Characterization of a rabbit gene encoding a clofibrate-inducible fatty acid ω-hydroxylase: CYP4A6

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 296, No. 1, July, pp. 66-72,1992

Characterization of a Rabbit Gene Encoding a Clofibrate-Inducible Fatty Acid a-Hydroxylase: CYP4A6’12 A. Scott Muerhoff,

Keith

J. Griffin,

and Eric F. Johnson3

Division of Biochemistry, Department of Molecular and Experimental The Scripps Research Institute, L.u Jolla, California 92037

Received January

17,1992, and in revised form February

lo,1992

CYP4A6 mRNAs are induced in the rabbit liver and kidney following treatment with the antihyperlipidemic drug clofibrate. As a first step toward the elucidation of the mechanism controlling the induction of this and other CYPIA genes by clofibrate and other peroxisome proliferators, we have cloned and characterized the CYP4A6 gene. Genomic DNA containing the first 12 exons encoding CYP4A6 was isolated as three recombinant X phage, two of which were overlapping. The sequence of more than 1000 bp of the 5’ upstream region as well as of the first 12 exons has been determined. These 12 exons encode all but approximately 80 bp at the 3’ terminus of CYP4A6. Intron/exon junctions within the coding region of the gene are conserved relative to the rat CYP4Al and CYP4A2 genes. Primer extension analysis indicates that transcription is initiated 33 bp upstream of the start codon. The CYP4A6 promoter region, like that of the rat CYP4Al and CYP4A2 genes, does not contain a consensus TATA box. However, a consensus Spl recognition element is apparent at -46 bp upstream of the transcription start site. In addition, a sequence related to one of two regulatory elements that control the induction of the rat acyl-CoA oxidase gene by ciprofibrate is present upo lssz Academic Press, I~C. stream of the CYP4A6 promoter.

i This work was supported by USPHS Grant HD04445 (E.F.J.) and a postdoctoral fellowship (91-68) from the American Heart Association, California Affiliate (A.S.M.). Facilities for computer-assisted sequence analysis, automated nucleotide sequencing, and the synthesis of oligonucleotides are supported in part by GCRC Grant MO1 RR00833 and by the Sam and Rose Stein Charitable Trust. ’ Sequence data for the CYP4A6 5’-flanking region and exon 1 have been deposited with the EMBL/GenBank Data Libraries. 3 To whom correspondence should be addressed.

66

Medicine,

The cytochrome P450 IVA gene subfamily (CYP4A)4 encodes a number of enzymes which catalyze the w-hydroxylation of saturated and unsaturated fatty acids, including arachidonic acid, as well as products of the lipoxygenase and cyclooxygenase pathways. The four members of this family that have been characterized in rabbits exhibit diverse regulation (l-3). The abundance of mRNAs encoding CYP4A6 and CYP4A7 is increased in both liver and kidney by clofibric acid (3). Of these, CYP4A6 is the most highly induced in the liver, whereas the expression of CYP4A4 in this tissue is unaffected by clofibric acid.5 CYP4A4 is, however, selectively elevated in lung and liver during pregnancy (4-7). The expression of CYP4A5 in liver and kidney is not greatly affected either by treatment with clofibric acid or by pregnancy.5 In addition, these four enzymes exhibit distinct substrate profiles. CYP4A4 is the only one of the four to exhibit high activity with CYP4A4, CYP4A6, and prostaglandin substrates. CYP4A7 each exhibit a high capacity to metabolize arachidonic acid, whereas arachidonic acid is a poor substrate for CYP4A5.= Clofibric acid and other antihyperlipidemic drugs such as ciprofibrate, as well as the plasticizer di(2-ethylhexyl)phthalate (8) and the chlorophenoxy acetic acid herbicide 2,4,&trichlorophenoxyacetic acid (9), are com-

’ Abbreviations used: CYP, cytochrome P450; CYP4A is used to describe a subfamily of related cytochrome P450 genes; CYP4A6 refers to a unique member of this subfamily. Gene designations used in this manuscript conform to the uniform system of nomenclature described by Nebert et al. (31). PPAR, peroxisome proliferator-activated receptor; HNF-1, hepatic nuclear factor 1; HNF-4, hepatic nuclear factor 4; ARP1, apolipoprotein AI regulatory protein 1; PCR, polymerase chain reaction; bp, base pairs; Pipes, piperazine-Nfl-bis(2-ethanesulfonic acid). ’ L. J. Roman, J. E. Clark, B. S. Masters, A. S. Muerhoff, K. J. Griffin, and E. F. Johnson, manuscript in preparation.

