Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter
Elizabeth M. Ellis1,3,*,
Cara M. Slattery2,* and
John D. Hayes2,3
1 Department of Bioscience and Pharmaceutical Sciences, University of Strathclyde, Glasgow G1 1XW
2 Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK
3 To whom correspondence should be addressed Email: elizabeth.ellis{at}strath.ac.uk and john.hayes{at}cancer.org.uk
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Abstract
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Rat aflatoxin B1 aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B1. The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is
8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to
2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of
900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the core ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.
Abbreviations: AFAR1, aflatoxin B1 aldehyde reductase 1; AFB1, aflatoxin B1; AKR, aldo-keto reductase; CAT, chloramphenicol acetyltransferase; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle medium; FISH, fluorescent in situ hybridization; FITC, fluorescein isothiocyanate; MARE, Maf recognition element; NF-E2, nuclear factor-erythroid 2; Nrf, NF-E2 p45-related factor; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SSC, 30 mM sodium citrate, 0.3 M NaCl, pH 7.0; TRE, 12-O-tetradecanoylphorbol-13-acetate responsive element; TSS, transcriptional start site; UTR, untranslated region.
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Introduction
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The ability of xenobiotics to inhibit tumourigenesis has attracted considerable interest as a means of reducing the incidence of neoplastic disease (1). This type of intervention therapy is referred to as chemoprevention. Experimentally, the administration of coumarins, dithiolethiones, flavonoids, hydroquinones, indoles, isothiocyanates, lactones, phenolic antioxidants and mono- and di-terpenes has been shown to protect rodents against a diverse number of chemical carcinogens that are widespread in the environment (24). Such carcinogens include the mycotoxin aflatoxin B1 (AFB1), produced by Aspergillus flavus, and polycyclic aromatic hydrocarbons generated as products of combustion.
One mechanism by which cancer chemopreventive compounds exert their effects is through the induction of cytoprotective antioxidant proteins and drug-metabolizing enzymes (2). The protective systems that are inducible by chemopreventive compounds include aldo-keto reductases (AKR), microsomal epoxide hydrolase, ferritin, glutamate cysteine ligase, glutathione S-transferases (GST), glutathione reductase, heme oxygenase-1, malic enzyme, NAD(P)H:quinone oxidoreductase-1 (NQO1) and UDP-glucuronosyl transferases (510). Induction of these proteins in animals or cell lines is associated with temporary pleiotropic resistance to oxidative stress and to electrophilic compounds (11).
It is thought that induction of gene expression by chemopreventive agents occurs primarily through the antioxidant response element (ARE) (11,12). This enhancer was first identified in the 5'-flanking region of the rat GSTA2 and NQO1 genes (13,14), where it was found to be responsible for both basal expression and inducibility by ß-naphthoflavone and tert-butylhydroquinone. On the basis of deletion analyses of the ARE in the promoter of GSTA2, and comparison with the ARE in the NQO1 promoter, Rushmore et al. (13) proposed that the sequence required for enhancer function is 5'-A/GGTGACNNNGC-3'; the core ARE is often referred to as 5'-GTGACNNNGC-3' (15). Subsequently, similar cis-acting elements have also been identified in the promoters of mouse Gsta1 (16), rat GSTP1 (17), human NQO1 (18), mouse heme oxygenase-1 (19) and the human glutamate cysteine ligase catalytic (GCLC) subunit gene (20).
Compelling evidence has been presented that the regulation of ARE-driven genes is mediated by the nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2) (21). In the liver and small intestine from adult Nrf2 knockout mice, the basal expression of GST and NQO1 is lower than in the same organs from wild-type mice (10,22,23). Furthermore, many cancer chemopreventive agents fail to induce GST, NQO1 and GCLC in adult Nrf2 KO mice (2224). Also, macrophages from Nrf2 knockout mice are unable to induce antioxidant proteins in response to oxidative stress (25).
One of the most dramatic examples of a protein that is responsive to chemopreventive agents is provided by rat aflatoxin aldehyde reductase (AFAR1 or AKR7A1) (26,27). This enzyme, which is a dimeric member of the AKR superfamily (2830), appears to play a pivotal role in the detoxication of AFB1 by protecting against formation of protein adducts (26,31). It is capable of reducing the dialdehyde form of the mycotoxin to a less toxic dialcohol (31), and catalyses the reduction of a range of harmful aldehydes and diketones to alcohols (32). Amongst the proteins in rat liver that respond to chemopreventive agents, AFAR1 is one of the most inducible. Following treatment of animals with phenolic antioxidants, coumarin, dithiolethiones or ß-naphthoflavone, the hepatic levels of AFAR1 protein and/or mRNA are typically increased between 10- and 30-fold (7,26,27,3336). Nuclear run-on experiments have shown the AFAR1 gene to be transcriptionally activated 50-fold 6 h after treatment of the rat with 1,2-dithiole-3-thione, whereas other inducible genes were transcriptionally activated <5-fold at this time point (35,36). Together, these data indicate that transcription of rat AFAR1 is increased by chemopreventive agents more quickly, and to a greater extent, than other detoxication genes.
