The peroxisome proliferator activated receptors (PPARs) are members of the nuclear hormone receptor gene superfamily of ligandactivated transcription factors [Issemann and Green, 1990]

Chew, Choy Hoong

Najimudin, Nazalan

Tengku-Muhammad, Tengku Sifzizul

English

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The 4th Annual Seminar a/National Science Fellowship 2004[BI019] Molecular cloning and characterisation of the 5 -untranslated region andpromoters in human peroxisome proliferator-activated receptor alpha (hPPARa.)Chew Choy Hoong, Nazalan Najimudin and Tengku Sifzizul Tengku-Muhammad :J-g.1 %School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, PenangIntroductionThe peroxisome proliferator activatedreceptors (PPARs) are members of the nuclearhormone receptor gene superfamily of ligand-activated transcription factors [Issemann andGreen, 1990]. So far, three distinct subtypes ofPPARs have been identified, PPARa, PPARyand PPARp/8, with each subtype encoded bya separate gene and has a specific tissuedistribution [lssemann and Green, 1990;Braissant et aI, 1996]. The PPARa, the firstisoform to be identified, is expressed mainlyin tissues that have high ratio of fatty acidoxidation such as liver, heart and kidney[Issemann and Green, 1990; Braissant et al.,1996]. PPARa has attracted considerableattention since it was demonstrated to beregulators of lipoprotein metabolism [Staels etal., 1998], vascular inflammation [Chinetti etal., 2000], atherosclerosis [Chinetti et al.,2000; Pineda-Torra et ai, 2002] andcarcinogenesis [Neve et al., 2000; Collett etal., 2000]. Moreover, recent studies suggestedimpoliant roles for PPARa in skin woundhealing, and epidermal maturation and repair[Wahli,2002].In order to understand the structure andmolecular mechanisms governing hPPARaregulation, we carried out experiments toidentify, and characterise the 5 -untranslatedregion (UTR) and promoter regions of thehPPARa gene. In this study, we managed toidentify six differentially spliced variants inthe 5 -untranslated region (UTR) of humanPPARa gene, due to four di fferent promoterusages and alternative splicing [Chew et al.,2003]. Since promoter A has beencharacterised [Pineda-Torra et al., 2002], threeother promoters designated as promoter B, Cand D were cloned, sequenced and theirpromoter activities analysed. By carrying outtransient transfection analysis in humanhepatocarcinoma HepG2 cells, we provedthese three promoters are functionalpromoters, capable of inducing the basalluciferase gene expression, with promoter B96being the most potent and strongest promoterof the three promoters.Materials and MethodsIsolation oftotal cellular RNATotal cellular RNA was isolated from thehuman hepatoma HepG2 cell line using Tri-Reagent LS (Molecular Research Center)according to the manufacturer's instruction.The concentration of each total cellular RNAwas then quantified using GeneQuant(Amersham Pharmacia Biotech).Rapid amplification of cDNA ends (5 RLM-RACE) and sequencing ofRACE products5 RLM-RACE was performed on 5 Ilgtotal RNA using the GeneRacer™ Kit(Invitrogen@ Life Technologies) according tothe manufacturer's instruction. The first strandcDNA was generated using primers suppliedby the manufacturer and subjected to two PCRreactions. Specifically, the synthesised cDNAwas used as the template in the amplificationprocess using the internal gene-specific primerGSP I (5 -GCAGGAACTCTTCAGATAACGG-3), located atthe 5 end hPPARa exon 3 and theGeneRacer™ 5 Primer. PCR cyclingparameters were as follows: initialdenaturation at 94°C for 3 min followed by 30cycles of I min at 94°C, I min at 65°C, 1 minat 60°C, and I min at noc with a finalelongation at noc for 10 min. The primaryPCR