Tissue-Selective, Bidirectional Regulation of PEX11{alpha} and Perilipin Genes through a Common Peroxisome Proliferator Response Element

Most cis-acting regulatory elements have generally been assumedto activate a single nearby gene. However, many genes are clusteredtogether, raising the possibility that they are regulated througha common element. We show here that a single peroxisome proliferatorresponse element (PPRE), located between the mouse PEX11 ${alpha}$ andperilipin genes, confers on both genes activation by peroxisomeproliferator-activated receptor alpha (PPAR ${alpha}$ ) and PPAR ${gamma}$ . A functionalPPRE 8.4 kb downstream of the promoter of PEX11 ${alpha}$ , a PPAR ${alpha}$ targetgene, was identified by a gene transfection study. This PPREwas positioned 1.9 kb upstream of the perilipin gene and alsofunctioned with the perilipin promoter. In addition, this PPRE,when combined with the natural promoters of the PEX11 ${alpha}$ and perilipingenes, conferred subtype-selective activation by PPAR ${alpha}$ and PPAR ${gamma}$ 2.The PPRE sequence specifically bound to the heterodimer of RXR ${alpha}$ and PPAR ${alpha}$ or PPAR ${gamma}$ 2, as assessed by electrophoretic gel mobilityshift assays. Furthermore, tissue-selective binding of PPAR ${alpha}$ and PPAR ${gamma}$ to the PPRE was demonstrated in hepatocytes and adipocytes,respectively, by chromatin immunoprecipitation assay. Hence,the expression of these genes is induced through the same PPREin the liver and adipose tissue, where the two PPAR subtypesare specifically expressed.

INTRODUCTION

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Introduction

Eukaryotic genes are regulated by transcription factors thatbind to specific DNA elements located in their vicinity. Thebinding site and the transcription factor for a given gene aregenerally supposed to act only on the gene and thus assure thespecificity of its regulation. Recent genome analysis, however,revealed that an unexpectedly large number of genes of highereukaryotes form clusters (3). They may reside in the same oropposite orientation, sometimes sharing promoters and, in somecases, even overlapping in part. Such an arrangement of geneswould raise a hitherto unassumed possibility that two or moregenes can be regulated through a common cis element, becausethe regulatory element of a given gene would inevitably be closeto another gene. We report here that the mouse PEX11 ${alpha}$ and perilipingenes, which are positioned in tandem on the genome, are regulatedthrough a common recognition site for peroxisome proliferator-activatedreceptors (PPARs).

The PPAR family has three subtypes, ${alpha}$ , ${gamma}$ , and ${delta}$ (or ß),within the nuclear hormone receptor superfamily (18). PPAR ${alpha}$ ishighly expressed in the liver and regulates the expression ofseveral genes involved in lipid metabolism (14). PPAR ${gamma}$ has twoisoforms ( ${gamma}$ 1 and ${gamma}$ 2) generated by alternative transcription start(77); PPAR ${gamma}$ 1 is expressed in many tissues, including the adiposetissue and immune cells (10), whereas PPAR ${gamma}$ 2 is exclusively expressedin adipose tissue, playing a central role in adipocyte differentiation(71). PPARß/ ${delta}$ is ubiquitously expressed and is involvedin the physiology of certain tissues as well as lipid homeostasis(7, 51, 74). PPAR binds to peroxisome proliferator responseelements (PPREs) located in the vicinity of target genes byheterodimerizing with another nuclear hormone receptor, retinoidX receptor (RXR) (32). A PPRE is comprised of two AGGTCA orrelated half sites separated by a single nucleotide, termeddirect repeat 1. For optimal binding, PPAR requires an extendedhalf site constituted by AGGTCA and four extra residues on the5' side (28, 31, 46, 49).

PPAR ${alpha}$ was first identified as a transcription factor that mediatesthe action of certain drugs that cause peroxisomes to proliferatein rodent liver (29). Peroxisomes are ubiquitous organellesbounded by a single membrane, and mammalian peroxisomes havemany important metabolic functions, such as ß-oxidationof very-long-chain fatty acids and synthesis of cholesteroland ether lipids (35). Peroxisomes in rodent liver markedlyproliferate on the administration of various hypolipidemic agentscollectively termed peroxisome proliferators (8, 55). Concomitantly,peroxisomal and mitochondrial ß-oxidation enzymesas well as microsomal ${omega}$ -oxidation enzymes are induced. Disruptionof the PPAR ${alpha}$ gene of mice results in refractivity to peroxisomeproliferation in the liver upon administration of peroxisomeproliferators (36). PPAR ${alpha}$ was shown to activate the expressionin the liver of several genes for enzymes involved in peroxisomaland mitochondrial fatty acid ß-oxidation, such asacyl coenzyme A (CoA) oxidase (AOx) (47, 72), peroxisomal bifunctionalenzyme (76), and 3-ketoacyl-CoA thiolase (32, 45). On the otherhand, some adipogenesis-related genes, such as those for adipocyteP2 (70) and phosphoenolpyruvate carboxykinase (69), are knownas PPAR ${gamma}$ targets. However, no gene involved in peroxisome biogenesishas been reported to date as a target of PPAR ${alpha}$ , despite the dramaticincrease in the number of hepatic peroxisomes in rodents causedby the PPAR ${alpha}$ ligands.