ooo3-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

STRUCTURE

pounds that are capable of inducing fatty acid w-hydroxylases in several species. As these compounds also cause peroxisomes to proliferate in sensitive species, where they also appear to be nongenotoxic carcinogens, these compounds are often referred to as peroxisome proliferators (10). The mechanism by which peroxisome proliferators alter the expression of peroxisomal enzymes or CYP4A enzymes remains unclear. Clofibric acid induces both CYP4Al and CYP4A2 in the rat (ll), and this process has been shown to reflect a stimulation of gene transcription (12). Clofibrate also induces the transcription of the acyl-CoA oxidase (EC 1.3.3.6), an enzyme involved in peroxisomal fatty acid P-oxidation (13). Gene transfer experiments have demonstrated that the induction of the rat peroxisomal acyl-CoA oxidase is governed by two distinct regulatory sequences in the 5’-flanking region of the gene (14). As we show here, a sequence highly related to one of these two sequences is found upstream of the CYP4A6 promoter. Peroxisome proliferators are known to activate a transcription factor, the peroxisome proliferator activated receptor (PPAR), which is a member of the superfamily of ligand-activated receptors that include the steroid and thyroid hormone receptors (15). It is not yet known, however, if this receptor regulates directly or indirectly the expression of either the acyl-CoA oxidase or the CYP4A genes, nor if the regulation of these two genes occurs by a common mechanism. In order to examine the mechanism of induction in greater detail, we have cloned and characterized the CYP4A6 gene, and compared the sequence of the promoter region with that of the rat CYP4Al and CYP4A2 genes and with the regulatory elements mediating the induction of the rat acyl-CoA oxidase by peroxisome proliferators. The extensive 5’-flanking sequence which was cloned will facilitate future experiments to define regulatory elements that function in the induction of the CYP4A6 gene by clofibrate. EXPERIMENTAL

PROCEDURES

Screening of a rabbit genomic library. A rabbit genomic library was screened according to established procedures (16) using probes derived from rabbit CYP4A cDNAs (2,3). The library was constructed in XEMBL-3 using insert DNA isolated from the spleen of a strain III/J inbred rabbit and partially digested with Mb01 (17). Positive phage were subjected to successive rounds of plating until cloned. Recombinants that hybridized to a probe corresponding to exons 1 and 2 were identified by sequence analysis of subcloned restriction fragments using an exon l-specific primer. Additional recombinants containing exons 6 through 12 were identified by hybridization with an oligonucleotide derived from the sequence of exon 6 that selectively hybridizes with the CYP4A6 cDNA when compared to the other CYP4A cDNAs. Preparation of recombinant plasmids. Overlapping restriction fragments derived from EcoRI, BamHI, or Sal1 digestion of purified, recombinant phage DNA were isolated after electrophoresis through agarose using Geneclean (Bio 101, La Jolla, CA) as described by the manufacturer. The fragments were inserted into pBluescript vectors (Stratagene,

OF CYP4A6

67

La Jolla, CA) using T4 DNA ligase (Pharmacia) and propagated in Escherichia coli strain DH5a (transformation competent, BRL, Gaithersburg, MD). Additional subclones were generated from these plasmids to facilitate restriction mapping or sequence analysis. Plasmid DNA was purified using a Nucleotide sequence analysis. procedure that includes precipitation with cetylammonium bromide (18). DNA sequencing utilized dideoxy chain termination methods (19), a modified T7 DNA polymerase (Pharmacia, Piscataway, NJ), and specific primers synthesized to correspond to sequences within the CYP4A6 cDNA. The oligonucleotides were synthesized on an Applied Biosystems Model 380B DNA synthesizer (Foster City, CA). In some cases, fragments generated using restriction sites in or near exons were sequenced using primers for the pBluescript plasmids (Stratagene). The products were analyzed by polyacrylamide gel electrophoresis (6% acrylamide) Additional in the presence of 7.8 M urea followed by autoradiography. sequence information was determined using an Applied Biosystems Model 373A automated sequencer. Estimation of intron sizes. The sizes of introns l-8 were determined by detailed restriction mapping of the phage genomic DNA inserts. The sizes of introns 9, 10, and 11 were determined by the method of Brudzinski and Gelehrter (20). This method utilizes exon-specific oligonucleotide primers in order to generate DNA fragments by the polymerase chain reaction that correspond to the sizes of the intervening sequences. The templates used were the 3.7-kbp BamHI fragment of X44, which includes exons 10 and 11 and 142 bases from the 5’ end of exon 12 and the overlapping EcoRI fragment of X44 which includes exons 6-11. DNA amplification reactions were performed using the GeneAmp PCR kit (Perkin-Elmer-Cetus, Norwalk, CT) as described by the manufacturer. Each reaction contained 100 ng of template DNA and 42 pmol of each primer in a final reaction volume of 0.1 ml. The solution was covered with one drop of mineral oil and amplification was carried out using an Ericomp thermal cycler (San Diego, CA). Cycling times were 1 min at 94°C (denaturation), 1 min at 34°C (annealing), and 3 min at 72°C (extension). Thirty cycles of denaturation, annealing, and extension were performed, concluding with a 7-min extension at 72°C. The amplified DNA samples were electrophoresed through 1% agarose gels in TAE buffer (16) and sizes of the fragments were then determined by comparison to double-stranded DNA standards (Lambda DNA, HindIII/ EcoRI digested, Sigma, St. Louis, MO). Total cellular RNA was isolated by the Primer extension analysis. acid-guanidine isothiocyanate method (21) from 0.5 g of liver obtained from individual rabbits. Rabbits were injected ip daily for 3 days with either 400 mg/kg body weight of clofibric acid (Sigma, St. Louis, MO) neutralized with NaOH (22) or the equivalent volume of 10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl. An antisense oligonucleotide primer corresponding to nucleotides 112-129 (5’CCACTGGCGGTGCAGGTA-3’) of the CYP4A6 cDNA (2) was endlabeled with 32P using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) as described (16). The primer (40 ng) was annealed to 50 pg of rabbit liver RNA template and extended using AMV reverse transcriptase (BRL) exactly as previously described (16). The products were analyzed on a sequencing gel. A sequence ladder generated from the CYP4A6 cDNA using an exon 6-specific primer served as a sizing standard.

