Molecular characterisation of six alternatively spliced variants1 and a novel promoter2 in human peroxisome proliferator-activated receptor α

BBRC Biochemical and Biophysical Research Communications 305 (2003) 235–243 www.elsevier.com/locate/ybbrc

Molecular characterisation of six alternatively spliced variants1 and a novel promoter2 in human peroxisome proliferator-activated receptor a Choy-Hoong Chew, Mohd Razip Samian, Nazalan Najimudin, and Tengku Sifzizul Tengku-Muhammad* School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia Received 11 April 2003

Abstract Peroxisome proliferator-activated receptor a (PPARaÞ is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 50 untranslated region (UTR) of hPPARa gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 50 -untranslated region of human PPARa gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2 kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARa4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment )341/+34, but lower in the region )341/)1147 as compared to the fragment )341/+34, indicating the presence of an element conferring transcriptional activation between positions )341 and +34 or alternatively, the presence of transcriptional repression between positions )341 and )1147 in the promoter B of hPPARa. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: PPARa; Variants; Promoter; Alternative splicing; Exons

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor gene superfamily of ligand-activated transcription factors [1]. So far, three distinct subtypes of PPARs have been identified, PPARa, PPARc, and PPARb=d, with each subtype encoded by a separate gene and having a specific tissue distribution [1,2]. The PPARa, the first isoform to be identified, is expressed mainly in tissues that have high ratio of fatty acid oxidation such as liver, heart, and kidney. PPARc, highly expressed in adipose tissue, is a potent regulator of lipid metabolism and *

Corresponding author. Fax: +604-656-5125. E-mail address: [email protected] (T.S. Tengku-Muhammad). 1 Sequences deposited into GenBank Accession No. AY258326– AY258331. 2 Sequences deposited into GenBank Accession No. AY258332.

adipocyte differentiation and PPARb=d is found ubiquitously in various tissues, including brain [1,2]. Each isoform also shows ligand selectivity. Natural (leukotrine B4) or synthetic (fibrate Wy 14,643) specific ligands have been identified for PPARa [3]. In addition, fatty acids, peroxisome proliferators, and fibrate hypolipidemic drugs also specifically activate PPARa [4]. In contrast, 15-deoxy-12,14-prostaglandin J2 and thiazolidinedione antidiabetic agents are selective ligands for PPARc [5,6]. The PPAR possesses three major structural domains common to other members of the nuclear receptor gene family; the NH3 -terminal domain, the DNA-binding domain (DBD), and the ligand-binding domain (LBD). The DBD is very highly conserved amongst the PPAR isoforms and consists of two zinc fingers that facilitate binding to the cis-acting elements. Lower level of

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00731-9

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conservation is seen in the LBD which explains for ligand selectivity and binding specificity of the PPAR subtypes [7]. The NH3 -terminal of the isoforms shows low sequence identity and is thought to be responsible for differences in the biological functions of the subtypes [8]. To date, at least nine exons have been identified in the hPPARa gene and the open reading frame consists of the 30 end of Exon 3, Exons 4–7, and the 50 end of Exon 8 [9]. Currently, four exons have been identified in the 50 -UTR of hPPARa namely Exon 1a, Exon 1b, Exon 2, and the 50 end of Exon 3 [10]. The same group has also identified the presence of 2 alternatively spliced variants at the 50 -UTR, indicating the existence of at least two different variants of hPPARa. PPARa has attracted considerable attention since it was demonstrated to be regulator of lipoprotein metabolism [11], vascular inflammation [12], atherosclerosis [13,14], and carcinogenesis [15,16]. Moreover, recent studies suggested important roles for PPARa in skin wound healing, and epidermal maturation and repair [17–21]. PPARa plays a pivotal role in the development of diseases, and, cell differentiation and maturation by regulating the target genes in a manner very much similar to other nuclear receptors. In the absence of ligands, PPARa exists as monomers and upon ligand activation, PPARa forms a heterodimeric complex with the retinoic X receptor (RXR) and binds to a specific sequence in the 50 region of the target genes, known as the peroxisome proliferator response element (PPRE), thereby regulating the transcription of genes [1]. In order to understand the physiological role of PPARa, it is crucial that we gain insights into the regulation of PPARa gene expression in human. However, studies on the regulation of PPARa gene expression have been overlooked so far, until recently, new Exon 1b and 2 spliced variants of 50 -UTR of hPPARa were identified [10]. In this study, further characterisation of the 50 -flanking region of human PPARa gene was successfully carried out. We, hereby, report the presence of six differentially spliced variants in the 50 -untranslated region (UTR) of hPPARa gene due to different promoter usages and alternative splicing. The transcriptional start sites for each variant and another three new novel exons, namely Exon A, Exon B, and Exon 2b, were also mapped and a new functional promoter was cloned, sequenced, and characterised.