PEX11 is one of the PEX genes (15) encoding the factors requiredfor the biogenesis of peroxisomes. At least 26 PEX genes havebeen identified, based on genetic complementation using peroxisome-deficientmutants of yeast and mammalian cells (43, 53, 63, 64, 66). PEX11was shown to promote peroxisome division in yeast and mammaliancells (1, 2, 19, 42, 50, 58, 61). Disruption of the PEX11 genein Saccharomyces cerevisiae and Candida boidinii resulted indecreased numbers of peroxisomes that were larger than normal(19, 42, 58). Although it was suggested that peroxisomal fattyacid metabolism affects peroxisome abundance (12), a recentstudy showed that overexpression of the PEX11 gene is sufficientto promote peroxisome proliferation without affecting peroxisomalmetabolism (39). In mammals, three subtypes of the PEX11 gene( ${alpha}$ , ß, and ${gamma}$ ) have been identified and mapped on differentchromosomes. The expression of PEX11 ${alpha}$ and PEX11 ${gamma}$ is tissue specific,being most prominent in the liver, whereas PEX11ßis ubiquitously expressed (37, 61). Interestingly, PEX11 ${alpha}$ expressionis induced by peroxisome proliferators, though the expressionof PEX11ß and PEX11 ${gamma}$ is not affected by these agents(1, 2, 37, 61, 65). Thus, the proliferation of peroxisomes causedby these agents is possibly attributable to the induction ofPEX11 ${alpha}$ expression, and hence we inferred that PEX11 ${alpha}$ is a targetgene of PPAR ${alpha}$ .

In the present study, we searched for a functional PPRE in thevicinity of the mouse PEX11 ${alpha}$ gene and identified one in the downstreamregion. Moreover, upon inspection of the mouse genome database,we found that the gene for perilipin is located downstream ofPEX11 ${alpha}$ , close to the PPRE. Perilipin is expressed mainly in adipocytesand steroidogenic cells and coats the surfaces of intracellularlipid droplets (22, 62). Perilipin was suggested to regulatethe lipolysis of triacylglycerol by blocking the access of hormone-sensitivelipase to stored lipids (13). Perilipin is produced during adipocytedifferentiation (22), where PPAR ${gamma}$ 2 plays a central regulatoryrole. Here, we show that the PEX11 ${alpha}$ and perilipin genes are regulatedby PPAR ${alpha}$ and PPAR ${gamma}$ , respectively, through a common PPRE.

MATERIALS AND METHODS

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Materials and Methods

RNA analysis. Reverse transcription (RT)-PCR was carried out using hepaticRNA derived from wild-type and PPAR ${alpha}$ -null mice fed a diet withor without Wy14,643 for 2 weeks (36). The procedure for RT-PCRwas as described previously (68). Concentrations of RNA sampleswere normalized based on the intensity of the signal for a ribosomalprotein, 36B4, a control unaffected by peroxisome proliferators.Adequate PCR cycles were set for individual genes so that theband intensities could be compared in the exponential amplificationphase.

Isolation of mouse PEX11 ${alpha}$ gene. Screening was carried out using a mouse genomic ${lambda}$ phage librarywith a cDNA fragment of rat PEX11 ${alpha}$ as a probe. Rat PEX11 ${alpha}$ cDNAwas cloned from a rat cDNA library by PCR. We obtained a positivegenomic clone (clone D) containing a 2.2-kb upstream regionof the PEX11 ${alpha}$ gene. To obtain the genomic DNA fragments encompassingmore upstream regions than clone D, we isolated two restrictionfragments (kb -12.5 to -7.5 and -9.5 to -1.5) from a mouse BACclone, RP23-171D12, by walking from the 5' end of clone D.

Plasmids. Genomic DNA fragments of the PEX11 ${alpha}$ gene were subcloned intothe luciferase reporter plasmid pGVP, containing a SV40 promoteror pGVB basic vector (Toyo Ink). When the genomic fragmentswere placed on the downstream side, they were inserted in arestriction site downstream of the poly(A) addition site ofthe luciferase gene. Site-directed mutagenesis was carried outusing a QuickChange (Stratagene) mutagenesis kit according tothe manufacturer's protocol. The mutant clones were tested forthe presence of the desired mutation and the absence of anyunexpected mutations by nucleotide sequencing.

pAOXPPREluc, a mouse PPAR ${alpha}$ expression vector, pNCMVPPAR ${alpha}$ , andan empty plasmid, pCMVNot, were constructed as described previously(46). Expression vectors for mouse PPAR ${gamma}$ 2 (70) and PPAR ${delta}$ (5) wereprovided by B. M. Spiegelman and P. A. Grimaldi, respectively,and the inserts were subcloned into a cloning vector, pCMX,provided by K. Umesono with the permission of R. M. Evans. PPAR ${gamma}$ 1cDNA was obtained by RT-PCR and subcloned into pCMX. The ß-galactosidaseexpression vector, pCMVß, was a gift from G. MacGregor(41).

Cell culture and DNA transfection. HeLa cells were cultured in 96-well culture plates with F-12medium containing 10% fetal bovine serum (FBS) at 37°C under5% CO₂. Transfection was carried out by the calcium phosphatemethod (59). To each well, 0.1 µg of a reporter plasmid,0.1 µg of a PPAR expression vector, and 0.175 µgof an empty vector (pCMVNot or pCMX) were cotransfected. After4 h, the calcium phosphate precipitates were removed, and thecells were cultured for 24 h in the same medium supplementedwith the ligands (100 µM Wy14,643; 1 µM BRL49,653;and 10 µM carbaprostacyclin, for PPAR ${alpha}$ , PPAR ${gamma}$ , and PPARß/ ${delta}$ ,respectively) or vehicle (dimethyl sulfoxide [DMSO]).