In addition to its induction by xenobiotics, AFAR1 is over-expressed in livers of selenium-deficient rats (37), as well as in rat hepatomas caused by exposure to AFB1 (26). The enzyme is also regulated in liver during development, being expressed at only low levels in fetal animals, and reaching peak hepatic levels at pubsecence before decreasing in adult animals (38).
The fact that AFAR1 is markedly transcriptionally activated by synthetic antioxidants and dithiolethiones indicates it is a prototypic target for chemopreventive agents that induce gene expression. In order to identify those features of AFAR1, which are responsible for its induction by chemopreventive agents, and to account for its patho-physiological regulation, we have characterized the gene and its promoter. The cloning of the 5'-flanking region of AFAR1 has allowed its promoter to be compared with those of other inducible genes that are members of the ARE-gene battery. Also, isolation of AFAR1 has allowed us to compare its gene structure with those of other AKR members.
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Materials and methods
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Chemicals and enzymes
Unless otherwise stated, chemicals were purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK). Restriction enzymes and Pfu DNA polymerase were obtained from sources described in previous publications from our laboratories (7,8,23,38). Dulbecco's Modified Eagle Medium (DMEM) with glutamax, sodium pyruvate, 1000 mg/ml glucose and pyroxidine; DMEM with 0.11 g/l sodium pyruvate with pyroxidine; heat-inactivated fetal bovine serum (FBS); penicillin/streptomycin; trypsin and versene were all purchased from Gibco BRL Life Technologies (Paisley, Scotland, UK). The digoxigenin-11-dUTP was from Boehringer-Mannheim (Lewes, East Sussex, UK).
Oligonucleotides
The oligonucleotides used in this study for DNA sequencing, polymerase chain reaction (PCR) amplification, or determination of the transcriptional start site (TSS) are listed in Table I. They were obtained from the Cancer Research UK, Clare Hall Laboratories (Potters Bar, Herts, UK).
Bacterial strains, plasmids and library
The Escherichia coli strain NM522, that was purchased from Novagen Inc. (Witney, Oxon, UK), was used for the propagation of most plasmids. JM109 cells from Promega (Southampton, UK) were used as the host for the pCAT®3-Basic vector (pCAT3B) and pCAT®3-Promoter vector (pCAT3P). One ShotTMTop10 cells from Invitrogen (Paisley, Scotland, UK) were used as the host for the pCR®-Blunt plasmid. The XL1 Blue MRA strain was obtained from Stratagene (Cambridge, UK) and was used to propagate the
-DASHTM phage library.
The pBluescript® KS (-) phagemid (Stratagene) was used as a general cloning vector. The pCR®Blunt (Invitrogen) was used to clone blunt-ended PCR products, and pCAT®3-Basic and pCAT®3-Promoter vectors (Promega) were used to examine the function of the 5'-flanking region of AFAR1.
Screening of a rat genomic library
A Fischer rat genomic library in the
DASH vector (Stratagene) was screened using the 400 bp EcoRISmaI restriction digest fragment from pEE60 that represents the 5' one-third portion of the cDNA encoding AFAR1 (27). Positive plaques were purified through several rounds of screening. Once purified, the inserts were excised, digested with EcoRI, and the restriction fragments were subcloned into pBluescript.
DNA sequence determination and computer-aided sequence analysis
Dr Andrew Cassidy performed automated sequencing of AFAR1 on ABI Prism 3100 equipment using the oligonucleotides listed in Table I. To assist with analysis of the 5'-upstream region of rat AFAR1, a computer search of the transcription factor database TRANSFAC was performed using the MatInspector software tool (http://transfac.gbf.de/) (39).
Fluorescent in situ hybridization
Swedish FISH (Department of Cell and Molecular Biology-Genetics, Göteborg University, Gothenburg, Sweden) performed fluorescent in situ hybridization analysis as a commercial service. Rat Fischer 344 fibroblast cells (FR1D) that had been treated with ICRF145 to elongate the chromosomes were employed for the fluorescent in situ hybridization (FISH) experiments (40,41). Identification of the chromosomal location of rat AFAR1 by FISH was achieved using a probe comprising a 6.1 kb genomic DNA EcoRI restriction fragment representing part of intron 1 through to exon 7 of the gene (i.e. the pEM4A plasmid, see below for details). A 1 µg portion of genomic DNA was labelled by nick translation with digoxigenin-11-dUTP using a kit supplied by Life Technologies Inc. (Gaithersburg, MD, USA) to yield rat AFAR1 probes of between 200 and 400 bp. These were denatured in buffer containing 50% (v/v) formamide, 2x SSC (30 mM sodium citrate, 0.3 M NaCl, pH 7.0) and 10% (w/v) dextran sulfate. After denaturation, the probe containing mixture was placed on slides of metaphase rat chromosomes that had been denatured by treatment with 70% (v/v) formamide and 2x SSC at 72°C for 2 min. Once hybridization had been allowed to occur (37°C for 48 h in a humid box), the slides were washed for 15 min in 50% (v/v) formamide, 2x SSC. Finally, the hybridized probe was detected using FITC-antidigoxigenin (Oncor Inc., Gaithersburg, MD, USA) and counterstained with 0.1 µg/ml DAPI (4,6-diamidino-2-phenylindole) in an antifade solution. Microscopy was performed with a Leica DM RXA. Chromosomes stained with DAPI were screened through a UV-A filter (excitation at 340380 nm) to allow the banding patterns to be viewed. The FITC signals were visualized through a band pass G filter (excitation at 490 nm). The images were captured using QW-FISH software for microscopy.