RESULTS AND DISCUSSION Isolation and characterization of CYP4A6 genomic clones. Four positive, recombinant phage were identified that contained CYP4A6 specific sequences. These were extensively characterized by restriction mapping and sequencing. Two of these clones, X27 and X4, were found to be overlapping in that they both contained exon 1 of the CYP4A6 gene (Fig. 1). Clone X44, which contains exons

68

MUERHOFF,

GRIFFIN,

AND

JOHNSON

of the cDNA, suggesting the occurrence of a 13th exon of approximately 80 bp that contains the polyadenylation II I I I II X27 signal sequence. This comparison was based on the alignment of the sequence determined for the CYP4A6 gene with the sequence reported by Yokotani et al. (3) for their 1 234 5 rabbit kidney CYP4A6 cDNA, designated KDRS, which contains a longer 3’ untranslated region than the 4A6 cDNA characterized in our laboratory (2). Attempts to locate exon 13 within the phage inserts using a synthetic oligonucleotide were negative. The rat CYP4Al gene also displays 13 exons with an intervening sequence in the 3’ 6789 1011 12 untranslated region of the gene; however, the position of this intervening sequence differs between the 4Al and x44 : 4A6 genes in its distance from the termination codon, 5 ,2 bp versus 566 bp, respectively. The site of Characterization of the promoter region. FIG. 1. Restriction maps of three recombinant phage containing portions of the CYP4A6 gene. Exons are indicated by the solid boxes and transcription initiation was determined by primer extenare identified by a number. Restriction sites mapped by digestion with sion analysis using an antisense oligonucleotide correEcoRI (E), BamHI (B), and XbaI (X) are indicated by the corresponding sponding to nucleotides 112-129 of the cDNA (2), and letter. RNA prepared from the liver of either a clofibrate-treated or an untreated rabbit as a template. Primer extension 6-12, does not appear to overlap clone X4 as determined products of 139, 160, 161, 162, and 247 nucleotides were from restriction enzyme mapping and Southern blot detected for mRNA from either source (Fig. 3). The three analysis (23) using probes derived from the ends of each products corresponding to 160, 161, and 162 nucleotides insert. The remaining recombinant contained an insert were more prominent when RNA from clofibrate-treated similar to that of X44. rabbits was employed as the template as compared to RNA The sequences of the exons and adjacent intronic re- from untreated rabbits. These three products are most gions were determined from subclones terminating at or likely to correspond to transcripts arising from the near exon/intron junctions or by the use of specific primCYP4A6 gene as the amount of the transcripts obtained ers synthesized to correspond to the sequence of CYP4A6. reflects the differences in CYP4A6 mRNA abundance obThe genomic sequences obtained agreed with that of the served between control and clofibrate-treated animals. cDNA with the exception of a single nucleotide substi- The primer employed does not discriminate between tution in exon 9 where codon 380, CUG encoding leucine, CYP4A6 and the three additional rabbit CYP4A cDNAs differs from the AUG encoding methionine seen in both that have been characterized due to their high degree of published cDNA sequences(2,3). Exon/intron boundaries nucleotide sequence identity in the first exon, and therewere determined by alignment of the sequences obtained fore, the primer extension products of 139 and 247 nufrom the genomic subclones to that of the CYP4A6 cDNA cleotides seen in Fig. 3 may reflect transcripts generated (Fig. 2). The 5’ and 3’ intron boundaries displayed confrom the other CYP4A genes. sensus donor and acceptor sites, respectively, and the poThe sequence of approximately 750 bp upstream of the sitions of each intron within the coding region are confirst exon was determined for both X4 and X27, giving served with that observed for the rat CYP4Al and identical results (data not shown). As shown in Fig. 4, CYP4A2 genes. The sizes of the intervening sequences there is no consensus TATA box evident upstream of the (Fig. 2) were estimated from restriction digests or by PCR start site as has been reported for the rat CYP4Al and mapping employing exon-specific primers followed by CYP4A2 genes. A consensus recognition sequence for the electrophoretic analysis to determine the size of the PCR eukaryotic transcription factor Spl (24) is apparent, product. The size of intron 5 could not be estimated in however, at -46 to -37 bp upstream of the start site (Fig. this manner because the flanking exons were not present on overlapping genomic clones. An estimate of the size 4). A GC rich region also occurs in the rat CYP4Al gene at the same relative position (Fig. 4), but its similarity to of intron 5 could not be obtained by Southern blotting (23) due to the multiplicity of DNA fragments which hy- the consensus Spl recognition sequence is less apparent. bridize to probes derived from exons 5 and 6 of CYP4A6 This potential Spl recognition site overlaps with a short because of the complexity of the CYP4A gene subfamily segment of sequence that is highly conserved between the rat CYP4Al and CYP4A2 genes (25). Although a similar (data not shown). sequence is evident in CYP4A6, several nucleotides have The sequence obtained from exon 12 of the CYP4A6 gene diverged from that of the 3’ untranslated sequence been deleted (Fig. 4), suggesting that it is unlikely to re1