3T3-L1 and H4IIEC3 cells were cultured in 35-mm dishes withDulbecco's modified Eagle's medium (DMEM) containing 10% FBS.For differentiation experiments, 3T3-L1 cells were treated for2 days with a hormone cocktail containing 1 µM dexamethasone,0.5 mM 3-isobutyl-1-methylxanthine, and 5 µg of insulin/ml(57). The cocktail was then removed, and the cells were culturedfor 3 days in the same medium supplemented with 5 µg ofinsulin/ml. Transfection was performed using LipofectAMINE (Invitrogen)5 days after initiation of the cocktail treatment. Cells weremaintained for 8 h with the DNA-liposome mixture in a serum-freemedium, Opti-MEM (Invitrogen), and then cultured for 20 h inDMEM containing 10% FBS and 5 µg of insulin/ml supplementedwith 1 µM BRL49,653 or DMSO. H4IIEC3 cells were stablytransfected with the plasmids by a modified calcium phosphatemethod (47). For the transfection of linearized plasmids, reporterplasmids were cut at a unique Aor51HI site just behind the downstreamfragment of PEX11 ${alpha}$ . The linearized plasmids were purified usingNucleoSpin Extract (MACHEREY-NAGEL). HeLa cells were culturedin 35-mm dishes with F-12 medium containing 10% FBS and transfectedas described above with 4 µg of a linearized reporterplasmid, 1 µg of the PPAR ${alpha}$ expression vector, and 1 µgof pCMVß. After 6 h of transfection, the cells weresubjected to a glycerol shock with 0.5 ml of 15% glycerol inHEPES-buffered saline for 30 s. The cells were washed with F-12medium and then cultured in the same medium containing 10% FBSsupplemented with 100 µM Wy14,643 or DMSO for 24 h.

Luciferase assays. In the 96-well plates, the cells were solubilized with 20 µlof cell lysis buffer (5 mM Tris-phosphate [pH 7.8]; 2 mM dithiothreitol;1 mM ethylenediamine-N,N,N',N'-tetraacetic acid; 10% glycerol;1% Triton X-100). Luciferase activity was measured using a PicaGene(Toyo Ink) reagent kit in a Lucy2 (Anthos) microplate luminometer.After transfection was performed in 35-mm dishes, cells wereextracted with 200 µl of the cell lysis buffer, and theluciferase assay was carried out using 10, 20, and 40 µlof cell extract for the experiments involving stable transformants,linearized plasmids, and 3T3-L1 cells, respectively, with thesame reagent kit in a Lumat LB9501 (Berthold) luminometer. Theluciferase activities in the experiments using 35-mm disheswere normalized for the efficiency of transfection on the basisof ß-galactosidase activity and are presented as relativevalues. All transfection experiments were carried out in triplicate,and the averages are shown together with the standard deviations.

Electrophoretic mobility shift assay (EMSA). A double-stranded oligonucleotide composed of 5'-TCGACTTCCCTTGTCACCTTTCACCCACATCCTAGAATCC-3'and 5'-TCGAGGATTCTAGGATGTGGGTGAAAGGTGACAAGGGAAG-3' and encompassingthe downstream PPRE of PEX11 ${alpha}$ was used as a probe (Underliningindicates the sequences corresponding to PPRE throughout). Themutant sequence of PEX11 ${alpha}$ PPRE was as described below. We alsoused a double-stranded oligonucleotide consisting of 5'-CGAACGTGACCTTTGTCCTGGTCCCCTTTTGCTCC-3'and 5'-TCGGGAGCAAAAGGGGACCAGGACAAAGGTCACGT-3' and containingthe known PPRE of rat AOx as a positive control (46). A mutantAOx PPRE composed of 5'-CGAACGTGACCTTCTCGAGGGTCCCCTTTTGCTCC-3'and its complement was also used. The probes were 3' end labeledwith Klenow DNA polymerase and [ ${alpha}$ -³²P]dCTP. The assay was carriedout with fusion proteins, maltose-binding protein-PPAR ${alpha}$ , andglutathione S-transferase (GST)-RXR ${alpha}$ , as described previously(46), or with PPAR ${gamma}$ 2 and RXR ${alpha}$ synthesized in a rabbit reticulocytelysate system (Amersham). Other experimental conditions wereas previously described (46).

ChIP assay. H4IIEC3 and 3T3-L1 cells were cultured in 10-cm dishes withDMEM containing 10% FBS. H4IIEC3 was grown to 80% confluency,followed by treatment with 100 µM Wy14,643 or DMSO for2 days. 3T3-L1 was differentiated as described above and culturedfor 5 days after initiation of the cocktail treatment. Approximately1 x 10⁷ cells were then processed for chromatin immunoprecipitation(ChIP) assay using a reagent kit (Upstate Biotechnology) asrecommended by the manufacturer. Immunoprecipitation was performedwith polyclonal antibodies against PPAR ${alpha}$ , PPAR ${gamma}$ , pan-RXR, andCBP (all from Santa Cruz Biotechnology) and a preimmune rabbitimmunoglobulin G (IgG). For PCR, primer pair 5'-GAATGGCCAAGAGCCCTGCTC-3'(positions + 2282 to + 2302, relative to the first residue ofthe putative polyadenylation signal of the PEX11 ${alpha}$ gene) and 5'-GCTCTGCTGACAAAGCTGGTC-3'(+2462 to + 2482) was used for the rat PEX11 ${alpha}$ /perilipin-PPRE,primer pair 5'-GAGTGGTCAAGACCTCTGCTC-3' (+2094 to + 2114) and5'-GCTCTGCTGACAAAGCCGGTC-3' (+2265 to + 2285) was used for themouse PEX11 ${alpha}$ /perilipin-PPRE, primer pair 5'-ACCTATGCATGGATGACCACTA-3'(-1737 to -1716) and 5'-CACAGCAATTAAACAGTGAC-3' (-1543 to -1524)was used for a region distal to the rat PEX11 ${alpha}$ /perilipin-PPRE,and primer pair 5'-CTGTGCATGAGTGACCACTCG-3' (-1694 to -1674)and 5'-CTAAACAGTGACTAAGGAGTCATTA-3' (-1500 to -1476) was usedfor a region distal to the mouse PEX11 ${alpha}$ /perilipin-PPRE. A 5-µlaliquot out of the 50-µl solution of DNA recovered fromeach immunoprecipitate was used for PCR, and the products wereanalyzed on an agarose gel after 35 cycles of amplification.