Preparation of rat genomic DNA
For the preparation of genomic DNA,
1 g of liver (from 14-week-old male Fischer 344 rats, obtained from Harlan Olac, Bicester, Oxon, UK) was homogenized in 12 ml of 10 mM Tris, 100 mM NaCl, 25 mM EDTA buffer, adjusted to pH 8.0 with HCl, that contained 0.5% (w/v) SDS and 0.1 mg/ml proteinase K. The resulting mixture was placed in a shaking water bath and left to digest at 50°C overnight. Thereafter, the DNA was extracted using the phenolchloroform procedure (42).
Isolation of intron 1 from AFAR1
Nested PCR using genomic DNA from the livers of male Fischer 344 rats as template was performed with oligonucleotide primers that represent the 3' region of exon 1 (i.e. AFARF7 and AFARF8, see Table II) and an area close to the 5'-end of the insert in the
DASH clone
EM4 (i.e. 4AR1 and 4AR2, see Table II). In the first reaction, PCR was performed in a 50 µl reaction buffer containing 32 ng of genomic DNA, 2.5 µl of dimethylsulphoximine, 200 ng of each of the AFARF8 and 4AR1 oligonucleotides (Table II), 0.2 mM each of the four dNTPs, and 1.25 U of Pfu turbo DNA polymerase. During the first cycle of amplification the genomic DNA was denatured for 3 min at 94°C, the primers were annealed for 30 s at 45°C and extended for 5 min at 72°C. Thereafter, for the next 29 cycles of amplification, identical reaction conditions were employed with the exception that the time of DNA denaturation was reduced to 2 min. In the final cycle (cycle 30), the DNA was extended for 7 min at 72°C to yield the first amplification product. Examination of the resulting reaction mixture by electrophoresis in 0.8% (w/v) agarose gels revealed several DNA species, including a major band of
2.2 kb. This product was recovered from the agarose gel and was employed as the template for a second round of PCR using the oligonucleotides AFARF7 and 4AR2 (Table II) as primers. The second PCR was performed under identical conditions as the first PCR with the exception that AFARF7 and 4AR2 were annealed to the template DNA at 55°C. It yielded a unique PCR product that was
2.2 kb in size. Following purification using a Qiagen gel extraction kit, the amplified product was sequenced.
Determination of the transcription start site in AFAR1
The TSS in AFAR1 was determined by the 5'-RACE (rapid amplification of cDNA ends) method using the GeneRacerTM Kit from Invitrogen. Total RNA was isolated from the livers of rats by the method of Chomczynski and Sacchi (43). A 3 µg portion of the RNA was treated with calf intestinal phosphatase for 1 h at 50°C before the enzyme was removed by extraction with an equal volume of phenolchloroform. RNA was precipitated by addition of sodium acetate along with 2 µg of mussel glycogen. After centrifugation, the RNA was resuspended and treated with tobacco acid pyrophosphate, for 1 h at 37°C, to remove the 5' cap structure from full-length transcripts. Following another precipitation step, the GeneRacerTM RNA Oligo (provided in kit) was ligated to the 5' end of the mRNA using T4 RNA ligase. The ligated mRNA was then converted to cDNA with avian myeloblastosis virus-reverse transcriptase using the GeneRacerTM Oligo dT primer. The 5' end was amplified from this template using the GeneRacer 5' primer (homologous to the GeneRacer RNA oligo) and the primer RACE2 (Table II), which hybridizes to AFAR1 cDNA. This resulted in a 500 bp product, which, after agarose gel electrophoresis, was excised and purified using a SNAP column (Invitrogen). A 4 µl aliquot of the purified PCR product was ligated into pCR®Blunt and sequenced to reveal exactly where the full-length mRNA had ligated to the RNA oligo.
