 binding to the single consensus half sites seen in these elements. The binding of ARP-1 to the PPREs that contain two divergent half sites (Fig. 6B, Z, BIF, HMG) is greatly reduced compared to the other elements.

These results demonstrate that the extended binding site is not required for the binding of PPARRXR to idealized DR1 sequences. PPREs that contain divergent half sites are less able to bind competitors such as RXR and ARP-1 homodimers, while PPARRXR binding is maintained to response elements with divergent DR1 sequences 3` of the conserved extended binding site.

To assess the specificity of these elements in a complex mixture of transcription factors, mouse liver total protein was assayed for binding to the Z element and the APOCIIIB element (Fig. 7). The binding studies with the individual receptors described above suggested that the APOCIIIB element would be less likely to bind PPARRXR than the Z element when in competition with other nuclear receptors. Similar amounts of binding activity were observed with liver extracts for both oligonucleotides (C). The addition of anti-PPAR serum to the binding assay results in the supershifting (SS) of a major proportion of the Z binding activity. In contrast, no detectable supershifting of the APOCIIIB binding activity is observed. Therefore, the Z element is likely to be occupied by PPARRXR heterodimers in liver, and the APOCIIIB element is more likely to be occupied by other proteins.


Figure 7: Binding of mouse liver proteins to the CYP4A6 Z element and the apolipoprotein CIIIB element. 100 µg of total liver protein was assayed for binding to double-stranded oligonucleotides corresponding to the CYP4A6-Z element and the APOCIIIB element. Shown is the assay in the absence of added serum (-), in the presence of preimmune serum (Pi), and in the presence of anti-PPAR serum (I). The protein/DNA complex is indicated (C), and the protein/DNA/antibody complex (supershift) is indicated (SS).




DISCUSSION

Previous studies have identified three PPREs in the 5`-flanking sequences of the CYP4A6 gene; each of these contain directly repeated imperfect copies of the nuclear receptor binding sequence AGGTCA(5, 6) with a spacing of one nucleotide (DR1). The Z element has been shown to mediate the major proportion of the response to peroxisome proliferators by CYP4A6 reporter constructs in transient expression assays. This element binds PPARRXR much more efficiently than the other CYP4A6 PPREs (6) (Fig. 6). A reporter construct containing the direct repeat of the Z element, in which the 5`-extended half site was deleted, did not display a response to peroxisome proliferators when cotransfected with expression vectors for PPAR, suggesting that sequences 5` of the DR1 were necessary for the response to peroxisome proliferators. In vitro DNA binding studies revealed the requirement of six additional nucleotides 5` of the DR1 for the binding of PPARRXR. This additional sequence was not required for the binding of RXR or ARP-1 homodimers. Mutagenesis of the CYP4A6 reporter constructs confirmed that the ability of the CYP4A6 gene to respond to peroxisome proliferators is predominantly determined by binding of PPARRXR to the Z element; however, it appears that binding of PPARRXR to the -27 and X elements may contribute to this response.

The additional sequence immediately 5` of the DR1 motif is partially conserved between PPREs and appears to differ from the AGGTCA motif, which constitutes the recognition sites for other members of this family of receptors. The consensus derived from the known PPRE sequences (C(A/G)(A/G)A(A/T)CT) and the mutational analysis described here suggests that the 5`-extended binding site for PPARRXR may be similar to the A-T-rich 5`-extended binding site seen for the monomeric nuclear receptors Rev-ErbA, ROR, and NGFI-B(24, 26, 28) . This binding is thought to involve a region that is immediately adjacent to the second zinc finger of the receptor known as the AT box (28). The AT box of the PPAR is highly conserved with that of Rev-ErbA-. This is the first observation of this type of sequence requirement for binding by a putative heterodimeric receptor complex. It is tempting to speculate that the novel second zinc finger(29, 30, 31) that is characteristic of the PPAR subfamily of nuclear receptors may prevent the binding of PPAR monomers to single half sites. The requirement of this extended binding site for the binding of PPARRXR heterodimers would imply that PPAR occupies the 5`-half site of the DR1. Studies on the binding of RXR heterodimers with the thyroid hormone and retinoic acid receptors have shown a strict requirement for RXR to occupy the 5`-half site of direct repeat(32, 33, 34, 35) . Further studies are in progress to determine the structure of the PPARRXRDNA complex.