Materials and methods Isolation of total cellular RNA. Total cellular RNA was isolated from the human hepatoma HepG2 cell line using Tri-Reagent LS (Molecular Research Center) according to the manufacturerÕs instructions. The concentration of each total cellular RNA was then quantified using GeneQuant (Amersham Pharmacia Biotech). Rapid amplification of cDNA ends (50 RLM-RACE) and sequencing of RACE products. 50 RLM-RACE was performed on 5 lg total RNA

using the GeneRacer Kit (Invitrogen Life Technologies) according to the manufacturerÕs instruction. The first strand cDNA was generated using primers supplied by the manufacturer and subjected to two PCRs. Specifically, the synthesised cDNA (1 ll) was amplified using the internal gene-specific primer GSP1 (50 -GCAGGAACTCTTCA GATAACGG-30 ), located at the 50 end hPPARa Exon 3 and the GeneRacer 50 Primer. PCR cycling parameters were as follows: initial denaturation at 94 °C for 3 min followed by 30 cycles of 1 min at 94 °C, 1 min at 65 °C, 1 min at 60 °C, and 1 min at 72 °C with a final elongation at 72 °C for 10 min. The primary PCR mix was then subjected to a nested PCR under the same conditions with the GeneRacer 50 Nested Primer and GSP2 (50 -GCAGAGTGGGCTTTCCGTGT-30 ) designed immediately upstream of GSP1. Oligonucleotides GeneRacer 50 Primer and GeneRacer 50 Nested Primer were provided in the GeneRacer Kit. The PCR products were size fractionated on 1.5% (w/v) agarose gel and selected bands were then gel purified using QIAquick gel extraction kit (QIAGEN). The purified bands were directly ligated into pGEM-T Easy vector (Promega) and transformed into JM109. Recombinant plasmids were then isolated and purified for DNA sequencing using Plasmid Midi Kit (QIAGEN). Sequencing reactions were carried out using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystem). Cloning of the human PPARa promoter and construction of reporter plasmids. The promoter sequence of hPPARa variant was amplified from human genomic DNA as template with Platinum Pfx DNA polymerase (Invitrogen Life Technologies) to minimise PCR generated mutations, using oligonucleotides GSP3 (50 -TGTTCTGCAAC CAGTCTTGTC-30 ) and GSP4 (50 -GAGCTCAAATATCTGGTG GAG-30 ) designed against the 50 end of Exon B and 1.2 kb upstream of the transcriptional start site of Exon B, respectively. The promoter fragments with different sizes were amplified from the cloned promoter as template using primers containing XhoI and HindIII to facilitate the cloning into the pGL3 basic vector (Promega) in the correct orientation. Amplification of region )341/+34 was performed using primers TrBfor1 (50 -CCCCCCCTCGAGCTGAGGTCAGGAGTTCGAGAC30 , with restriction site shown in bold) and TrBrev2 (50 -GTGTGTAAG CTTGAGCTCAAATATCTGGTGGAG-30 , with restriction site shown in bold), while region )765/+34 was obtained by utilising primers TrBfor2 (50 -CCCCCCCTCGAGGATTACAGGTGTGAGC CACTG-30 , with restriction site shown in bold) and TrBrev2. The region )1147/+34 of the promoter was successfully amplified using primers TrBfor3 (50 -GTGTGTAAGCTTGAGCTCAAATATCTGGT GGAG-30 , with restriction site shown in bold) and TrBrev2. After restriction digestion with XhoI and HindIII, these PCR fragments were subcloned into pGL3 basic vector (Promega), creating the reporter vectors pGL3-B1a4 ()341/+34), pGL3-B2a4 ()765/+34), and pGL3B3a4 ()1147/+34), respectively. Cell culture and transient transfection assays. Human hepatoma HepG2 and Hep3B cell lines were grown in EagleÕs Minimum essential medium with EarleÕs BSS medium (Gibco) and DulbeccoÕs modified EagleÕs medium (Gibco), respectively, supplemented with 10% (v/v) fetal bovine serum, 2 mM L -glutamine, 100 U/ml penicillin, and 100 lg/ ml streptomycin (Hyclone). The cultures were maintained at 37 °C in a humidified atmosphere containing 5% (v/v) CO2 in air. The cells were transfected with individual promoter fragment using Lipofectin transfection reagent (Invitrogen Life Technologies) according to the manufacturerÕs instructions. Briefly, a day before transfection, the cells were subcultured into 6-well plates at a density of 2  105 /ml. On the day of transfection, complexes of lipid-DNA containing 2 lg of promoter-firefly luciferase construct and 0.5 lg pRL-TK plasmid (a Renilla luciferase reporter control vector used for monitoring transfection efficiency) were prepared with 5 ll Lipofectin solution in the final volume of 200 ll. The complexes were incubated at room temperature for 10 min before the addition of 800 ll of medium. The complexes were then added to the cells (1 ml/well) and incubated for 4 h. Subsequently, the medium was replaced with fresh cultured medium and maintained for a further 36 h. The luciferase activities in the cell