Isolation of the 5'-flanking region of AFAR1
A GenomeWalkerTM kit (Clontech) was used to isolate the promoter of AFAR1 that employs PCR to amplify the region of interest from libraries of adapter-ligated rat genomic fragments. The first PCR reaction used the outer adapter primer (AP1) provided in the kit and the gene-specific primer GSP1 (Table II) that was designed to anneal to the 5' end of the rat AFAR1 cDNA. Five different libraries were provided in the kit, each cut with a different restriction enzyme. PCR was performed on each of the five libraries as recommended by the manufacturer. For the first seven cycles of amplification, each round entailed DNA denaturation at 94°C for 25 s and primer elongation at 72°C for 4 min. For the next 32 cycles, the DNA was again denatured at 94°C for 25 s, but elongation was performed at 67°C for 4 min. Analysis of the resulting PCR products by agarose gel electrophoresis revealed the presence of multiple amplified products. Aliquots of these reaction mixtures were subjected to a secondary amplification using the inner adapter primer (AP2) provided in the kit and the gene-specific primer GSP2 (Table II). The second PCR was performed under conditions similar to those used for the first amplification. Electrophoretic examination of the products of the second PCR showed the presence of two major bands. These were excised separately and following cloning into pT7Blue3 they were found to represent products of 645 and 954 bp; the plasmid containing the largest of these was called pEE88.
Generation of gene expression reporter constructs
Reporter constructs were generated in which portions of the 5'-flanking region of AFAR1 were ligated into a vector containing the bacterial chloramphenicol acetyltransferase (CAT ) gene. The 954 bp fragment in pEE88 was amplified by PCR using the primers 954F2 and 954R1 to generate products with PstI and SalI sites at the 5' and 3' ends, respectively (Table II). This was performed under conditions similar to those used to isolate intron 1 (see above) with the exception that the primers were annealed for 30 s at 56°C and extended for 2 min at 72°C. Following agarose gel electrophoresis, the amplified product was purified using a Qiagen gel extraction kit and subcloned into the PstISalI site of pBluescript. The insert from this plasmid, p954BS, was excised by digestion with SacI and XhoI and ligated into pCAT3Basic that had been digested with the same enzymes. The resulting construct was called p954CATB.
Deletions from the 5' end of the upstream flanking region of AFAR1 were made by PCR using p954BS as template with four different top strand oligonucleotides (i.e. -677CAT, -448CAT, -326CAT and -240CAT) in combination with the bottom strand oligonucleotide +55CAT designed to the 3' end of the insert (Table II). To aid cloning, a KpnI site was included in each of the 5' primers, and a XhoI site to the end of the 3' primer. The different lengths of AFAR1 5'-flanking region were generated by PCR over 30 cycles, in which each amplification round entailed denaturation of DNA for 1 min at 94°C, annealing of oligonucleotides for 1 min at either 55 or 59°C, and primer extension for either 1 or 1.5 min at 72°C. The products were resolved by polyacrylamide gel electrophoresis, and the appropriately sized bands purified and cloned into pCR-Blunt. After transformation, positive clones were identified, the inserts cut out with KpnI and XhoI and subcloned into pCAT3Basic. All deletion plasmids were sequenced to ensure the correct insert was present.
Transfection of rat liver cells and gene expression reporter assays
Rat liver RL34 cells were grown in DMEM (with 0.11 g/l sodium pyruvate and pyroxidine) supplemented with 10% fetal calf serum as adherent monolayers maintained at 37°C in 5% CO2 and 90% relative humidity. They were seeded at a density of
5 x 105 cells/well, and left for 24 h before transfection using lipofectin (Gibco). In this procedure, a 6 µl aliquot of lipofectin was mixed with 100 µl of OptiMEM® (reduced serum medium) and incubated at room temperature for 45 min. Meanwhile, 1 µg of each pCAT reporter plasmid and 1 µg of ß-gal reporter plasmid were added to 100 µl of OptiMEM. The two solutions were mixed and incubated at room temperature for 1015 min. Thereafter, 0.8 ml of OptiMEM was added, and 1 ml of the resulting solution was overlaid on the cells which had been washed previously with PBS. After incubation for 6 h at 37°C, the DNA solution was removed and fresh media added. Cells were incubated at 37°C overnight, to allow recovery from transfection. After this period the cells were harvested and gene reporter assays performed.
The CAT reporter enzyme assay was performed by incubating lysates of RL34 cells with acetyl coenzyme A and [14C]chloramphenicol for 2.5 h at 37°C. The chloramphenicol was extracted with ethyl acetate, and the different products resolved by thin-layer chromatography. The TLC plate was dried and placed in a phosphorimager overnight. The resulting image was analysed by Molecular Imager software.
The pCMV-ß-galactosidase (pß-gal) plasmid was used to determine transfection efficiency. The spectrophotometric assay of Sambrook et al. (42) was used to measure the ß-galactosidase activity in RL34 cells.