mix was then subjected to a nested PCRunder the same conditions with theGeneRacer™ 5 Nested Primer and GSP2 (5 -GCAGAGTGGGCTTTCCGTGT-3 )designed immediately upstream of GSP 1.Oligonucleotides GeneRacer™ 5 Primer andGeneRacer™ 5 Nested Primer were providedin the GeneRacer™ Kit. The PCR productswere size fractionated on 1.5% (w/v) agarosegel and selected bands were then gel purifiedusing QIAquick gel extraction kit (QIAGEN).The purified bands were directly ligated intoThe 4th Annual Seminar ofNational Science Fell01l'ship 2004pGEM-T Easy vector (Promega) andtransformed into JMI09. Recombinantplasmids were then isolated and purified forDNA sequencing using Plasmid Midi Kit(QIAGEN). Sequencing reactions werecarried out using the ASI PRlSM@ BigDye™Terminator Cycle Sequencing Ready ReactionKit (PE Applied Biosystem).Cloning and construction of reporter-plasmidsThe promoter regions were amplified fromhuman genomic DNA as template withPlatinum® Pfx DNA polymerase (Invitrogen®Life Technologies) to minimise PCRgenerated mutations. These products werethen used as template for generating promoterfragments which represent different regions ofthese three promoters. The promoters'fragments with different sizes were amplifiedfrom the cloned promoter as template usingprimers containing XhoI and HindIIl. tofacilitate the cloning into the pGL3-Basicvector (Promega) in the correct orientation.For promoter B, reporter-promoter plasmids,pGL3alphBI (carrying regions -341/+34),pGL3alphB2 (-765/+34) and pGL3alphB3 (-1147/+34), were constructed. For promoter C,reporter-promoter constructs were cloned andnamed pGL3alphC 1 (carrying regions -413/+41), pGL3alphC2 (-967/+41),pGL3alphC3 (-1364/+41), while in promoterD, reporter-promoter plasmids designatedpGL3alphDI (-395/+28), pGL3alphD2 (-672/+28) and pGL3alphD3 (-934/+28) wereconstructed.Cell culture and transient transfection assaysHuman hepatoma HepG2 cell line weregrown in Eagle's Minimum essential medi.umwith Earle's BSS medium (Gibco)supplemented with 10% (v/v) fetal bovineserum, 2mM L-glutamine, 100 U/ml penicillinand 100 Ilg/m I streptomycin (Hyclone). Thecultures were maintained at 37°C in ahumidified atmosphere containing 5% (v/v)CO2 in air. The cells were transfected withindividual promoter fragment, together withpRL-TK containing Renilta luciferase(Promega) which served as internal control,using Lipofectin transfection reagent(Invitrogen® Life Technologies) according tothe manufacturer's instructions for 36 hI's. Theluciferase activities in the cell extracts werethen determined using the Dual-Luciferase97Reporter Assay System (Promega). The fireflyluciferase activity was normalised to theRenilta luciferase activity, with eachtransfection being carried out in triplicate andmeasurements were repeated at least twice.Results and DiscussionIdentification of Alternatively SplicedVariants ofthe 5 UTR ofhPPARa geneUsing RLM-RACE PCR approach, sixRACE-PCR products of different sizes(100bp, 200bp, 230bp, 300bp, 330bp and600bp) were successfully amplified (figurenot shown). Sequencing analysis showed thatthe 3 end of all RACE-PCR products shared100% identity with the 5 end of Exon 3 ofhuman PPARo: gene, where the gene-specificprimers were designed, indicating that allRACE-PCR products were specific andrepresent the 5 -UTR of the human PPARo:.Interestingly, sequence analysis amongst thesix RACE-PCR products revealed differentcombination of exons which strongly indicatesthe existence of 6 differentially splicedvariants in the 5 -UTR of the hPPARo: gene,designated as hPPARo: I, hPPARo:2,hPPARo:3, hPPARo:4, hPPARo:5 andhPPARo:6 (Figure IA) [Chew et aI., 2003].In addition to these six alternativelyspliced variants, we have also