RESULTS

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Results

PEX11 ${alpha}$ is a target gene of PPAR ${alpha}$ . To confirm that the PEX11 ${alpha}$ gene is a target of PPAR ${alpha}$ , we firstinvestigated the induction of PEX11 ${alpha}$ mRNA expression by Wy14,643,one of the peroxisome proliferators, using hepatic RNA fromwild-type and PPAR ${alpha}$ -null mice (36) (Fig. 1). AOx, a known targetgene of PPAR ${alpha}$ (47), was used as a reference. 36B4, a gene forribosomal protein, was examined as a control that was not affectedby peroxisome proliferators. In the wild-type mice, PEX11 ${alpha}$ expressionwas induced by the peroxisome proliferator, consistent withother reports (2, 61), but no induction was seen in the PPAR ${alpha}$ -nullmice. These results closely paralleled those for AOx. Thus,PEX11 ${alpha}$ is a bona fide target gene of PPAR ${alpha}$ .

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FIG. 1. Induction of mouse PEX11 ${alpha}$ gene expression by a peroxisome proliferator. RT-PCR was carried out using hepatic RNA of wild-type and PPAR ${alpha}$ -null mice fed with or without Wy14,643, a peroxisome proliferator. AOx, a known target gene of PPAR ${alpha}$ , was used as a positive control. 36B4 was used as a control for a gene that is not induced by Wy14,643.

A PPRE is located downstream of PEX11 ${alpha}$ . To seek the PPRE of the PEX11 ${alpha}$ gene, we isolated a DNA fragment(clone D) containing the region of mouse PEX11 ${alpha}$ from a mousegenomic ${lambda}$ phage library, using rat PEX11 ${alpha}$ cDNA as a probe. CloneD contained a 2-kb upstream region, a 5-kb region of the PEX11 ${alpha}$ structural gene consisting of three exons and two introns, anda 12-kb downstream region (Fig. 2A). In addition, two DNA fragmentscontaining regions further upstream (-12.5 to -7.5 kb and -9.5to -1.5 kb from the transcriptional initiation site, respectively)were isolated from a mouse genomic BAC clone (RP23-171D12).

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FIG. 2. Gene loci of PEX11 ${alpha}$ and perilipin. (A) Maps of the PEX11 ${alpha}$ and perilipin genes. Exons are shown by shaded boxes, together with the numbers. A DNA fragment derived from a mouse genomic library (clone D) is shown by an open box, together with the relative distance from the transcriptional initiation site of the PEX11 ${alpha}$ gene. The asterisk indicates the location of PPRE identified in the present study. The human genome map was obtained from the NCBI database (http://www.ncbi.nlm.nih.gov). (B) Sequence comparison of the downstream regions of mouse and human PEX11 ${alpha}$ genes. Matching nucleotides are shown by asterisks. The PPRE is boxed, together with arrows indicating the half sites, and a broken line following the second arrow shows the additional nucleotides required for optimal binding of PPAR. The numbers are relative to the first residue of the polyadenylation signal of the PEX11 ${alpha}$ gene. The alignment was made with the GENETYX-Mac (SDC, Tokyo, Japan) program, using the human genomic sequence of the PEX11 ${alpha}$ region (GenBank accession no. AC079075).

To search for a PPRE in the PEX11 ${alpha}$ promoter region, we constructeda luciferase reporter plasmid containing a 2.2-kb upstream region(positions -2.2 to +43), as well as several constructs containingpartial fragments of the region deleted from the 5' side stepwise.These were introduced into the HeLa human cervical cancer cellline together with a PPAR ${alpha}$ expression vector (Fig. 3A). Onlybasal levels of luciferase activity were observed with all theconstructs tested in the presence of PPAR ${alpha}$ and/or Wy14,643 comparedwith that for a positive control containing the PPRE of ratAOx, which exhibited marked activation by PPAR ${alpha}$ under these assayconditions. The promoter region of the PEX11 ${alpha}$ gene appeared tobe devoid of a functional PPRE, since we observed only a slightincrease in luciferase activity with the PEX11 ${alpha}$ constructs bythe ligand in the presence of PPAR ${alpha}$ , but a similar increase wasalso observed with the vector without a promoter sequence (Fig.3A, inset). The slight activation by PPAR ${alpha}$ and the ligand wasprobably due to a cryptic PPRE-like sequence in the vector.In fact, the luciferase vector containing only a basal promoterof SV40 (pGVP) (Fig. 3B) or the herpes simplex virus thymidinekinase gene also exhibited a similar activation by PPAR ${alpha}$ andthe ligand (data not shown).

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FIG. 3. Transfection analysis of the genomic region of mouse PEX11 ${alpha}$ . The luciferase (Luc) reporter gene constructs are depicted on the left, and the results of the reporter assay are depicted on the right. The numbers in the maps of the constructs are relative to the transcriptional initiation sites of the PEX11 ${alpha}$ gene. (A) Analysis of the region proximal to the PEX11 ${alpha}$ gene promoter. pAOXPPREluc, containing the functional PPRE and promoter region of the rat AOx gene (46), was used as a positive control. Luciferase activity is shown as a relative value, with the activity of pAOXPPREluc in the absence of both ligand and PPAR ${alpha}$ expression vector as 1. The inset shows luciferase activity of a negative control vector without a promoter sequence (pGVB). (B) Analysis of various genomic regions of the PEX11 ${alpha}$ gene. Triangles indicate the positions of boundaries of regions included in the reporter vectors. Luciferase activity is given as a value relative to the activity of pGVP in the absence of both ligand and PPAR ${alpha}$ ex-pression vector. (C) Effect of the PEX11 ${alpha}$ downstream region combined with the native promoter. Luciferase activity is shown as a value relative to the activity of pGVB in the absence of both ligand and PPAR ${alpha}$ expression vector.