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Results
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Cloning of the rat AFAR1 gene
Two genomic clones that hybridized to the 400 bp EcoRISmaI pEE60 rAFAR1 cDNA probe were isolated through screening
5 x 105 plaques from a
-DASH library. These were designated
EM4 and
EM6, and each contained inserts of
12 kb. Upon digestion with EcoRI, the two
clones yielded distinct electrophoretic restriction fragment patterns, although several of the bands migrated similarly. Four of the major EcoRI fragments from both
EM4 and
EM6 were subcloned into pBluescript. Initial DNA sequencing of the subclones revealed that the inserts in
EM4 and
EM6 overlapped. The insert from
EM4 contained most of rat AFAR1, whereas that from
EM6 contained the 3' two-third portion of the gene, as well as additional downstream sequence (Figure 1). As
EM4 appeared to extend significantly further into the 5' region of rat AFAR1 than did
EM6, attention was focused on the former clone.
The largest EcoRI fragment obtained from
EM4 was 6.1 kb in length and represented the major portion of AFAR1. This fragment was subcloned, to yield pEM4A, and was subjected to automated DNA sequencing. The resulting information has been submitted to GenBank and given accession numbers AY230491AY230497. The plasmid pEM4A was found to contain 770 bp from intron 1, all of exons 2 (188 bp), 3 (105 bp), 4 (97 bp), 5 (100 bp), 6 (130 bp) and 7 (380 bp), and also all of introns 2 (194 bp), 3 (212 bp), 4 (237 bp), 5 (2254 bp) and 6 (915 bp). The subclones pEM4B, pEM4C and pEM4D, obtained from
EM4, have also been subjected to automated DNA sequencing and found to be 4.5, 1.45 and 1.3 in length, respectively. Restriction mapping of
EM4 and the pEM4 subclones suggested they are ordered as follows: 5'-A-C-B-D-3'. Thus, the 4B, 4C and 4D subclones do not encode rat AFAR1, and presumably represent intragenic untranslated region (UTR) sequences.
Determination of intron 1exon 1 boundary
The 5' portion of intron 1 that was not represented in pEM4A was isolated by nested PCR. This was achieved using oligonucleotides that anneal to exon 1 and the 5'-end of sequence in
EM4. Specifically, 4AR1 and AFARF8, followed by 4AR2 and AFARF7 (Table II), were used to amplify this region from genomic DNA. The amplified product was estimated by agarose gel electrophoresis to be 2.2 kb in size, and it was partially sequenced to confirm that it contained the correct flanking regions of exon 1 (from pEE60) and intron 1 (from pEM4A). Together, the electrophoresis and the DNA sequencing data suggest that intron 1 is
2.9 kb in length (Figure 1).
Determination of the transcriptional start site
The full-length 5'-UTR in the mRNA for rat AFAR1 was determined by 5'-RACE as described in the Materials and methods section. Sequencing of the amplified product that was cloned into pCR®Blunt showed that the complete 5'-UTR of AFAR1 is 73 nucleotides long. The first 20 nucleotides from the 5' end (5'-AGGGCTGAGGACTGCTGGAA-3') were not represented in pEE60. The remaining 5'-UTR is identical to the proximal 53 nucleotides in the 5'-UTR of the original AFAR1 cDNA (27). The 5'-RACE data, together with the sequence information from pEE60 and pEM4A, indicate that the full-length transcript from rat AFAR1 is 1275 nucleotides in length.
Rat AFAR1 gene structure
The nucleotides representing exons 17 in rat AFAR1 are presented in Figure 2. The DNA sequence across the putative exon 1intron 1 boundary in the amplified genomic fragment and across similar boundaries in pEM4A revealed that the introns in rat AFAR1 all contain classical splice site consensus sequences. These sites are also conserved in the human AKR7A2 gene (Table III). A bioinformatic search of the human genome database for AKR7A3 revealed that it also contained classical splice site consensus sequences (Table III).


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Fig. 2. Structure of exons 17 in rat AFAR1. The structure of the gene was deduced from sequencing pEM4A, pEE60 and the 5'-RACE product amplified using GeneRacer 5'-primer and the RACE2 oligonucleotide (Table II). The AFARF7 and AFARF8 oligonucleotides used to define the size of intron 1 are located between +188 and +204 and between +226 and +242, respectively.
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Table III. Comparison of the donor and acceptor sequences at the intronexon boundaries of rat AFAR1 and human AKR7A2 and AKR7A3 genes
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Exons 26 of rat AFAR1 are identical in size to those reported by Praml et al. (44) for the human AKR7A2 gene (Table IV). Furthermore, use of bioinformatics showed that exons 26 of the human AKR7A3 gene are identical length to those in rat AFAR1 and human AKR7A2 (Table IV). Although exon 1 of rat AFAR1 appears longer than those of AKR7A2 and AKR7A3, it must be emphasized that the transcriptional start sites of the latter two human genes has not been established and therefore the size of exon 1 in AKR7A2 and AKR7A3 is uncertain. Most importantly, the 3' boundary of exon 1 in rat AFAR1, determined in this study by nested PCR, is conserved in AKR7A2 and AKR7A3.