C.-H. Chew et al. / Biochemical and Biophysical Research Communications 305 (2003) 235–243 extracts were then determined using the Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase activity was normalised to the Renilla luciferase activity, with each transfection being carried out in triplicate and measurements were repeated at least twice.

Results and discussion Identification of alternatively spliced variants of the 50 -UTR of the hPPARa gene The aim of our study was to further characterise the 50 -UTR of the hPPARa gene. In order to amplify and identify the exon(s) of the 50 -UTR of the gene, two genespecific primers, GSP1 and GSP2, were designed against the 50 end of Exon 3. PCR amplification was carried out using gene-specific primers coupled with the primers provided in the kit (GeneRacer 50 Primer and GeneRacer 50 Nested Primer). Using this approach, six RACEPCR products of different sizes (100, 200, 230, 300, 330, and 600 bp) were successfully amplified (data not shown). Sequence analysis showed that the 30 end of all RACE-PCR products shared 100% identity with the 50

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end of Exon 3 of human PPARa gene, where the genespecific primers were designed, indicating that all RACE-PCR products were specific and represent the 50 UTR of the human PPARa. Interestingly, sequence analysis amongst the six RACE-PCR products revealed different combinations of exons which strongly indicate the existence of six differentially spliced variants in the 50 -UTR of the hPPARa gene, designated as hPPARa1, hPPARa2, hPPARa3, hPPARa4, hPPARa5, and hPPARa6 (Fig. 1A). The sequences of the 50 -UTR variants of hPPARa gene have been deposited in Genbank (Accession No. AY258326–AY258331). In order to determine the spliced junctions and exons of the 50 -UTR of hPPARa variants, the published PPARa mRNA sequences obtained from GenBank (Accession Nos. S74349, NM_005036, L02932, and BC000052) were first compared and aligned with a contiguous genomic DNA region taken from the sequences overlapping human PPARa DNA from GenBank (Accession Nos. AY206718, AF323915, AL049856, Z94161, and AL078611). These data together with previously reported studies [9,10] were utilised to determine the

Fig. 1. (A) The six alternatively spliced variants identified from this study. Alternative splicing mechanisms and different promoter usages give rise to variants with unique combinations of exons. (B) Schematic representation of the 50 -UTR of hPPARa gene. The new exons identified are indicated as shaded boxes. Numbers in bold and in boxes represent the sizes of the introns which span the exons. Arrows indicate the presence of promoter in front of Exon A, Exon B, Exon 2a, and Exon 3.

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spliced variants and exons of the 50 -UTR of the published PPARa mRNA and subsequently used to compare and align with the sequences of the six RACE-PCR products obtained from this study. The 50 -UTR of human PPARa was previously reported to be coded by Exon 1, Exon 2, and the 50 end of Exon 3 [9]. Recently, an additional Exon 1b located between Exon 1 (later it was renamed as Exon 1a) and Exon 2 (Fig. 1B) has been identified [10]. In this study, we have successfully identified three new novel exons of the human PPARa gene, designated as Exon A, Exon B, and Exon 2b (Fig. 1B). Exon A was identified at the 50 end of two variants, hPPARa3 and hPPARa5 (Figs. 1A and 2). It is interesting to note that the last 4 nucleotides (50 -CCAG-30 ) at the 50 end of previously identified Exon 1a of the human PPARa [10,22] may actually belong to the first 4 nucleotides of the 30 end of the newly identified Exon A, thus, conforming to the splice consensus sequence GT-AG. By contrast, if the nucleotides, 50 CCAG-30 , belong to Exon 1a, the 30 splice acceptor of Exon 1a will be TG and the 50 splice donor of Exon A will be CC, thereby, opposing the rule of GT-AG splicing mechanism (Table 1). Exon B, which is positioned between Exon 1a and 1b, encompasses the 50 -end of variants hPPARa4 and hPPARa6 (Figs. 1A and 2). The presence of variant hPPARa4 was predominant in all PCRs (data not shown), indicating that the promoter located at the 50