EB

B

X

E

E

STRUCTURE

69

OF CYP4A6

-1070 ACATCTGCCCGCTCCTTGGMATGCACAGTAAMCACACAGCMTTGTGCMGGGCTCCTMCAGTTGGGGGGCTGCCACATGGACATACAMCATTTCAGTMGGTTGCTGTGAGAGAMA -930 CACATAMGTTCTTTGCTCAGMCACAGTAAATACTCMCGTTAGCT~CAGCACAGCAGGGGCCCAGTGATCAC~~GCTCAG~CTGGACMGG~GACTCMCMCC~C

-790 ATTCCAAACAGCAAACCGCACTCCCTGGCTTCAMGTTCTTTCTGCTCCTTGTTCTTTTCACCTTGTGCTGTAGGTGCACMCAGGTTCTGAGACMGTAGWICAMGGCCAGGGTCCM AcylCoA Oxidase -650 GAGTCCCTGGTCATGGAGAGAGGCTGCACAGCGAGATGGMCGCAAACACTGMCTAGGGCAAAGTTGAGGGCAGTGGGGTG~TGCTTGTCTGCCCATCTCCCTGAGGTTTTCCACA -510 GCGCCCAGMTTAGGGCACAAAAAAGGAGCTGTGGCACCTCGGGGGTTATACTGMTCACCTTTMGGAGG~GGCGAGG~GGGTGTTGGTCACATTTCTGAGGCTGTTGGAGGGA -370 GGCAGTTTGCGMTCCAATAAAAAATGGTGCCAATCCCACCTTCAACTGGCMTGACTGCTTTCCTCCGAAAACCATCGCTTCACCTCCCTGTCCTCMCGTCTTGATGGATCAAGTCM -350 GGCAGTMTAMTGCAGCCACACTTATTGMGTTCAMTAAACTAGCCTGCGMTTCCCTCAAGTTGGGGCTCATATCTGTTCTTGCTAATGTGCCGCCCAACACTGAGCATACTGCCTG -210 GCACATAGTAGGTGCTCATTAMTGTTTGGGMTAACATGGAGATGGGCAGGTGTCCAC~CATTGTCACTGCCAGCTGAT~GCTTTTCTGGCTTTCTTCCCCCAGGGTGTTGTGM CYP4Al +1 -70 CTTCTTCCCAGGGTTAATGATCTCTGCTCTGTGAGGTTGACTTCTGTCCCCACCCTTCGCTCTCCCCATAGGTGGGCGGGGCMCGCTCCT~CTTGGGCMGGGTCAGTGACAGG.G HNF-1 HNF-4 SPl 70 AGGGACAGTCACAGGTCCAGAGCCGCTGCACCATGAGCGTGTCTGCACTGMCCCCACCCGGCTCCCGGGCAGCCTCTCCGGGCTCCTCCMGTGGCGGGCCTGCTGGGCTGCCTCCTGC MSVSALNPTRLPGSLSGLLQVAGLLGCLLL

< 210 TGCTGCTCMGGCAGCTCAGCTCTACCTGCACCGCCAGTGGCTGCTCAGAGCCCTCCAGCAGTTCCCGTGCCCACCCTTCCACTGGCTCCTGGGGCACAGCCGAGAGgtaggaaggegcc LLKAAPLYLHRPULLRALPQFPCPPFHULLGHSRE 250 ggacaggeccggagcaggg...

INTRON 1, 2.7Ktp

. ..cagctccaatcactacttagacctttaaggccagaactatctccctttggttgtcctacagttcattccactccaagaa

CtagaactgaatgatgacttttaatgatttctctttcttttggtgacagTTCC~TGGCCATGAGTTACMGTGATGCT~TGGGTGGAG~TTCCCMGTG~TTGTCCTCGCTG FPNGHELQVMLKUVEKFPSACPRU < GCTATGGGGGAGCAGAGCCCACCTCCTGATCTAT~CCCTGACTACATGMGGTGATTCTGGGGA~TCAGgtaagcgtgcaacctccccccagttggagcagccctgccctgttgggct LUGSRAHLLIYDPDYNKVILGRSD

tct . ..INTRON

2,

0.7Kbp . ..ccgctattggtgccgtcattggccaacacttgtaattacttgtctttcagACCC~GCTCMGGTTCCTACAGATTCCTGGCTCCCTGGATTG PKAQGSYRFLAPUIG

ggtatgtacgactaaattaggactgaacccacttcccagttaagctt...INTRON

3,

D.SKbp...