Chromosomal localization of rat AFAR1
Figure 3 shows the localization of the AFAR1 pEM4A probe distally on RNO5 at band 5q36 (for nomenclature, see ref. 45). The background staining is minimal and most cells studied produced double hybridization spots on both RNO5 chromosomes. In maximally elongated chromosomes, generated after treatment of cultures of RF1D cells with ICRF145, the q36 band could be subdivided allowing AFAR1 to be allocated to 5q36.5. This is an important observation because rat chromosome 5q36.5 is syntenic with human chromosome 1p35 (46), where the human AKR7A2 gene resides (44).

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Fig. 3. FISH analysis of AFAR1. Localization of the AFAR1 probe pEM4A in rat chromosome 5 is visualized by the yellow dots that are indicated by arrows. The label is observed in metaphase chromosomes, as well as two spots of hybridization in the resting nuclei (top left hand region in both panels).
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Isolation of rat AFAR1 5'-upstream region
As indicated above, neither
EM4 nor
EM6 contained the upstream flanking region of rat AFAR1. This was isolated by PCR genome walking using the gene-specific primers GSP1 (corresponding to nucleotides +85 to +61 of the cDNA) and GSP2 (corresponding to nucleotides +54 to +27) along with adapter primer oligonucleotides AP1 and AP2 (supplied with the genome walking kit). By using this technique, two clones were isolated from different libraries. The largest of these contained 954 bp upstream from the 5' end of GSP2. The smaller clone of 645 bp was identical to the 3' portion of the 954 bp product. PCR analysis of rat genomic DNA using a primer designed from within the clone of the putative 5'-flanking region (954F1: -629 to -613) and a primer designed from exon 1 (AFARR6: +210 to +226) gave a product of 950 bp, as estimated by migration during agarose gel electrophoresis. This confirmed that the genome walking clones are located immediately upstream from the rat cDNA in the genome.
It is important to note that the PCR primers used to verify the position of the genome walking clones and those used to amplify the 5'-end of intron 1 overlap. Thus, from analysis of genomic DNA, there is no evidence that an intron exists within the 5' UTR or amongst the nucleotides encoding the first 67 amino acids (numbering includes the initiator methionine).
Identification of a functional promoter in the 5'-flanking region of AFAR1
Figure 4 shows the 5'-flanking sequence of rat AFAR1. This region does not contain a perfect TATA box. However, a closely related sequence, 5'-TTAATAAA-3', situated between nucleotides -32 and -25 from the TSS, appears to fulfil this function.

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Fig. 4. Sequence of the 5'-flanking region of rat AFAR1. The region lying immediately upstream of AFAR1 was isolated by genome walking PCR. The DNA was sequenced on an ABI Prism 3100 instrument, and the nucleotides are numbered (as depicted in the left-hand side margin) from the A nucleotide (in bold) transcriptional start site situated 73 residues 5' from the ATG initiation codon. The imperfect TATA box, at -32 to -25, is shown in bold. The ARE-related sequences are shown underlined, and those in bold share similarity with a TRE. The nucleotides underlined with a hatched bar are those described previously in the cDNA (27). The nucleotides between +27 and +54, and between +59 and +83 (shown in italics) represent GSP2 and GSP1, respectively, used in 5'-RACE to isolate the promoter.
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DNA sequencing of the AFAR1 promoter revealed the presence of a large number of ARE-related motifs. A potential ARE (5'-ATGATTCAGCA-3') is found in reverse orientation between nucleotides -681 and -671 that can also be considered a putative MARE or NF-E2-binding site (47). It is also identical to the dominant half of the GPEI element found in the promoter of rat GSTP1 (48) (Table V); GPEI is responsible for the over-expression of GSTP1 in hepatic pre-neoplastic nodules (49). Four imperfect putative ARE enhancers (5'-CTGAGTGAGCG-3', 5'-GTGAGCGAGTG-3', 5'-GTGAGTGAACG-3' and 5'-GTGAGTTCATT-3') are located, in the forward orientation, between nucleotides -389 and -355; one of these is embedded in the NNN trinucleotides of the core ARE. It is noteworthy that three copies of the hepta-nucleotide motif 5'-GAGTGAG-3' occur within this 35 bp region. In addition to these five AREs, a further 11 half-ARE motifs (5'-TGAC/G-3') were observed at nucleotides -803, -672, -648, -584, -538, -463, -303, -275, -219, -164 and -115; the numbering of these motifs is from the 5' T. It is interesting that three of these half-AREs [at -648, -584 and -275] possess a G nucleotide immediately 5' of the T, to produce a 5'-GTGAG-3' penta-nucleotide motif. As shown in Table V, a remarkable total of 16 ARE-related sequences are present in the 5'-flanking region of AFAR1 between nucleotides -804 and -106 from the TSS.