successfullyidentified three new novel exons of the humanPPARo: gene, designated Exon A, Exon BandExon 2b (Figure IB) [Chew et aI., 2003].The presence of alternatively splicedvariants in human PPARo: mRNA is acommon phenomenon amongst PPARisoforms. For example, four variants resultingfrom four different promoters usages andalternative splicing at the 5 -UTRs have beenidentified in human PPARy mRNA [Fajas etaI., 1997, Fajas et aI., 1998, Sundvold andLien, 2001], while seven alternatively splicedvariants were detected in monkey PPARymRNA [Zhou et aI., 2002]. In addition, fourdifferentially spliced variants induced by fourdifferential promoter usages were alsodetected in mouse PPAR~ mRNA [Larsen etaI., 2002]. Meanwhile, in porcine PPARo:mRNA, two alternatively spliced mRNA werefound in the coding region of PPARo:[Sundvold et aI., 2001].Promoter DThe 4th Annual Seminar ofNational Science FellolVship 2004(A)hPPARcd I Exon 3 IhPPARa2 I Exon 2a I Exon 3 IhPPARa3 I Exon A I Exon la I Exon 3hPPARa4 I ExonB I Exon 3 IhPPARa5 I Exon A I Exon la I Exon 2a Exon 3 IhPPARa6 I ExonB Exon Ib I Exon 2a Exon 2b I Exon 3 I(8)Promoter A Promoter B Promoter C"rt~" A E,," " "f, E,," B E"" Ih ,~~~" 2, "" 2b , t.,_ r+ ATe(-~,,~,: f- --~....--'--) -1~~f--r:1r-';---,,..: -'----II -1.2 kb I I -20 kb I -5 kb I I -0.6 kb II -II kb II -9.5 kbFIGURE 1 (A) The six alternatively spliced variants identified from this study. Alternative splicingmechanisms and different promoters usage give rise to variants with unique combinations of exons. (B)Schematic representation of the 5 -UTR ofhPPARo: gene. The new exons identified are indicated as shadedboxes. Numbers in bold and in boxes represents the sizes of the introns which span the exons. Arrowsindicate the presence of promoter in front of Exon A, Exon B, Exon 2a and Exon 3.Determination ojtranscriptional start sitesRLM-RACE method allows only fulllength 5 -capped transcripts to be amplifiedvia the elimination of truncated mRNA,thereby rendering it a safe, reliable andaccurate method for mapping of the 5 ends oftranscripts and transcriptional start sites ofgenes [Maruyama et al., 1994, Volloch etal.,1994]. The transcriptional start sites of Exon 3and Exon 2a were situated 31 bp and 124 bpupstream of the ATG initiation stalt codon inthe processed mRNA, respectively, whileexons A has two different transcriptional staltsites, each individually marks the stalt sitevariants hPPARa3 and hPPARa5. Thetranscriptional stalt site of hPPARa3 andhPPARa5 are situated 172 bp and 264 bpupstream of the ATG initiation stalt codon,respectively; with the hPPARa598transcriptional stalt site located 8 nucleotidesupstream of the transcriptional start site ofvariant PPARa3 in Exon A. Meanwhile, ExonB also has two transcriptional stalt sites(variant PPARa4 and variant PPAR(6)mapped to the positions 251 bp and 540 bpupstream of their respective ATG start codon.The transcriptional stalt site for PPARa4 is 48bp upstream of the transcriptional start site inPPARa6 [Chew et al., 2003].Multiple transcriptional start sites withinthe same promoter region are in fact acommon phenomenon and frequentlyobserved PPAR family, i.e. in the mousePPARa and PPARp gene [Larsen et al., 2002,Gearing et al., 1994].The 4th Annual Seminar ofNational Science Fellowship 2004Cloning and analysis of the promoter regionsB, CandDWe determined the transcriptional startsites for each variant of the human PPARo:gene, which suggested the presence of fourpromoters responsible for transcribing thesevariants. Since promoter A was identified andcharacterised (Pineda-Torra et al., 2002),promoters B, C and D were chosen to befurther characterised. Direct PCRamplifications using human genomic DNA