tttttgccttttgtccttgatggaccttcttctttctccccaagtctctct

ctgtacccccaaattgtcccaggtcatccttttgatcctgtcacccacacacacacatgcacactcatctctggcccagGGTATGGTTTGCTCCTGCTGAATGGGCAGACGTGGTTCC~G YGLLLLNGPTUFQ CACCGGCGCATGCTCACCCCAGCCTTCCACTACGACATCCTGMGCCCTACGTGGGGCTCATGGCGGACTCCGTCCAAATCATGCTGgtgagtccgtctgtctgtcacctccacacaccc HRRMLTPAFHYDILKPYVGLMADSVQIML actcccagcacgcagtcacagactccgctgtcaaactcatgtcccccacagtcatcagacacagccacacaaagtagcactcaggcattgataggaagcag...lNTRON

4,

l.ZKbC,

. . . ..ttcatttacaatatttgctttacaagacaatttgttacattacatctgctctgtgtgagggtttctaaacaaacacctgggggttcaggaaagaaccgcagctccctgcccaggc cttgggtggaatcaaggccaaggcgcagcctccccacag~CAMTGG~GCAGCTGGTCAGCCAG~CTCCTCCCTGGAGGTCTTCCMGACATCTCCCT~T~CCCTG~CACCATC DKUEPLVSPDSSLEVFPDISLMTLDTI

<

ATGMGTGTGCCTTCAGCCACCAGGGCAGCGTCCAGTTGGACAGgttagtgataatccttttgtgtgtttgcttttagaaagctcttatttaatgaatacaaatttcacaggtacagctt MKCAFSHPGSVQLDR ??Kbp . ..actctgacctcgtccacgtcctctccctcagGMTTCCCAGTCCTACATCCA~GCTGTTGGGGACCTG~CAACCTGTTCTT NSQSYIQAVGDLNNLFF < TTCCCCAGTGAGGMCGTCTTTCATCAGAGTGACACCATCTACAGGCTGAGCCCTGAAGGCCGCTTGTCCCACCGTGCCTGCCAGCTCGCCCAC~GCACACAGgtgctgcctcctcctc SRVRNVFHQSDTIYRLSPEGRLSHRACQLAHEHTD ttggaatatagcggttc...INTRON

5,

ctgtgtccccctccttgagcgagcactgataaaggagctgacatcaactcataacggagacgg...INTRON

6, 0.3Kbp . ..acccacacacccactcccacgctcaccctgacctc

FIG. 2. Sequence of the CYP4A6 5’ flanking region, exons, and adjacent sequences. Intronic sequences are shown in lower case and the estimated intron sizes are given. The predicted amino acid sequence of CYP4A6 is indicated below the exonic sequences. Primers used for sequence analysis or primer extension analysis are indicated by the long arrows. The site of transcription initiation is indicated by +l. Spl, HNF-1, and HNF-4 elements, CCAAT boxes, and the ll-bp element conserved between the CYP4A6 and the CYP4Al genes are underlined. The region within the CYP4A6 5’-flanking sequence exhibiting homology with the acyl-CoA oxidase A-element is also indicated.

70

MUERHOFF,

GRIFFIN, AND JOHNSON >

cagggcacccgggttggggcaggtggggctctcagctgctccggtacccactgctctggcacegACCGAGTGATCCAGCA~G~GGCTCAGCTGCAGCAG~GGGG~GCTG~~G RVIPPRKAPLPPEGELEK

<

GTCAGGAGGMGAGGCGCTTGGACTTCCTGGACGTCCTCCTCTTTGCCMGgtcagtgtgtgcagggcaggcccgagcttggcctggaagcacagagccagggacagagcagagcccgtg VRRKRRLDFLDVLLFAK ccctctctcccgctgtggcctcccccagATGGAGMCGGGAGCAGCCTGTCC~CCAG~CCTCCGCGCC~GGTG~CACGTTCAlGTTC~GGGCCAC~CACCACGGCCAGCGGCAT MENGSSLSDGDLRAEVDTFMFEGHDTTASGI

<

CTCCTG;ATCTTCTATGCCCTGGCCACGCACCCC~GCATCAGCACCGGTGCCGCGAGGAGATCCAGGGCCTCCTGGGG~CG~GCCTCCATCACCTGgtgagtgagagcccaagggtg SUlFYALATHPEHQHRCREElQGLLGDGASITW aggcagggggccctc...INTRON 8, D.ZKbp . ..agaggggccctgcctgtctgggagaagctgggggcccagcccttccccctcttctgcctgggctttgttttcagG~G~CCT E H -> GGACCAGATGCCCTACACCACCATGTGCATCMGGAGGC~T~~CTCTACCCACCAGTGCCAGGTGTCGGCAGACAGCTCAGCTCACCTGTCACCTTCCCT~T~~CGCTCCCTCCC DPMPYTTMCIKEAMRLYPPVPGVGRPLSSPVTFPDGRSLP CMGGgtacgaactgcccaccctcacctaagckctcccaaggacacatggaggatccgacgtcctgggagctcctgcagctccaagcctctctg...lNTR~ KG > gtgggatcagccttttctctctgtctcttcctgccccaaagGTGTCATAGTCACGCTCTCCATCTACGCCCTTCACCACMCCC~GGTGTGGCC~CCCAGAGgtacgagggtcccg VIVTLSIYALHHNPKVUPNPE