To locate the functional PPRE of the PEX11 ${alpha}$ gene, luciferasevectors containing various genomic regions around PEX11 ${alpha}$ wereconstructed and introduced into HeLa cells together with a PPAR ${alpha}$ expression vector. The SV40 basal promoter was present in thesereporter vectors (Fig. 3B). Only with the reporter plasmid containingthe downstream region, kb +5.5 to +9.8, did we observe a significantdegree of ligand-dependent transactivation by PPAR ${alpha}$ . This constructwas also activated by PPAR ${gamma}$ 1 but not PPARß/ ${delta}$ (data notshown). On the other hand, there was no functional PPRE in theupstream region up to kb -12.5.

We next examined the effect of the downstream region, usinga luciferase reporter plasmid containing the PEX11 ${alpha}$ native promoter(Fig. 3C). In this plasmid, a downstream fragment (kb +3 to+9.7) was placed behind the luciferase gene, keeping the distancebetween the transcriptional initiation site and the downstreamregion as close as possible to the natural distance. We observeda ligand-dependent transactivation by PPAR ${alpha}$ with the constructcarrying the downstream region but not with that containingonly the promoter. These results indicate that the functionalPPRE of the PEX11 ${alpha}$ gene is located in the downstream region.

To determine the precise location of the PPRE, we examined aseries of deletion constructs of the downstream region startingfrom the kb +9.8 position (Fig. 4A). The downstream region couldbe deleted to kb +9 without a significant decrease in activationby PPAR ${alpha}$ . Further deletion of the sequence between kb +7.5 and+9 resulted in a marked decrease in transactivation, indicatingthat the PPRE of the PEX11 ${alpha}$ gene is located in this region. Whenthe sequence from kb +7.5 to +9 was deleted stepwise from theupstream side (Fig. 4B), the transcriptional activity droppedto basal level, between kb +8.3 and +8.5. Hence, the PPRE ofPEX11 ${alpha}$ seemed to be located within this 200-bp region. We nexttested the kb +8.3 to +8.5 fragment for activation by PPAR ${alpha}$ (Fig.4C). Activation by PPAR ${alpha}$ was indeed observed, indicating thatthis region contained a functional PPRE. Wy14,643, however,did not further promote activation by PPAR ${alpha}$ , and hence this regionby itself seemed to be insufficient for responsiveness to theperoxisome proliferator. A limited region between kb +8.5 and+9 containing several candidate motifs for transcription-factorbinding was found to confer responsiveness when combined withthe kb +8.3 to +8.5 fragment (M. Shimizu and T. Osumi, unpublisheddata).

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FIG. 4. Identification of the PPRE. The luciferase (Luc) reporter gene constructs are depicted on the left, and the results of the reporter assay are depicted on the right. The numbers in the maps of the constructs are relative to the transcriptional initiation sites of the PEX11 ${alpha}$ gene. The lowercase letters in the sequences are mutated residues. (A) Deletion analysis of the PEX11 ${alpha}$ downstream region from the 3' side. (B) Analysis of the kb +9.5 to +7.5 region by sequential deletion from the 5' side. The region was inserted in an orientation opposite to the natural one for convenience in construction. (C) Sequence-specific transactivation by PPAR ${alpha}$ conferred by the kb +8.3 to +8.5 region of PEX11 ${alpha}$ . WT and mt indicate reporter plasmids containing the wild-type and mutated forms of putative PPRE, respectively. (D) PPAR ${alpha}$ -selective transactivation of the PEX11 ${alpha}$ gene. For PPAR ${alpha}$ and PPAR ${gamma}$ 2, 100 µM Wy14,643 and 1 µM BRL49,653, respectively, were used as ligands. (E) The PPRE is sufficient for transactivation by PPAR ${alpha}$ . Double-stranded DNA fragments (WT, 5'-TCGACTTCCCTTGTCACCTTTCACCCACATCCTAGAATCC-3'; mt, 5'-TCGACTTCCCTTGTCACCTTTCGGGCACATCCTAGAATCC-3') were inserted in pGVP, upstream of the SV40 promoter. The underlined letters in the sequences correspond to the PPRE. (F) Transfection study of linearized plasmids. Plasmids were cut with a restriction enzyme, Aor51HI, at a unique site just behind the kb +9.7 position.

A PPRE-like sequence, TCACCTTTCACCC, was found in the kb +8.4downstream region. Within this motif, we mutated three basesthat closely matched the PPRE consensus and abolished activationby PPAR ${alpha}$ (Fig. 4C). Transactivation was also suppressed by thismutation in a construct composed of the native promoter anda longer downstream region of the PEX11 ${alpha}$ gene (Fig. 4D, upperpanel). Moreover, a 40-nucleotide fragment around the PPRE-likemotif was sufficient for activation by PPAR ${alpha}$ (Fig. 4E). Thus,this motif is a genuine PPRE and the primary target of PPAR-mediatedtransactivation of the PEX11 ${alpha}$ gene. Interestingly, PPAR ${gamma}$ 2 didnot support transactivation from the PPRE in this context (Fig.4D, lower panel), indicating that the function of this elementwas selective for PPAR subtypes. The constructs shown in Fig.4D were next stably transfected into a rat hepatoma cell line,H4IIEC3, which is susceptible to induction by peroxisome proliferatorsin culture (48). In these transformants, a significant inductionby Wy14,643 was observed with the wild type but not the mutantconstruct (data not shown).