end of Exon B was predominantly active in human HepG2 cells. In the case of variant hPPARa6, an additional exon was found spliced in between Exon 2 and Exon 3. Interestingly, this exon was exactly similar to part of the mRNA available in GenBank (Accession No. BC000052). Thus, this newly identified exon was named Exon 2b and the previously known Exon 2 [22] was renamed Exon 2a. In addition, two different Exon 3 were also identified amongst six hPPARa variants (Table 1). hPPARa3, hPPARa4, and hPPARa5 contain Exon 3 similar to Exon 3 as previously reported [9]. By contrast, Exon 3 of hPPARa2 and hPPARa6 lacks three nucleotides at its 50 end (50 -TAG-30 ) which corresponds to Exon 3 of PPARa mRNA submitted to GenBank (Accession Nos. NM_005036, BC000052, and S74349). Table 1 illustrates that all the splice donor/acceptors of the 50 -UTR of human PPARa gene conform to the splice consensus sequence GT-AG. Fig. 1B shows the genomic organisation of the 50 -UTR of hPPARa gene consisting of seven different exons and four different promoters (shown by black arrows) resulting in variable 50 terminal regions. The presence of alternatively spliced variants in human PPARa mRNA is a common phenomenon amongst PPAR isoforms. For example, four variants resulting from four different promoter usages and alternative splicing at the 50 -UTRs have been identified in human PPARc mRNA [23–25], while seven alterna-

Fig. 2. Genomic sequences corresponding to the alternatively spliced Exon A, Exon B, and Exon 2b. Nucleotides in uppercase and lowercase letters correspond to exon and intron sequences, respectively. The acceptor and donor splice sites are depicted in bold. Arrows indicate the transcriptional start sites of the respective variants.

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Table 1 Exon/intron boundaries of the 50 -UTR of human PPARa gene Exon (bp)

Donor (50 –30 )

Acceptor (50 –30 )

Exon

A (55) 1a (83) B (209) 1b (156) 2a (84) 2b (100)

CGGCCCAG:gtgcc CACCACAG:gtaaa CAGGACAG:gtgag ATTCATAG:gtagg GATAAGAG:gtaca TAGCTCAG:gtgcg

tgccag:GCTGAAGC cacag:TTCTGGAG aacag:TTCTAGCC cacag:TTCTGGAG gccag:TGCCACAC cccagtag:CTTGGAGC cccag:TAGCTTGGAGC

1a 2a 1b 2a 2b 3 (hPPARa2 and 6) 3 (hPPARa3, 4, and 5)

Nucleotides in exons are indicated in uppercase letters, whereas the flanking nucleotides in introns are in lower case.

tively spliced variants were detected in monkey PPARc mRNA [26]. In addition, four differentially spliced variants induced by four differential promoter usages were also detected in mouse PPARb mRNA [27]. Meanwhile, in porcine PPARa mRNA, two alternatively spliced mRNAs were found in the coding region of PPARa [28]. The functional significance of the spliced variants identified at the 50 -UTR of human PPARa gene remains to be understood. For many genes, alternative splicing results in considerable diversity in the untranslated region. For example, eight alternatively spliced variants have been identified in the 50 -UTR for human growth hormone receptor mRNA in liver, but the functional significance of these variants is poorly understood. Recently, the role of the 50 -UTR has been suggested in controlling protein synthesis by alternative splicing and the choice of the translation initiation codon, which is controlled by mRNA-specific elements such as the GC content and the size of the 50 -UTR [29]. For example, the 50 -UTR of mouse retinoic acid receptor b2 (RARb2), another subfamily of nuclear receptor superfamily, is involved in the regulation of translational efficiency and tissue specific translational control [29]. Thus, this suggests a similar role for the 50 -UTR of human PPARa gene. However, the functional significance of the variants remains to be determined. A comparison of the 50 -UTR of human PPARa gene with that of mouse [30] using ClusterW1.82 revealed a significantly low degree of sequence homology among the species. The homology of Exon 1a, Exon 1b, and Exon 2a of hPPARa gene is 45.2%, 21.2%, and 46.4% identity, respectively, to the corresponding exons of mouse PPARa (mPPARaÞ gene. Interestingly, comparisons and alignment of mPPARa cDNA and its genomic sequence published in GenBank (Accession No. AK081709, BC016892, NM_011144, AK035676, X75287, X75288, and X75289) showed the presence of another unreported exon upstream of Exon 1a of mPPARa gene, thus, suggesting the possibility of an ‘‘Exon A’’ upstream of Exon 1a in mouse PPARa gene. An alignment of the respective exon in mouse to the human Exon A identified in variants hPPARa3 and hPPARa5 revealed 76.3% identity, thus, strongly indi-