9,

L

1.3Kbp..atgg

gaaggagggcaggggatgagccccggaeccccacacctcctcatttgactcccatctctatgggctctgtcccctcccgggttggaggcagagcttgaccccacccgtcctggccctggtg < ctctcctcagGTGTTTGACCCTTCCCGCTTCGCACCGGGTTCTGCTCGCCACAGCCACGCTTTCCTGCCCTTCTCAGGAGGACCACGgtgagtgtccgtgtgcgctgacagagtcggggg VFDPSRFAPGSARHSHAFLPFSGGPR INTRON 11, l.OKbp..tgcccagctcagaggaggggcctgaagcgcttctccttgcactgtccctgcctggcccagCMCTGCATCGGC atgatggggagtctcagggctca... N C I G K -> GCMTTTGCCATGMTGAGCTGAAGGTGGCCGTGGCCCTCACCCTCGTGCGCTTCCAGCTGCTGCCAGATCCCAMAGAGTCCCGGACCAAAAACCACGTCTTGTGCTGMGTCCAGCM PFAMNELKVAVALTLVRFELLPDPKRVPDQKPRLVLKSSN > CGGWTCCACCTGCGTCTGAGGMGCTCCGCTCCGCTMCCCTGGTGGGGACMGAGCAGGCTCTGGGGGCCTTCTGCCAGGCGTCCTGGCTTCCTGTCAcCTGCCCATGCCCCCTGCCTGTCTG GIHLRLRKLR* > CCCACATCCTGCTTTCTATCCACCAGCACTTCTTCCACCTGTCTGCCTTGCTGCCTCTTGGCCTCCAGGCTGTCTGTCCTCTCGCACCTTCCTCTGGGCCACTGACCTGTCTGTCTACTG TCCGCTTCCTGCCAGCATCTCTGACCGTGCACCTMCGCTGGTCACTGGCAGCCCTCTGCTGCCTCCCTTCCGCTGGTCCTTTCCCTCTGCTGCCGTCCGTGGATGCAGTTCCGACAGCT > GCTGCACTATCTCGGCTGTGATGGTTCTCCTCTTGCAGTCCTGGTGGTCGGAGTCTGMACACCTGTCACTGCTGGGAGT~GCMGCTGCCAGCTGGGCCAGGCTCCCTCTGCGGTTC TAGGAGCGMTCCCCGCCTCGGCTCTTCAGATCTGTGTTCCTGGCACCCTGCAGMCTGGCCCCTCTCTCCTCCTTAAAGCCAGCAGCATCTTTCTCTCTCTCTCTCTCTCTCTCTCTCT CTCTCTgtccacatctcccctctcacaaa...INTRON

12,

??Kbp...

ctcttgtgattacatcaaagccactcagctaacctaggcgagtttgccctggccatctttcacttaatc

acatctctccaaattccctttttgcaagagatggggtcacacactcaaggattccataggttccatgtgtccatcttagagctattggtcattgtaacccagtcctgactgtcaccccat

FIG. 2-Continued

fleet a functional sequence. In addition, a sequence matching the first 11 of 12 nucleotides that constitute a consensus HNF-4 binding site (26) occurs just upstream of the transcription start site (Fig. 4). This sequence is highly conserved in the rat CYP4Al gene at this position. However, the rat CYP4A2 gene does not exhibit either apparent Spl or HNF-4 recognition sequences at these positions. An additional segment of sequence identity of 11 bp is observed in the rat CYP4Al and CYP4A6 gene at -382 to -372 and -221 to -211, respectively (Fig. 2). This sequence is not observed in the 5’ flanking region of the rat CYP4A2 gene. The sequence from -106 to -94 in the CYP4A6 gene (Fig. 2) matches the consensus recognition sequence for hepatocyte nuclear factor 1 (HNF1) (27) at 9 of 13 residues. There are also two CCAAT boxes located at -464 and -448 in the CYP4A6 5’ flanking region (Fig. 2).