We further investigated whether the PPRE indeed worked withthe PEX11 ${alpha}$ promoter when located downstream, because the PPREwas separated from the transcriptional initiation site of thePEX11 ${alpha}$ gene by more than 8 kb. In a supercoiled circular plasmid,the PPRE may act from the upstream side or a sterically closeposition, even when it is placed downstream of the reportergene. Therefore, we linearized the reporter plasmids shown inFig. 4D by cutting at a point just behind the downstream fragmentof PEX11 ${alpha}$ and transfected them into HeLa cells together witha PPAR ${alpha}$ expression vector (Fig. 4F). We again observed significanttransactivation with the wild type but not the mutant construct,thereby confirming that the PPRE worked from the downstreamside.

Perilipin gene is transactivated by PPAR ${gamma}$ 2 through the PPRE. The transcriptional initiation site of the perilipin gene islocated 1.9 kb downstream from the PEX11 ${alpha}$ -PPRE in the mouse genome(Fig. 2A). Production of perilipin is induced during adipocytedifferentiation (22), and the level of perilipin mRNA is increasedby BRL49,653, a PPAR ${gamma}$ ligand, in the differentiated adipocytes(56). Hence, we inferred that the perilipin gene is regulatedby PPAR ${gamma}$ 2 through the same PPRE. To examine this model, the regionfrom positions -2877 to +56 of the perilipin gene containingthe PPRE and native perilipin promoter was inserted into a reportervector and introduced into HeLa cells together with a PPAR ${gamma}$ 2expression vector (Fig. 5A, lower panel). A ligand-dependenttransactivation by PPAR ${gamma}$ 2 was observed, and mutation of PPREcompletely abolished this activation. This construct was activatedby PPAR ${gamma}$ 1 and marginally by PPAR ${alpha}$ (Fig. 5A, upper panel) but notat all by PPARß/ ${delta}$ (data not shown).

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FIG. 5. Transactivation of the perilipin gene through the common PPRE. The numbers in the maps of the constructs are relative to the transcriptional initiation site of the perilipin gene. Luciferase (Luc) activity is shown as a relative value, with the activity of pGVB in the absence of both a ligand and a PPAR expression vector as 1. The lowercase letters in the sequences are mutated residues. (A) PPAR ${gamma}$ 2-selective transactivation of the perilipin gene. For PPAR ${alpha}$ and PPAR ${gamma}$ 2, 100 µM Wy14,643 and 1 µM BRL49,653, respectively, were used as ligands. (B) Ligand-dependent function of the PPRE in adipocytes. Reporter plasmids were introduced into 3T3-L1 preadipocytes and adipocytes. Adipocytes were the cells treated with the hormone cocktail as described in Materials and Methods. Preadipocytes were the cells cultured for the same period as adipocytes without the hormone cocktail treatment.

To investigate whether the PPRE works in adipocytes, we introducedthese plasmids into 3T3-L1 preadipocytes and adipocytes (Fig.5B). A transactivation by BRL49,653 was observed in the differentiatedadipocytes where PPAR ${gamma}$ 2 is highly expressed, but the reporterexpression was not significantly activated in the preadipocytes.We also observed a significant luciferase expression withoutBRL49,653 in the adipocytes, possibly because of endogenousligands of PPAR ${gamma}$ , such as fatty acids. The reporter expressionwas completely abolished in the mutant construct, thereby indicatingthat the perilipin gene is transactivated by PPAR ${gamma}$ 2 through thePPRE.

PPAR/RXR heterodimer binds to the PPRE. We next investigated the binding of the PPAR ${alpha}$ /RXR ${alpha}$ heterodimerto the PPRE by EMSA, using probes encompassing the PPREs ofthe PEX11 ${alpha}$ and AOx genes (Fig. 6A). An assay was carried outusing the fusion proteins, maltose-binding protein-PPAR ${alpha}$ , andGST-RXR ${alpha}$ (46). Shifted bands were observed with both probes onlywhen both PPAR ${alpha}$ and RXR ${alpha}$ were added to the reaction (Fig. 6A,lanes 4 and 10). This binding was abolished by the wild-typebut not the mutant competitors (Fig. 6A, lanes 5 to 8 and 11to 14). These results indicate that the heterodimer PPAR ${alpha}$ /RXR ${alpha}$ directly binds to the PPRE. We also examined the binding ofPPAR ${gamma}$ 2/RXR ${alpha}$ to the PPRE by EMSA (Fig. 6B) using the proteins synthesizedin a rabbit reticulocyte lysate system. PPAR ${gamma}$ 2 and RXR ${alpha}$ , whenadded together, exhibited binding to the PPRE, but PPAR ${gamma}$ 2 orRXR ${alpha}$ alone did not (Fig. 6B, lanes 2 to 4). This binding wasabolished only by the wild-type competitors (Fig. 6B, lanes5 to 8). Thus, the PPAR ${gamma}$ 2/RXR ${alpha}$ heterodimer specifically boundto the PPRE.

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FIG. 6. Binding of the PPAR/RXR heterodimer to the PPRE in vitro. For competition experiments, unlabeled oligonucleotides in 100-fold excess were used. Sequences of probes and competitors were used as described in Materials and Methods. Competitors a, b, c, and d were wild-type and mutant PEX11 ${alpha}$ and wild-type and mutant AOx, respectively. (A) Specific PPAR ${alpha}$ /RXR ${alpha}$ binding to the PEX11 ${alpha}$ /perilipin-PPRE. EMSA was performed using MAL-PPAR ${alpha}$ and GST-RXR ${alpha}$ fusion proteins. (B) Specific binding of PPAR ${gamma}$ 2/RXR ${alpha}$ to the PPRE. PPAR ${gamma}$ 2 and RXR ${alpha}$ were synthesized in a rabbit reticulocyte lysate system.