cating that both mouse and human PPARa may share similar 50 -UTR genomic organisation. However a thorough comparison between the two species remains to be further elucidated. Determination of transcriptional start sites RLM-RACE method allows only full length 50 -capped transcripts to be amplified via elimination of truncated mRNA, thereby rendering it a safe, reliable, and accurate method for mapping of the 50 ends of transcripts and transcriptional start sites of genes [31–33]. The transcriptional start sites of Exon 3 and Exon 2a were situated 31 and 124 bp upstream of the ATG initiation start codon in the processed mRNA, respectively, while Exon A has two different transcriptional start sites, each individually marking the start site variants hPPARa3 and hPPARa5. The transcriptional start sites of hPPARa3 and hPPARa5 are situated 172 and 264 bp upstream of the ATG initiation start codon, respectively; with the hPPARa5 transcriptional start site located 8 nucleotides upstream of the transcriptional start site of variant PPARa3 in Exon A. Meanwhile, Exon B also has two transcriptional start sites (variant PPARa4 and variant PPARa6) mapped to the positions 251 and 540 bp upstream of their respective ATG start codon. The transcriptional start site for PPARa4 is 48 bp upstream of the transcriptional start site in PPARa6. Multiple transcriptional start sites within the same promoter region are in fact a common phenomenon and frequently observed in many genes. Multiple transcriptional start sites were identified in the mouse PPARa gene [30]. Interestingly, two major transcriptional start sites and at least two more minor transcriptional start sites were mapped in the mouse PPARa gene [30]. This finding is further supported by the presence of multiple transcriptional start sites within the same promoter reported in mouse PPARb gene [27] and also in Xenopus laevis bTrCP gene [34] whereby a single promoter produces four and two transcriptional start sites, respectively. Furthermore, two transcriptional start sites were determined in the promoter regions of human PPARc1 and PPARc2 mRNA [23]. A group hypothesised that

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multiplicity of transcriptional initiation sites was caused by the presence of several more or less TATA boxes in the promoter region [34]. However, research conducted

proved that deletion of TATA from promoters resulted in a variety of different initiation sites [35–37]. In the cases of the transcriptional start site for Exon B, no

Fig. 3. Nucleotide sequence and putative transcriptional factor binding sites of promoter B. Numbers are relative to the transcriptional start site (+1) of variant PPARa4. The putative sites are underlined with (r), indicating the sequence occurs in reverse orientation.