astemplate and gene-specific primers werecarried out to amplify these putative promoterregions. Products with sizes of 1.2 kb(promoter B), 1.5 kb (promoter C) and 1.0 kb(promoter D) were successfully amplified.A computer search of the putativepromoter regions using the Matisnspectorprogramme in TRANSFAC database(http://transfac.gbf.de/TRANSFAC/index.html) revealed theexistence of multiple potential binding sitesfor transcription factors that may regulate theexpression of the human PPARo: gene.Binding sites for transcription factors such asOct-I, AP-I, Spl, C/EBP, STAT, GATA-I,GATA-2, HNF-3~ and RORo:I wereidentified within the promoters B, C and Dregions of human PPARo: gene (figures notshown).Characterisation and analysis of promotersB, C and D activitiesThe relative strength of the differentpromoters and different fragments of thepromoters was compared by transienttransfection in Hep3B and HepG2 cells, usingvarious fragments of promoters' sequences.Fragments containing regions -341/+34(pGL3alphB I), -765/+34 (pGL3alphB2) and -1147/+34 (pGL3alphB3) of promoter B,fragments bearing the regions -413/+41(pGL3alphC I), -967/+41 (pGL3alphC2) and-1364/+41 (pGL3alphC3) of promoter C andfragments -395/+28 (pGL3alphDI), -672/+28(pGL3alphD2) and -934/+28 (pGL3alphD3)of promoter D were cotransfected into thecells with pRL-TK plasmid which served asan internal control for transfection efficiency.Results obtained from this study proved thatall three promoters B, C and D are functionalpromoters capable of driving the basaltranscription of the luciferase gene.99Promoter activities for promoter B werethe highest in HepG2 cells compared topromoter C and D, thereby indicating thatpromoter B is the highest promoter of thethree, followed by promoter D being astronger promoter than promoter C (Figure 2).Overall, the smallest promoter fragmentsof the three promoters, pGL3alphB I (-341/+34), pGL3alphCI (-413/+41) andpGL3alphDI (-395/+28), proved to have thestrongest activity compared to their respectivefragments in HepG2 cells (Figure 2). Thiscould indicate the absence of elementsconferring transcriptional repression upstreamof the stalt sites, or alternatively, the presenceof an element enhancing transcription betweenpositions -341 to +34 of promoter B, positions-413 to +41 of promoter C, and positions -395to +28 of promoter D, or a combination ofboth.The 4th Annual Seminar ofNational Science Fellowship 2004--------III.... 60'"'"'"<:~ 50'"...~'v" 40...l'"::::.~ 30Cl:::;.:,l;:~ 20ii:::l~ 10<:>'l:~ O+-__"--~-'--.L-~-"---"-~-"--'-~........<..L-_._.........,LL.._._..........LL-~v ~ ~>:- ~>:- ..v. -$' ..v. ,'i>I ~'i>I ~'i>I ~,<~u \X",4x \:o\X \x ~\x "'~x \X' "",'?' \X Q'"..' ;.,,, !'Ie, :-,b'.f~"; ~"\ ~" ~'" ," ~., J> ~'"~' :<:0<0'" ....'0.:. (} ~ ~'Q' :<:0"1'" ~., ,,"!O" :<:0" :<:0"" ~~~ ~<>. ~~ ~~ ~~ 'S~~ ~~ ~~ ~~v., V v., v., V v., V V~u QU QU QU QU ,," QU ~u ~u~Promotcr-Lucifcrase DNA ConstructsFIGURE 2 Analysis of promoters B, C and D activities using transient transfection assays. Thepromoter fragments of promoters B, C and D were fused into pGL3 basic vector and co-transfected withpRL-TK into HepG2. Values (mean ± SD) represent firefly luciferase activity normalised to a Renillaluciferase activity which served as internal control. Luciferase activities are shown relative to the activity ofthe pGL3 basic vector. .ConclusionsSix variants, named hPPARa1, hPPARa2,hPPARa3, hPPARa4, hPPARa5 andhPPARa6, were successfully identified at the5'-UTR of human PPARa mRNA as a resultof alternatively splicing and the use of fourdifferent promoters. Each of thetranscriptional stm1 sites of the variant wasdetermined and mapped, leading to thediscovery of four promoters in the humanPPARa gene which are responsible fortranscribing these alternatively splicedvariants. In addition, three new novel exonsdesignated Exon A, Exon Band Exon 2b wereidentified in this study. Three new novelhuman PPARa promoters were also clonedand characterised. Demonstrated by transienttransfection assay, these putative promoters B,C and D were all proven to be functionalpromoters, with promoter B being thestrongest of the three promoters.