PPAR ${alpha}$ and PPAR ${gamma}$ bind to the PPRE tissue selectively in vivo. To confirm that the PPAR ${alpha}$ /RXR or PPAR ${gamma}$ /RXR heterodimer binds tothe PPRE in vivo in a tissue-selective manner, we performeda ChIP assay. H4IIEC3 and 3T3-L1 cells were used as representativesof hepatocytes and adipocytes, respectively. H4IIEC3 derivedfrom a rat hepatoma and the rat sequence of the region at aposition corresponding to the PEX11 ${alpha}$ /perilipin-PPRE is differentfrom that of mouse (Fig. 7A). Hence, we first confirmed thatthe rat genomic sequence encompassing the putative PPRE alsoconferred the PPAR-dependent transactivation in the gene reporterassay (data not shown). In addition to the binding of PPAR ${alpha}$ ,PPAR ${gamma}$ , and RXR, recruitment of CREB-binding protein (CBP), arepresentative coactivator of nuclear hormone receptor, wasalso examined as an indicator of transactivation.

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FIG. 7. Tissue-selective binding of PPAR ${alpha}$ and PPAR ${gamma}$ to the PPRE in vivo. (A) Schematic diagram of the region around the PEX11 ${alpha}$ /perilipin-PPRE in the genomes of mouse and rat. Arrows indicate the regions amplified by PCR in the ChIP assay. (B) Selective binding of PPAR ${alpha}$ to the PPRE in the liver. H4IIEC3 cells were treated with 100 µM Wy14,643 (Wy) or DMSO (DM) for 2 days and processed for the ChIP assay. Immunoprecipitation was performed with the antibodies indicated or preimmune rabbit IgG. (C) PPAR ${gamma}$ selectively binds to the PPRE in the adipose tissue. The ChIP assay wasperformed on the 3T3-L1 preadipocytes (Pre) and adipocytes (Ad).

In H4IIEC3 cells, we observed significant bands for PPAR ${alpha}$ , RXR,and CBP but not for PPAR ${gamma}$ with the PPRE primer pair (Fig. 7B).These bands were negligible either with the distal region primersor for a control IgG. No dependence on the PPAR ${alpha}$ ligand was observedeven for CBP, possibly reflecting the apparently ligand-independenttransactivation by PPAR ${alpha}$ often observed in the culture system.In 3T3-L1 cells, strong bands were observed for PPAR ${gamma}$ , RXR, andCBP but not for PPAR ${alpha}$ with the PPRE, and only in the differentiatedadipocytes were strong bands not observed with the distal primers(Fig. 7C). These results support the view that PPAR ${alpha}$ and PPAR ${gamma}$ selectively bind to the PPRE in the liver and the adipose tissueand activate the transcription of PEX11 ${alpha}$ and perilipin genes,respectively.

The PEX11 ${alpha}$ /perilipin-PPRE is conserved in the human genome. We compared the nucleotide sequences around the PPRE in themouse and human genomes. In the human genome database, we founda PPRE-like sequence in the downstream region of the human PEX11 ${alpha}$ gene (Fig. 2B). This sequence matched the PPRE of the mousePEX11 ${alpha}$ gene except for an optional base between the two halfsites. Furthermore, not only the PPRE but also the sequencesaround it were highly conserved between the two species. Hence,the human PEX11 ${alpha}$ gene was also suspected to be transactivatedby PPAR ${alpha}$ . We examined whether expression of the human PEX11 ${alpha}$ wasinduced by peroxisome proliferators, using human hepatoma, HepG2.Cells were cultured in the presence or absence of ciprofibrate,a peroxisome proliferator, and the expression of endogenousPEX11 ${alpha}$ was monitored by RT-PCR. The expression was not increasedby the drug (data not shown), though under the same conditions,carnitine palmitoyl transferase 1 and mitochondrial HMG-CoAsynthetase were induced, in accordance with reports by otherinvestigators (27, 34). Peroxisome numbers are not significantlyincreased by peroxisome proliferators in human liver (40). Theresponse of the PEX11 ${alpha}$ gene to peroxisome proliferators is probablyrestricted by a unique mechanism in human liver.

DISCUSSION

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Discussion

We found a functional PPRE located downstream of the mouse PEX11 ${alpha}$ gene, 8.4 kb from the promoter. On the other hand, we failedto observe PPAR-dependent transactivation with the upstreamregion up to kb -12.5 as well as the region of the PEX11 ${alpha}$ structuralgene that includes the introns. Using a linearized reporterplasmid, we observed transactivation by PPAR ${alpha}$ via the downstreamPPRE, thereby indicating that the PPRE indeed functioned fromthe downstream location at this distance. The PPRE identifiedis located in the vicinity of the perilipin gene and supportedthe activation of transcription from the perilipin promoter.We also found that the PEX11 ${alpha}$ gene was selectively transactivatedby PPAR ${alpha}$ and that the perilipin gene was transactivated by PPAR ${gamma}$ through this common PPRE. Consistently, the expression of PEX11 ${alpha}$ is induced in the liver and the expression of perilipin is inducedin the adipose tissue, where PPAR ${alpha}$ and PPAR ${gamma}$ 2 are, respectively,selectively expressed. In the ChIP assay, endogenous PPAR ${alpha}$ andPPAR ${gamma}$ indeed bound selectively to the PPRE in hepatocytes andadipocytes, respectively. Based on these observations, we proposea mechanism of tissue-selective regulation of PEX11 ${alpha}$ and perilipin(Fig. 8). The PPRE functions as a cis element for PEX11 ${alpha}$ geneactivation by PPAR ${alpha}$ in the liver and for perilipin gene expressionby PPAR ${gamma}$ in the adipose tissue. We observed transactivation selectivefor PPAR subtypes only with the natural PEX11 ${alpha}$ and perilipinpromoters. When the SV40 promoter was used, the PPRE workedwith both PPAR ${alpha}$ and PPAR ${gamma}$ (data not shown). Thus, the combinationwith specific promoters seems to determine the selectivity forPPAR subtypes. Transcription machineries acting on differentpromoters are not always the same, and tissue-specific promoter-bindingfactors have been described (25). The selectivity of PEX11 ${alpha}$ -perilipinPPRE to PPAR subtypes might be determined by the combinatorialinteractions between PPAR subtypes and promoter-binding factors,probably also involving coactivators.