CYP4A6 is induced by clofibrate in liver and kidney (3). As shown in Fig. 5, the 5’ flanking region of CYP4A6 exhibits sequence similarity with a portion of the rat acylCoA oxidase gene which has been shown to control the inductive response of the latter to the peroxisome proliferator, ciprofibrate (14). Footprinting experiments indicate that two regions, A and B (Fig. 5), within this segment of the acyl-CoA oxidase gene, are protected whether nuclear proteins are extracted from cells incubated with or without ciprofibrate. The A and B segments, either alone or in combination, were placed immediately upstream of the minimal promoter of the acyl-CoA oxidase gene in plasmids harboring the CAT reporter gene. When H4IIEC3 rat hepatoma cells were transfected with these constructs, the A region was found to confer a l&fold increase in transcription in the absence of ciprofibrate and a 32-fold increase in the presence of ciprofibrate. In

STRUCTURE

247 nt

160 nt

139 nt

FIG. 3. Primer extension analysis. The primer extension products generated from RNA templates isolated from individual untreated (control) or clofibrate-treated rabbits were analyzed on a sequencing gel. An autoradiographic image is shown. The sizes of the products were determined from the sequencing ladder shown in the adjacent lanes. The sixes of the pertinent products are indicated on the left. Details are described under Experimental Procedures.

contrast, the B element suppressed slightly the expression from the basal promoter in the presence or absence of ciprofibrate. However, when both elements were placed together upstream of the basal promoter, transcription was enhanced by only 2-fold in the absence of ciprofibrate, but transcription was increased by g-fold in its presence. The authors (14), therefore, surmised that the A segment

71

OF CYP4A6

was a positive regulator of transcription whereas the B segment was a negative regulator. The maximal ciprofibrate-dependent transcriptional activation occurred when both A and B segments were present together. While there is no identity with the B-element in the 5’ flanking region of the CYP4A6 gene, a segment of the 5’ flanking region of the CYP4A6 gene at -740 to -723 exhibits significant identity with the reverse orientation of the A element (Fig. 5), implicating this region as a potential controlling element regulating the induction of the CYP4A6 gene by peroxisome proliferators. Interestingly, as noted by Osumi et al. (14), this region contains significant sequence identity with an element found within the promoter region of the apolipoprotein AI and CIII genes. It has recently been demonstrated that the latter element can function as a responsive element for the transcription factors HNF-4 (26) and/or ARP-1 (28), which are members of the steroid hormone receptor superfamily. Another member of the steroid hormone receptor superfamily is activated by peroxisome proliferators, and it is referred to as the peroxisome proliferator-activated receptor or PPAR (15). It is not known whether or not the response element of the acyl-CoA oxidase gene interacts directly with this receptor. Based on the amino acid sequence of the zinc finger region of the PPAR, it has been suggested by analogy to other receptors that its DNA recognition sequence might be related to that of the thyroid hormone, retinoic acid, and vitamin D3 receptors (15). These three receptors have been shown to recognize a series of direct repeats closely related to the sequence AGGTCA where the selectivity of these receptors for a specific recognition site reflected the spacing between the two half-sites (29). A tandem repeat of this nature, AGGACA A AGGTCA, occurs in the A-element of the acylCoA oxidase gene, and a similar repeat, AGGACA A AGGCCA, is found at -740 in CYP4A6 (Fig. 5). Interestingly, the spacing between the two half-sites, 1 nucleotide, is different from that for the vitamin Ds, thyroid hormone, and retinoic acid receptors, 3, 4, and 5 nucleotides, respectively (29). However, a single nucleotide spacing has been reported between direct repeats recognized by the retinoid X receptor (30). Future experiments

9Pl

HNF-4

CCCCACCCTTCGCTCTCCCCATAGGn;GGCGGGGCAACGCTCCT~CT~C~~TCAGTGACA~ GCCTCACCTTTCTCCCTCCCACAGGTAGGCGGGCAATCCTTACA~ATTTAGGC~GA~~G~~AT TCTACTCTCCCTCCCACAAGTAGGTGTGTCTACCTCTACCTCCTCATGATTT~TGCA~TGTTCCACA~TTCAG

+1 GAGGGACAG...Exon +1 CTGTGAGAG...Exon +1 AGACCCTAG...Exon

1

CYP4A6

1

CYP4Al

1

CYP4A2

FIG. 4. A comparison of the promoter regions of the CYP4A6, CYP4A1, and CYP4A2 genes. The Spl consensus recognition sequence is overlined in CYP4A6 as is the GC box in CYP4Al. A sequence closely related to the HNF-4 consensus recognition sequence in CYP4A6 is also overlined. The 19-bp conserved element between CYP4AI and CYP4A2 is underlined. Regions in the CYP4A6 gene that are similar to sequences found in CYP4Al are underlined.