Reporter constructs containing sequential deletions of the 5'-flanking sequence of AFAR1 driving the bacterial CAT gene were transiently transfected into RL34 cells. After transfection, the cells were cultured in media for 24 h before CAT activity was measured. In transfected cells that were grown in the absence of xenobiotic, the basal CAT enzyme levels produced by all the constructs were significantly higher than the empty vector pCAT3B. This indicates that the 240 bp region upstream of the TSS is sufficient to drive transcription (Figure 5). However, the level of expression between the different constructs was found to vary significantly. The pCAT954 and pCAT677 constructs yielded similar levels of CAT activity. In comparison with these, pCAT448 gave significantly higher CAT activity, suggesting the presence of a negative regulating element between nucleotides -677 and -448. This may be attributed to a CCAAT box situated between nucleotides -526 and -522. The construct p326CAT gave lower CAT activity than pCAT448, suggesting the existence of a positive regulatory sequence between nucleotides -448 and -326. The multiple ARE-related sequences between nucleotides -389 and -355 are probably responsible for this effect. Lastly, basal CAT activity generated from pCAT240 was higher than that from pCAT326 suggesting the presence of a silencer element between nucleotides -326 and -240.

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Fig. 5. Mutational analysis of AFAR1 5'-flanking sequence. RL34 cells were co-transfected with each CAT reporter construct and pßGal. After 24 h, the cells were scraped off the plates, and CAT and ß-Gal activities measured. The CAT activity represents the mean and standard deviation of three to five separate transfections, and is reported relative to ß-Gal activity to allow correction for transfection efficiency. The CAT enzyme results are each presented as a fold-activity relative to the activity obtained form pCAT3B under basal conditions. The nucleotides from the AFAR1 promoter that are present in pCAT954, pCAT677, pCAT448, pCAT326 and pCAT240 are described in the Materials and methods section.
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Discussion
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AFAR1 mRNA levels are induced in rat liver and small intestine by a large number of xenobiotics including benzylisothiocyanate, butylated hydroxyanisole, coumarin, dithiolethiones, ethoxyquin, ß-naphthoflavone, oltipraz and trans-stilbene oxide. The fact that the reductase is markedly induced by cancer chemopreventive agents and catalyses the detoxification of reactive carbonyls suggests that it protects against tumourigenesis. One of the remarkable findings about AFAR1 is the magnitude and speed of transcriptional activation it undergoes following treatment of rats with the anticarcinogen 1,2-dithiole-3-thione (35,36). To date, the structure of neither the rat gene nor its promoter has been reported. In order to provide an understanding of the basis for the responsiveness of AFAR1 to chemopreventive agents, and to provide an insight into the relationship between AFAR1 and other AKR family members, we have undertaken cloning of the gene and its 5'-upstream region.
Genes for AKR7 enzymes are structurally similar but differ from those in other AKR families
Our study has shown that the rat AFAR1 gene is
8 kb in size (Table IV), and comprises 7 exons and 6 introns. Previous work has shown that human AKR7A2 is composed of 7 exons (44). During this study, we have found by searching the human database that AKR7A3 also comprises 7 exons (Table IV). Exons 26 in rat AFAR1, AKR7A2 and AKR7A3 are identical in length. Also, the 3'-end of exon 1 and the 5'-end of exon 7 are conserved in all three genes (Table IV).
FISH analysis using rAFAR1 as a probe mapped the gene to rat chromosome 5q36. This chromosome is known to be homologous to the short (p) arm of human chromosome 1 (45) that has been shown to be the location of AKR7A2 at 1p35-36.1 (44). The data indicate that the rat and human genes within the AKR7 family are not only structurally similar but also have common evolutionary origins. Interestingly, human chromosome 1p35 is frequently lost in brain, breast, cervix, colon and kidney tumours. It has therefore been proposed that AKR7A2, or a linked gene, helps prevent the development of cancer (44).
Recently, Kelly et al. (50) cloned a cDNA for a second rat AFAR protein, called rAFAR2 or AKR7A4. On the basis of primary structure and enzyme activity, these workers proposed that rAFAR2 is more closely related to human AKR7A2 than is rAFAR1, and that rAFAR1 is more closely related to human AKR7A3 than it is to AKR7A2. It remains to be determined whether AFAR2 is linked to AFAR1 on rat chromosome 5q36. However, this is probable as both AKR7A2 and AKR7A3 are located on the short arm of human chromosome 1.
Rat AFAR1 is structurally simpler than other mammalian AKR genes in that it is smaller and contains fewer exons. In contrast, the human aldehyde reductase gene AKR1A1 is
20 kb long and comprises 10 exons (51), the rat aldose reductase gene AKR1B4 is 14.1 kb long and contains 10 exons (52), and the rat dihydrodiol dehydrogenase gene AKR1C9 is 30 kb long and consists of 9 exons (53).
The promoter of AFAR1 contains numerous ARE- and TRE-related sequences
The 5'-flanking region of AFAR1 was isolated by a genome walking PCR strategy using gene-specific primers designed around the 5'-end of the cDNA. This resulted in amplification and the cloning of
900 bp immediately upstream of the TSS. The cloned fragment of DNA was found to support expression of a CAT reporter gene in transfection experiments, thereby supporting the hypothesis that it represents part of the promoter of AFAR1.