(MOSTE). This work was supported by theUSM Sh0l1 term grant.ReferencesBraissant, 0., Foufelle, F., Scotto, C., Dauca, M.,Wahli, W. (1996). Differential expression ofperoxisome proliferator activated receptors: tissuedistribution of PPARa, ~ and y in the adult rat.Endocrinology 137, 354-366.Chew C.H., Samian M.R, Najimudin N., Tengku-Muhammad T.S. (2003). MolecularCharacterisation of Six Alternatively SplicedVariants and a Novel Promoter in HumanPeroxisome Proliferator-Activated Receptor Alpha(PPARo:). Biochem. Biophys. Res. Coml11un. 305,235-243.Chinetti, G., Fruchart, lC., Staels, B. (2000).Peroxisome proliferator-activated receptors(PPARs): Nuclear receptors at the crossroadsbet\:veen lipid metabolism and inflammation.Injlal11l11 res. 49; 497-505.AcknowledgmentCCH acknowledged financial SUpp0l1 fromNational Science Fellowship (NSF), Ministryof Science, Technology and the EnvironmentCollett, G.P., Betts, A.M., Johnson, M.L,Pulimood, A.B., Cook, S., Neal, DE., Robson,C.N. (2000). Peroxisome proliferator-activatedreceptor 0: is an andogen-responsive gene in100The 4th Annual Seminar ofNational Science Fellowship 2004human prostate and is highly expressed in prostaticadenocarcinoma. Clin. Cancer Res. 6, 3241-3248.Fajas, L., Auboeuf, D., Raspe, E., Schoonjans, K.,Lefebrve, A.M., Saladin, R., Najib, J., Laville, M.,Fruchart, J.C, Deeb, S., Vidal-Puig, A., Flier, J.,Briggs, M.R., Staels, B., Vidal, H., Auwerx, J.(1997). The organization, promoter analysis andexpression of the human PPARy gene. J. 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Roleof peroxisome proliferator-activated receptors10/(PPAR) in atherosclerosis. Biochem. Pharmcol. 60,1245-1250. !Pineda-Torra, I., Jamshidi, Y., Flavell, D.M.,Fruchart, J.C, Staels, B. (2002). Characterizationof the human PPARo: promoter: identification of afunctional nuclear receptor response element. Mol.Endocrinol. 16,1013-1028.Staels B., Dallongeville J., Auwerx J., SchoonjansK., Leitersdorf E., Fruchart J.C (1998).Mechanism of action of fibrates on lipid andlipoprotein metabolism. Circulation 98, 2088-2093.Sundvold, H., Grindflek, E., Lien, S. (2001).Tissue distribution of porcine peroxisomeproliferator-activated receptor 0:: detection of analternatively spliced mRNA. Gene 273,105-113.Sundvold, H., Lien, S. (2001). Identification of anovel peroxisome proliferator activated receptor(PPAR) y promoter in man and transactivation bythe nuclear receptor RORo: 1. Biochem. Biophys.Res. Coml17un. 287,383-390.Volloch, V., Schweitzer, B., Rits, S. (1994).Ligation-mediated amplification of RNA frommurine erythroid cells reveals a novel class ofbeta-globin mRNA with an extended 5-untranslated region. Nucleic Acids Res. 22, 2507-2511.Wahli W. (2002), Peroxisome proliferatoractivated receptors (PPARs): from metaboliccontrol to epidermal wound healing. Swiss MedWkly. 132,83-91.

[BI019] Molecular Cloning And Characterisation Of The 5 -Untranslated Region And Promoters In Human Peroxisome Proliferator-Activated Receptor Alpha (hPPARα).

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[BI019] Molecular Cloning And Characterisation Of The 5 -Untranslated Region And Promoters In Human Peroxisome Proliferator-Activated Receptor Alpha (hPPARα).

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