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prominent TATA-box was found within the first 100 nucleotides upstream of the transcriptional start sites of this promoter region (Fig. 3), thereby explaining the multiplicity of transcriptional start sites. Cloning and analysis of the promoter region of Exon B As described earlier, Exon B-derived variant hPPARa4 was found to be the most highly expressed of all variants. Thus, a 1181 bp of the hPPARa sequence upstream of the transcriptional start site of hPPARa4 in Exon B (designated as promoter B, GenBank Accession No. AY258332) was amplified using primers GSP3 and GSP4, subsequently cloned and sequenced. A computer search of the putative promoter B using the MatInspector programme [38] revealed no consensus TATA box found within the first 100 nucleotides although it was observed at location )121 upstream of the transcriptional start site (Fig. 3). This region has the characteristics of namely GC-rich sequences and multiple Sp1 sites, which are also present in the mouse and human PPARa promoters (30, 10). A number of potential responsive elements, such as HNF3b, AP1, CCAAT, C/EBPb, CREB, STAT, TCFII, GATA, and Oct-1, are also present in this region. Sequence alignments of Promoter B using ClusterW1.82 revealed 41.6% identity to the corresponding mPPARa promoter. This finding again showed that the human PPARa promoter does not share a high homology with the mouse PPARa in accordance with the similar low % of identity between the promoter transcribing Exon A of hPPARa [10] and the corresponding promoter of mPPARa gene [30]. Analysis of hPPARa promoter B activity In order to determine the ability of the promoter to drive basal transcriptional activity, promoter sequences were fused into luciferase reporter gene and the relative strength of the promoter fragments were measured using transient transfection assay. Three promoter constructs containing regions )341/+34 (pGL3-B1a4), )765/+34 (pGL3-B2a4), and )1147/+34 (pGL3-B3a4) of promoter B (Fig. 4A) were transiently transfected into HepG2 and Hep3B cells as hPPARa is reported to be constitutively expressed in liver cell lines [1,2,10]. In both cell lines, the shortest promoter fragment (pGL3B1a4) showed the strongest promoter activity of the three fragments (Fig. 4B). This fragment increased the basal transcription activity to a 15- and 52-fold higher than the activity of the promoter-less construct (pGL3 basic) in Hep3B and HepG2 cells, respectively, suggesting that the region may contain binding sites for transcriptional factors that enhance the gene expression of hPPARa gene. In pGL3-B2a4 and pGL3-B3a4, the promoter activity was lowered significantly compared to the pGL3-B1a4, thereby indicating the presence of ele-

Fig. 4. (A) Schematic representation of the positioning of primers, Exon B, and promoter B fragments used for transient transfection. (B) Analysis of promoter B activity using transient transfection assays. The promoter fragments )341/+34 (pGL3-B1a4), )765/+34 (pGL3B2a4), and )1147/+34 (pGL3-B3a4) of promoter B were fused into pGL3 basic vector and co-transfected with pRL-TK into HepG2 and Hep3B cells. Values (mean  SD) represent firefly luciferase activities normalised to a Renilla luciferase activity which served as internal control. Luciferase activities are shown relative to the activity of the pGL3 basic vector.

ment(s) conferring transcriptional repression between positions )341 and )1147. Interestingly, the presence of potential STAT binding was found within these regions. STAT has been proven to negatively regulate the gene expression of PPARa [39,40]. Furthermore, binding sites for negative regulators such as AP1, STAT, Oct-1, TCFII, GATA-2, and COUP-II were also found abundantly within regions )341 to )1147 of promoter B (Fig. 3). In addition, the activities of the promoter fragments were substantially higher in HepG2 cells compared to Hep3B cells, suggesting that Hep3B cells may lack factors or HepG2 may contain factors needed to increase the basal transcription activity. However, a more thorough promoter analysis will be necessary to fully characterise the promoter of PPARa.

Conclusions Six variants, named hPPARa1, hPPARa2, hPPARa3, hPPARa4, hPPARa5, and hPPARa6, were successfully identified at the 50 -UTR of human PPARa mRNA as a result of alternatively splicing and the use of

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four different promoters. Each of the transcriptional start site of the variant was determined and mapped, leading to the discovery of four promoters in the human PPARa gene which are responsible for transcribing these alternatively spliced variants. In addition, three new novel exons designated Exon A, Exon B, and Exon 2b were identified in this study. A new novel human PPARa promoter was also cloned and characterised. Demonstrated by transient transfection assay, this putative promoter B was a functional promoter. The minimal promoter region ()341 to +34) was found to have the strongest promoter activity, indicating the presence of enhancing element(s) in these regions. However, promoter activity was reduced in the promoter region )341 to )1147 as compared to the promoter pGL3-B1a4, indicating this region as harbour putative binding sites for negative regulatory transcription factors such as STAT, GATA-2, and COUP-II.

[10]

[11]

[12]

[13]

[14]

[15]

Acknowledgments We thank Dr. D.P. Ramji for providing the Hep3B cell line. This work was supported by the USM short term grant. CCH acknowledged financial support from National Science Fellowship (NSF), Ministry of Science, Technology and the Environment (MOSTE).