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FIG. 8. Models of the tissue-selective regulation of PEX11 ${alpha}$ and perilipin genes. These genes are regulated through the common PPRE in a tissue-selective manner. In the liver, PPAR ${alpha}$ is highly expressed; hence, it initiates the activation of the PEX11 ${alpha}$ gene. On the other hand, PPAR ${gamma}$ 2 is limited to adipose tissue, working for perilipin gene induction.

Most PPREs reported to date (14) are located proximally to thetranscriptional initiation site. The location of the PEX11 ${alpha}$ PPRE,however, is not necessarily exceptional: a few PPREs have beenfound in distal positions, e.g., 4 to 5 kb upstream in the adipocyteP2 (70) and CYP4A1 genes (4), in an intron in the acyl-CoA-bindingprotein gene (23), and in the downstream region in the apolipoproteinE gene (20). Enhancers often function irrespective of positionrelative to the promoter and distance from the transcriptionalinitiation site (6). In fact, the wing margin enhancer of theDrosophila cut locus (30) and the T-cell receptor C ${alpha}$ gene enhancer(75) act from positions more than 50 kb upstream, and the ß-globinenhancer is functional in the downstream region (16, 17). Thedistance- and position-independent functions of enhancers havebeen attributed to the physical communication between the promotersand enhancers, bridged by transcription factor complexes (9).Evidence for such physical interactions has been emerging recently(11, 67).

The bidirectional function of a single PPRE is exceptional.Although tissue-selective activation by PPAR ${alpha}$ and PPAR ${gamma}$ was reportedfor the lipoprotein lipase gene, this activation involves onlya single gene (60). On the other hand, there are several precedentsfor bidirectional enhancers. In most of these cases, such aschicken ß-globin- ${varepsilon}$ -globin (44) and albumin- ${alpha}$ -fetoprotein(21) gene pairs, the target genes are related to each other,structurally and functionally, reflecting their originationby gene duplication. The functions of PEX11 ${alpha}$ and perilipin, however,are different, and their amino acid sequences are not related.The function of PEX11 remains obscure, made more confusing bya report that its primary function is in fatty acid oxidation,not peroxisomal division (73). In any case, PEX11 ${alpha}$ is expressedmainly in the liver, where lipid metabolism is one importantfunction, and is located on the membrane of peroxisomes, organellesinvolved in fatty acid catabolism. On the other hand, the expressionof perilipin is limited to the adipose tissue where lipid isstored. Based on these observations, the functions of PEX11 ${alpha}$ and perilipin would be the same in the area of lipid mobilization,and hence it would be reasonable that these genes are regulatedby partially overlapping mechanisms.

It has generally been postulated that an enhancer serves a singlegene. Recent genome analysis, however, revealed that many genesare located very close together, sometimes even sharing promoters(3). Enhancers as well as individual enhancer elements can activatetarget promoters located several kilobases apart. Hence, thefunctional distal enhancer element of a gene may be even closerto another gene. Based on these considerations, we expect thateven more gene pairs will be revealed in the future to be regulatedby common bidirectional enhancer elements. In such cases, combinationswith promoters and other proximal elements may confer regulatoryspecificity to each gene, as found in the present study of thePEX11 ${alpha}$ -perilipin gene pair.

The proliferative effect of hypolipidemic agents on peroxisomesin rodent liver was first observed in the 1960s (24). However,the protein that promotes peroxisomal division in response tothese agents is as yet unknown. One of the likely candidatesseems to be PEX11 ${alpha}$ , though peroxisomal metabolic activities werealso shown to affect the number of this organelle (12, 52, 54,73). In this study, we showed that the PEX11 ${alpha}$ gene is a bonafide target of PPAR ${alpha}$ and identified its functional PPRE. Theexpression of no other PEX gene has so far been reported tobe induced by peroxisome proliferators. Thus, it is temptingto speculate that the proliferation evoked by these agents isdirectly caused by PEX11 ${alpha}$ . It was reported, however, that peroxisomeproliferation is observed in PEX11 ${alpha}$ -null mice (37). In addition,a dynamin-related protein has recently been reported to be involvedin peroxisomal division (26, 33, 38). Future studies will berequired in order to determine which gene is the key factorin peroxisome proliferation.

ACKNOWLEDGMENTS

We thank B. M. Spiegelman, G. MacGregor, P. A. Grimaldi, thelate K. Umesono, and R. M. Evans for the plasmids. We also thankY. Emi for a mouse genomic library.

This work was supported in part by the 21st Century COE Programand a Grand-in-Aid for Scientific Research from the Japan Societyfor the Promotion of Science.

FOOTNOTES

* Corresponding author. Mailing address: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kamigori, Hyogo 678-1297, Japan. Phone: 81-791-58-0192. Fax: 81-791-58-0193. E-mail: osumi{at}sci.himeji-tech.ac.jp.

Present address: Genomics Research Institute, Utsunomiya University,Utsunomiya, Tochigi 321-8505, Japan.

REFERENCES

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