72

MUERHOFF,

GRIFFIN,

I

ACOX

CYP4A6

-516

-782

AND

JOHNSON

I

CATTTTACTGGAACCTAACCAAATAGGAGCAAAAGGGGAC I II TCACCTTGTGCTGTAGGTGCACAACAGGTTCTGAGACAAGTCC...MGAGTCCCTGGTCATGG 4HREM +HRE-,

III

I

II

I IIIIIIIIIIIIIII

-597

IIIIIIIIIIII

-103

FIG. 5. Sequence identity between the ciprofibrate responsive element of the acyl-CoA oxidase (ACOX) gene and a portion of the CYP4A6 5’ flanking region. Two segments determined to be important in mediating the transcriptional activation of the acyl-CoA oxidase gene by ciprofibrate are designated A and B (14). The conserved bases between the two genes are indicated by the vertical lines. Tandem repeats in CYP4A6 and ACOX related to recognition elements for the thyroid hormone, retinoic acid, and vitamin Ds receptors (29) but which differ in the spacing of the half sites are indi&d by HRE and arrows.

will address the functional significance of these observations. More than 8 kb of 5’ flanking DNA of the CYP4A6 gene has been cloned. Work is in-progress to generate Eonstructs containing sequences derived from the flanking region which will then be used to identify regulatory sequences in gene transfer experiments. It is anticipated that this will reveal the mechanism of the induction of this and other CYP4A genes by peroxisome proliferators and aid in the characterization of the proteins that regulate their expression. In addition, it should be possible to determine whether or not the PPAR directly regulates the induction of the CYP4A6 gene. REFERENCES 1. Yokotani, N., Sogawa, K., Matsubara, S., Gotoh, O., Kusunose, E., Kusunose, M., and Fujii-Kuriyama, Y. (1990) Eur. J. %&em. 187, 23-29. 2. Johnson, E. F., Walker, D. W., Griffin, K. J., Clark, J. E., Okita, R. T., Muerhoff, A. S., and Masters, B. S. (1990) Biochemistry 29, 873-879. 3. Yokotani, N., Bernhardt, R., Sagawa, K., Kusunose, E., Gotoh, O., Kusunose, M., and Fujii-Kuriyama, Y. (1989) J. Bid. &em. 264, 21,665-21,669.

12. Hardwick,

J. P., Song, B-J., Huberman, (1987) J. Biol. Chem. 262,801-810.

E., and Gonzalez,

F. J.

13. Reddy, J. K., Goel, S. K., Nemali, M. R., Carrino, J. J., Laffler,

T. G., Reddy, M. K., Sperbeck, S. J., Osumi, T., Hashimoto, T., Lalwani, N. D., and Rao, M. S. (1986) Proc. Natl. Acad. Sci. USA 83,1747-1751.

14. Osumi, T., Wen, J.-K., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 175,866-871.

15. Issemann, I. and Green, S. (1990) Nature 347,645-650. 16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 17. Pendurthi, U. R., Lamb, J. G., Nguyen, N., Johnson, E. F., and Tukey, R. H. (1990) J. Bid. Chem. 265,14,662-14,668. G., and Schneider, C. (1989) BioTechniques 18. Del Sal, G., Manfioletti, 7,514-519. 19. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Biol. 143, 161-178. 20. Brudzinski, C. J., and Gelehrter, T. D. (1989) DNA 8,691-696. 21. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-

159.

9. Bather, M. A., and Gibson, G. G. (1988) Chem. BioL Inter. 65,145156.

22. Yamamoto, S., Kusunose, E., Kaku, M., Ichihara, K., and Kusunose, M. (1986) J. Biochm. 100,1449-1455. 23. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. 24. Kadonaga, J. T., Jones, K. A., and Tjian, R. (1986) Trends Biochem. Sci. 11,20-23. 25. Kimura, S., Hanioka, N., Matsunaga, E., and Gonzalez, F. J. (1989) DNA 8,503-516. 26. Sladek, F. M., Zhong, W., Lai, E., and Darnell, J. E., Jr. (1990) Genes Dev. 4,2353-2365. 27. Courtois, G., Baumhueter, S., and Crabtree, G. R. (1988) Proc. Natl. Ad. Sci. USA 85, 7937-7941. 28. Ladias, J. A. A. and Karathanasis, S. K. (1991) Science 251,561565. 29. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) CeU 65,1255-1266. 30. Mangelsdorf, D. J., Umesono, K., Kliewer, S. A., Borgmeyer, U., Ong, E. S., and Evans, R. M. (1991) Cell 66,555-561.

10. Reddy, J. K., Azamoff, D. L., and Higinite, C. E. (1980) Nature 283,397-398. 11. Kimura, S., Hardwick, J. P., Kozak, C. A., and Gonzalez, F. J. (1989) DNA 8,517-525.

31. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sate, R., Waterman, M. R., and Waxman, D. J. (1991) DNA Cell Bid. 10, 1-14.

4. Williams, D. E., Hale, S. E., Okita, R. T., and Masters, (1984) J. Bid. &em. 259,14,600-14,608.

B. S. S.

5. Powell, W. S. (1978) J. Biol. Chem. 253,6711-6716. 6. Matsubara, S., Yamamoto, S., Sogawa, K., Yokotani, N., Fujii-Kuriyama, Y., Haniu, M., Shively, J. E., Gotoh, O., Kusunose, E., and Kusunose, M. (1987) J. Bid. Chem. 262,13,366-13,371. 7. Muerhoff, A. S., Williams, D. E., Leithauser, M. T., Jackson, V. E., Waterman, M. R., and Masters, B. S. S. (1987) Proc. Natl. Acad. Sci. USA 84,7911-7914. 8. Gibson, G. G. (1989) Xenobiotica 19, 1123-1148.