DNA sequencing of the portion of the AFAR1 promoter obtained by genome walking revealed the presence of a large number of ARE-related motifs between nucleotides -812 and -114 from the TSS. Using 5'-RTGASNNNGCR-3' as the ARE consensus (see Table V), eight sequences were found in the upstream region of AFAR1 that contained either 6 or 7 of the 8 nucleotides that are defined in the enhancer. Amongst these, several bore close similarity to the TRE. In particular, the sequence 5'-ATGATTCAGCA-3' found in reverse orientation between nucleotides -689 and -679 contains the tri-nucleotide TCA at the position occupied by NNN in the ARE consensus. As a consequence, it can be regarded as a potential MARE or NF-E2-binding site (47). In the context of AFAR1 protein being over-expressed in rat liver pre-neoplastic nodules initiated by AFB1, it is interesting that this motif shares complete identity with the dominant half of GPEI, the enhancer responsible for over-expression of GSTP1 during chemical hepatocarcinogenesis (48). It is also responsible for induction of GSTP1 in the livers of rats treated with lead (54). It remains to be demonstrated whether the 5'-ATGATTCAGCA-3' sequence between nucleotides -689 and -679 is indeed responsible for the increased levels of AFAR1 in hepatic foci and tumours produced by exposure to AFB1.
A remarkable finding of this study is the presence of four ARE-related motifs, two of which overlap, in the 5'-flanking region of AFAR1 between nucleotides -389 and -355. The sequence 5'-TGAGTGA-3', which resembles a TRE, occurs twice between -388 and -370, and the sequence 5'-GAGTGAG-3' occurs three times between -387 and -361. To our knowledge, none of the genes that are inducible by cancer che- mopreventive agents contain multiple direct repeats of similar sequences within such a short region of their promoters.
Multiple stress response elements have been identified previously in antioxidant genes. For example, the heme oxygenase-1 gene is regulated by five cis-acting elements, located over 6 kb of DNA, that are activated by CdCl2, NaAsO2 and hemin (5558). These StREs have the consensus 5'-A/GACCNTGACTCAGCA/GNAA-3' (55), and evidence suggests the elements function synergistically. Further work is required to establish the significance of each of the multiple 5'-GAGTGAG-3' motifs in determining either basal and/or inducible expression of the AFAR1 gene.
Identification of other potential enhancers besides the ARE and TRE in the promoter of AFAR1
A computer-aided search of the TRANSFAC database using MatInspector software identified several possible enhancers in the 5'-flanking region of AFAR1. In addition to the ARE enhancers, the following sites were identified: Oct1 (5'-GAATATG/TCA-3', at -79 and -877, both inverted); Sp1 (5'-GGGGGCGGGGC/T-3', at -72 in reverse); 5'-CCAAT (A/GA/GCCAATC/GA-3', at -896 and -520 in reverse). A CHOP1 site, also called GADD153, (5'-A/GA/GA/GCCAATC/GA-3') is present at -780. An XRE (5'-C/TGCGTGCA/CC/G-3', at -49), two BARBIE boxes (5'-ATNNAAAGCNG-3', at -286 and -131 inverse), two C/EBP sites (5'-TG/TNNGA/CAAG/T-3', at -180 and -91), an NF-
B-like site (5'-GGGGAC/GTTTCC-3', at -162) and an ERE half site (5'-AA/GGNNANNNTGACC C/T-3', at -672) were also identified. All these homologies were found to match with a probability of >0.86.
Presence of silencer elements in the promoter of AFAR1
Functional analysis of the 5'-flanking region of AFAR1 suggested the presence of several silencer elements between nucleotides -677 and -448, and between -326 and -240. This situation is reminiscent of the promoter of rat GSTP1 where, in addition to the GPEI enhancer, a cis-acting silencing element is responsible for the low constitutive expression of the gene in hepatocytes. It has been found that members of the CCAAT/enhancer-binding proteins mediate the negative regulation of GSTP1 (59). A similar phenomenon may occur in the case of AFAR1. Indeed, a CCAAT box can be observed in the promoter of AFAR1 between nucleotides -526 and -522.
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Concluding comments
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The present paper describes the rat AFAR1 gene and its promoter. Numerous ARE- and TRE-related sequences have been identified in the promoter that may be responsible for the induction of the gene by cancer chemopreventive agents, for its over-expression in selenium deficiency, and for its activation during hepatocarcinogenesis. The data contained herein will facilitate future attempts to show the involvement of Nrf1, Nrf2, large Maf or cJun in the regulation of AFAR1.
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Notes
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*These two authors contributed equally to the work 
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Acknowledgments
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It is a pleasure to acknowledge the help of Professor Masayuki Yamamoto and Dr Ken Itoh with experiments involving RL34 cells. We are deeply grateful to the Association for International Cancer Research for funding this work (98-023), and for providing a PhD studentship for CMS. E.M.E. was a Beit Memorial Fellow during part of this work.
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Received December 6, 2002;
accepted February 3, 2002.