This analysis of the effects of the extended binding site on the binding of PPARRXR to the consensus DR1 demonstrates that the extended binding site sequence is not absolutely required for the binding of PPARRXR. However, the presence of this sequence does seem to diminish the formation of RXR homodimers and promote the formation of the PPARRXR heterodimer.

The presence of one non-consensus and one consensus half site is seen in the DR1 of many natural elements. These elements display markedly reduced RXR binding. The binding of ARP-1 remains high to such elements, although much of this binding appears to be monomers of ARP-1 binding to the single consensus half site. The binding of PPARRXR to these elements is variable, and in some cases (the APOCIIIB element) binding is very weak. The APOCIIIB contains a T at the first position in the 5` half site that is also seen in the weak -27 element. It appears that this change may reduce PPARRXR binding. The -27 element displays marked PPARRXR synergism in binding, whereas the APOCIIIB element binds RXR homodimers and PPARRXR heterodimers equally well. This difference may be attributed to the fact that the -27 element contains a conserved 5`-extended binding site (6/7 match), and the APOCIIIB element contains a divergent 5`-extended binding site (2/7 match). In contrast, the FABP element binds very strongly to PPARRXR and contains a 6/7 match with the PPRE 5`-extended binding site consensus. Therefore, it appears that there is a stricter requirement for the extended binding site in elements with imperfect repeats.

The presence of two non-consensus half sites in the DR1 is a common feature of natural PPREs. The high efficiency PPREs from the CYP4A6 (Z), bifunctional enzyme, and the 3-hydroxy-3-methylglutaryl-CoA synthase genes are examples of elements that contain two non-consensus half sites. These elements display very weak binding to RXR homodimers and display greatly reduced ARP-1 binding when compared to the elements that contain perfect AGGTCA motifs. These elements can be considered to be very specific for PPARRXR and contain a 5`-extended binding site consensus match of 6/7.

The lower efficiency PPREs, X and -27, have mismatches in the half sites that are not observed in other PPREs and display reduced PPARRXR binding. However, they display selective PPARRXR binding over RXR homodimers as they contain conserved 5`-extended binding sites.

From the above observations, it appears that the upstream, extended binding site sequence allows the maintenance of PPARRXR binding in elements containing divergent repeated structures that bind poorly to competitors such as ARP-1 and RXR, thereby generating specificity from a seemingly promiscuous DR1 response element structure. This conclusion is supported by the finding that the CYP4A6 Z element binds mainly to PPAR containing complexes in mouse liver lysates, while the APOCIIIB element, which was shown to be more promiscuous in the binding of recombinant RXR and ARP-1, is mainly bound by proteins other than PPAR. It is therefore apparent that the sequence of the half site and the upstream extended binding site will modulate the specificity of a DR1 response element with respect to competition for other members of this receptor family (possibly including the monomeric receptors) and thus determine the regulation of genes in vivo.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant HD04445 (to E. F. J.) and postdoctoral fellowship American Heart Association of California Grant 93-96 (to C. N. A. P.). Facilities for computer-assisted analysis and the synthesis of oligonucleotides are supported in part by GCRC Grant M01 RR00833 and by the Sam and Rose Stein Charitable Foundation, respectively. 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: Division of Biochemistry, NX-4, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-8098; Fax: 619-554-6117; E-mail: johnson@scripps.edu.

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor ; RXR, retinoid X receptor ; PPRE, peroxisome proliferator-responsive element; ARP-1, apolipoprotein regulatory protein-1; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift analysis; FABP, fatty acid binding protein; GST, glutathione S-transferase; MBP, maltose binding protein; DR1, a direct repeat with a spacing of one nucleotide; PPAR-G, a mutant form of PPAR; Wy-14,643, 4-chloro-6(2,3-xylindino)2-pyrimidinylthiolacetic acid; individual cytochrome P-450s are designated according a uniform system of nomenclature (36), and the gene designations are preceded by the letters CYP.


ACKNOWLEDGEMENTS

We express appreciation to Dr. D. J. Mangelsdorf and Dr. R. Evans (Salk Institute, La Jolla, CA) for providing the RXR cDNA in the pRS expression plasmid and to Dr. S. K. Karathanasis (American Cyanamid Co.) for providing the ARP-1 cDNA.


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