[16]

References

[18]

[1] I. Issemann, S. Green, Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators, Nature 347 (1990) 645–650. [2] O. Braissant, F. Foufelle, C. Scotto, M. Dauca, W. Wahli, Differential expression of peroxisome proliferator activated receptors: tissue distribution of PPARa, b, and c in the adult rat, Endocrinology 137 (1996) 354–366. [3] P.R. Devchand, H. Keller, J.M. Peters, M. Vazquez, F.J. Gonzalez, W. Wahli, The PPARa-leukotriene B4 pathway to inflammation control, Nature 384 (1996) 39–43. [4] S.A. Kliewer, B.M. Forman, B. Blumberg, E.S. Ong, U. Borgmeyer, D.J. Mangelsdorf, K. Umesono, R.M. Evans, Differential expression and activation of a family peroxisome proliferator activated receptors, Proc. Natl. Acad. Sci. USA 91 (1994) 7355–7359. [5] B.M. Forman, P. Tontonoz, J. Chen, R.P. Brun, B.M. Spiegelman, R.M. Evans, 15-Deoxy-d 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARc, Cell 83 (1995) 803–812. [6] J.M. Lehmann, L.B. Moore, T.A. Smith-Oliver, W.O. Wilkison, T.M. Willson, S.A. Kliewer, An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator activated receptor c (PPAR c), J. Biol. Chem. 270 (1995) 12953–12956. [7] T.M. Willson, P.J. Brown, D.D. Sternbach, B.R. Henke, The PPARs: from orphan receptors to drug discovery, J. Med. Chem. 43 (2000) 527–550. [8] G. Castillo, R.P. Brun, J.K. Rosenfield, S. Hauser, C.W. Park, A.E. Troy, M.E. Wright, B.M. Spiegelman, An adipogenic cofactor bound by the differentiation domain of PPARc, EMBO J. 18 (1999) 3676–3687. [9] M.C. Vohl, P. Lepage, D. Gaudet, C.G. Brewer, C. Betard, P. Perron, G. Houde, C. Celliar, J.M. Faith, J.P. Despres, K.

[17]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

Morgan, T.J. Hudson, Molecular scanning of the human PPARa gene: association of the L162V mutation with hyperapobetalipoproteinemia, J. Lipid Res. 41 (2000) 945–952. I. Pineda-Torra, Y. Jamshidi, D.M. Flavell, J.C. Fruchart, B. Staels, Characterization of the human PPARa promoter: identification of a functional nuclear receptor response element, Mol. Endocrinol. 16 (2002) 1013–1028. B. Staels, J. Dallongeville, J. Auwerx, K. Schoonjans, E. Leitersdorf, J.C. Fruchart, Mechanism of action of fibrates on lipid and lipoprotein metabolism, Circulation 98 (1998) 2088–2093. G. Chinetti, J.C. Fruchart, B. Staels, Peroxisome proliferatoractivated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation, Inflamm. Res. 49 (2000) 497–505. K. Tordjman, C. Bernal-Mizrachi, L. Zemany, S. Weng, C. Feng, F. Zhang, T.C. Leone, T. Coleman, D.P. Kelly, C.F. Semenkovich, PPARa deficiency reduces insulin resistance and atherosclerosis in apoE-null mice, J. Clin. Invest. 107 (2001) 1025–1034. B.P. Neve, J.C. Fruchart, B. Staels, Role of peroxisome proliferator-activated receptors (PPAR) in atherosclerosis, Biochem. Pharmcol. 60 (2000) 1245–1250. G.P. Collett, A.M. Betts, M.I. Johnson, A.B. Pulimood, S. Cook, D.E. Neal, C.N. Robson, Peroxisome proliferator-activated receptor a is an andogen-responsive gene in human prostate and is highly expressed in prostatic adenocarcinoma, Clin. Cancer Res. 6 (2000) 3241–3248. P.R. Holden, J.D. Tugwood, Peroxisome proliferator-activated receptor a: role in rodent liver cancer and species differences, J. Mol. Endocrinol. 22 (1999) 1–8. M. Rivier, I. Safonova, P. Lebrun, C.E. Griffiths, G. Ailhaud, S. Michel, Differential expression of peroxisome proliferator activated receptor subtypes during the differentiation of human keratinocytes, J. Invest. Dermatol. 111 (1998) 1116–1121. B. Desvergne, W. Wahli, PPARs: nuclear control of metabolism, Endocr. Rev. 20 (1999) 649–688. K. Hanley, Y. Jiang, D. Crumrine, N.M. Bass, R. Appel, P.M. Elias, M.L. Williams, K.R. Feingold, Activators of the nuclear hormone receptors PPARa and FXR accelerate the development of the fetal epidermal permeability barrier, J. Clin. Invest. 100 (1997) 705–712. K. Hanley, Y. Jiang, S.S. He, M. Friedman, P.M. Elias, D.D. Bikle, M.L. Williams, K.R. Feingold, Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARa, J. Invest. Dermatol. 110 (1998) 368–375. W. Wahli, Peroxisome proliferator activated receptors (PPARs): from metabolic control to epidermal wound healing, Swiss. Med. Wkly. 132 (2002) 83–91. T. Sher, H.F. Yi, O.W. McBride, F.J. Gonzalez, cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor, Biochemistry 32 (1993) 5598–5604. L. Fajas, D. Auboeuf, E. Raspe, K. Schoonjans, A.M. Lefebrve, R. Saladin, J. Najib, M. Laville, J.C. Fruchart, S. Deeb, A. VidalPuig, J. Flier, M.R. Briggs, B. Staels, H. Vidal, J. Auwerx, The organization, promoter analysis and expression of the human PPARc gene, J. Biol. Chem. 272 (1997) 18779–18789. L. Fajas, J.C. Fruchart, J. Auwerx, PPARc3 mRNA: a distinct PPARc mRNA subtype transcribed from an independent promoter, FEBS Lett. 438 (1998) 55–60. H. Sundvold, S. Lien, Identification of a novel peroxisome proliferator activated receptor (PPAR) c promoter in man and transactivation by the nuclear receptor RORa1, Biochem. Biophys. Res. Commun. 287 (2001) 383–390. J. Zhou, K.M. Wilson, J.D. Medh, Genetic analysis of four novel peroxisome proliferator receptor-c splice variants in monkey macrophages, Biochem. Biophys. Res. Commun. 293 (2002) 274–283.

C.-H. Chew et al. / Biochemical and Biophysical Research Communications 305 (2003) 235–243 [27] L.K. Larsen, E.Z. Amri, S. Mandrup, C. Pacot, K. Kristiansen, Genomic organization of the mouse peroxisome proliferatoractivated receptor b=d gene: alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency, Biochem. J. 366 (2002) 767–775. [28] H. Sundvold, E. Grindflek, S. Lien, Tissue distribution of porcine peroxisome proliferator-activated receptor a: detection of an alternatively spliced mRNA, Gene 273 (2001) 105–113. [29] H.A. Meijer, A.A.M. Thomas, Control of eukaryotic protein synthesis by upstream open reading frames in the 50 -untranslated region of an mRNA, Biochem. J. 367 (2002) 1–11. [30] K.L. Gearing, A. Crickmore, J.A. Gustafsson, Structure of the mouse peroxisome proliferator activated receptor a gene, Biochem. Biophys. Res. Commun. 199 (1994) 255–263. [31] K. Maruyama, S. Sugano, Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligonucleotides, Gene 138 (1994) 171–174. [32] B.C. Schaefer, Revolution in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full length cDNA ends, Anal. Biochem. 227 (1995) 255–273. [33] V. Volloch, B. Schweitzer, S. Rits, Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of bglobin mRNA with an extended 50 -untranslated region, Nucleic Acids Res. 22 (1994) 2507–2511.

243

[34] M. Ballarino, M. Marchioni, F. Carnevali, The Xenopus laevis bTrCP gene: genomic organization, alternative splicing, 50 and 30 region characterization and comparison of its structure with that of human bTrCP genes, Biochim. Biophys. Acta 1577 (2002) 81–92. [35] C. Benoist, P. Chambon, In vivo sequence requirements of SV40 early promoter regions, Nature 290 (1981) 304–310. [36] S.L. McKnight, R. Kingsbury, Transcription control of a eukaryotic protein-coding gene, Science 217 (1982) 316–324. [37] R.F. Weaver, Molecular Biology, first ed., WCB/McGraw-Hill, New York, 1999. [38] K. Quandt, K. Frech, H. Karas, E. Wingender, T. Werner, MatInd and Matinspector: new fast versatile tolls for detection of consensus matches in nucleotide sequence data, Nucleic Acids Res. 23 (1995) 4878–4884. [39] Y.C. Zhou, D.J. Waxman, Cross-talk between Janus kinase-signal transducer and activator of transcription (JAK-STAT) and the peroxisome proliferator activated receptor a (PPARa) signalling pathways, Growth hormone inhibition of PPARa transcriptional activity mediated by STAT5b, J. Biol. Chem. 274 (1999) 2672– 2681. [40] Y.C. Zhou, D.J. Waxman, STAT5b down-regulates peroxisome proliferator-activated receptor a transcription by inhibition of ligand-independent activation function region-1 trans-activation domain, J. Biol. Chem. 274 (1999) 29874–29882.