A Novel cis-Acting Element in a Liver Cytochrome P450 3A Gene Confers Synergistic Induction by Glucocorticoids plus Antiglucocorticoids
1995; Elsevier BV; Volume: 270; Issue: 48 Linguagem: Inglês
10.1074/jbc.270.48.28917
ISSN1083-351X
AutoresLinda C. Quattrochi, A. Scott Mills, Joyce L. Barwick, Charles B. Yockey, Philip S. Guzelian,
Tópico(s)Hormonal Regulation and Hypertension
ResumoThe induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16α-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5′-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA. The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16α-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation. In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5′-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid. Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN. Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I. The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used. Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element. A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays. Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used. We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA. INTRODUCTIONCYP3A1, a member of the cytochrome P450 supergene family, is prominently induced in liver microsomes of rats or in primary cultures of adult rat hepatocytes treated with the synthetic glucocorticoid, dexamethasone and, paradoxically, by such antiglucocorticoids as pregnenolone 16α-carbonitrile (PCN)1(1Elshourbagy N.A. Guzelian P.S. J. Biol. Chem. 1980; 255: 1279-1285Abstract Full Text PDF PubMed Google Scholar, 2Heuman D.M. Gallagher E.J. Barwick J.L. Elshourbagy N.A. Guzelian P.S. Mol. Pharmacol. 1982; 21: 753-760PubMed Google Scholar, 3Schuetz E.G. Wrighton S.A. Barwick J.L. Guzelian P.S. J. Biol. Chem. 1984; 259: 1999-2006Abstract Full Text PDF PubMed Google Scholar, 4Schuetz E.G. Guzelian P.S. J. Biol. Chem. 1984; 259: 2007-2012Abstract Full Text PDF PubMed Google Scholar, 5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). Studies using isolated nuclei from rats treated with dexamethasone have demonstrated that transcriptional activation of the CYP3A1 gene is the primary mechanism underlying the induction process(6Telhada M.B. Pereira T.M. Lechner M.C. Arch. Biochem. Biophys. 1992; 298: 715-725Crossref PubMed Scopus (51) Google Scholar, 7Simmons D.L. McQuiddy P. Kasper C.B. J. Biol. Chem. 1987; 262: 326-332Abstract Full Text PDF PubMed Google Scholar, 8Pereira T.M. Lechner M.C. Eur. J. Biochem. 1995; 229: 171-177Crossref PubMed Scopus (16) Google Scholar). However, detailed pharmacologic analysis of these phenomena in culture disclosed that the time course of induction and the rank order and dose response of synthetic steroid inducers was different for CYP3A1 expression as compared to that for a typical glucocorticoid-responsive gene, tyrosine aminotransferase (TAT)(3Schuetz E.G. Wrighton S.A. Barwick J.L. Guzelian P.S. J. Biol. Chem. 1984; 259: 1999-2006Abstract Full Text PDF PubMed Google Scholar, 4Schuetz E.G. Guzelian P.S. J. Biol. Chem. 1984; 259: 2007-2012Abstract Full Text PDF PubMed Google Scholar, 5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). Moreover, tests of agonist-antagonist relationships demonstrated that rates of de novo synthesis of immunoreactive CYP3A1 protein and accumulation of CYP3A1 mRNA were stimulated synergistically when hepatocyte cultures were incubated in the presence of dexamethasone plus PCN, even though the expression of TAT was inhibited by this same protocol(3Schuetz E.G. Wrighton S.A. Barwick J.L. Guzelian P.S. J. Biol. Chem. 1984; 259: 1999-2006Abstract Full Text PDF PubMed Google Scholar, 4Schuetz E.G. Guzelian P.S. J. Biol. Chem. 1984; 259: 2007-2012Abstract Full Text PDF PubMed Google Scholar, 5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). Glucocorticoids up-regulate TAT by entering the hepatocyte and forming a complex with the glucocorticoid receptor (GR). This is followed by binding of the GR to two glucocorticoid-responsive elements (GRE) located in the 5′-flanking region of the TAT gene, an event that activates TAT gene transcription in a cooperative manner(9Jantzen H.-M. Strahle U. Gloss B. Stewart F. Schmid W. Boshart M. Miksicek R. Schutz G. Cell. 1987; 49: 29-38Abstract Full Text PDF PubMed Scopus (528) Google Scholar). Based on our initial studies, we suggested that the regulation of CYP3A1 by glucocorticoids may proceed by a mechanism that differs from classical GR binding to a GRE that might be located upstream of the CYP3A1 gene(4Schuetz E.G. Guzelian P.S. J. Biol. Chem. 1984; 259: 2007-2012Abstract Full Text PDF PubMed Google Scholar).A possible explanation for our unusual findings was that glucocorticoids and antiglucocorticoids might affect induction of CYP3A1 at different steps in gene expression including post-transcriptional events such as translation or degradation of CYP3A1 mRNA. To investigate the role of CYP3A1 transcription specifically, we established a system for transient expression of 1.5 kilobases of DNA 5′-flanking the CYP3A1 gene2 fused to a chloramphenicol acetyltransferase (CAT) reporter plasmid in primary cultures of adult rat hepatocytes that maintain synergistic inducibility of hepatocellular CYP3A1 mRNA by dexamethasone plus PCN(5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). We found that CAT expression from this transfected CYP3A1 chimeric plasmid was induced by treatment of the cultures with dexamethasone or with PCN and was induced synergistically by treatment with dexamethasone plus PCN while expression of a transfected control plasmid containing the classical glucocorticoid inducible MMTV gene was induced by dexamethasone, was not induced by PCN, and was only slightly induced by dexamethasone plus PCN(5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). These results establish that the unique features of CYP3A1 regulation by glucocorticoids largely involve effects on CYP3A1 gene transcription. Preliminary analysis of deletions of the 1.5 kilobases of CYP3A1 DNA defined a 165-base pair (bp) fragment approximately 200 bp upstream of the transcriptional start site that maintains dexamethasone and PCN inducibility and PCN synergy when transiently expressed in cultured hepatocytes(5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). In the present study, we have exploited the advantages of this culture system to closely define the functional glucocorticoid controlling DNA that flanks the CYP3A1 gene. Finding that the sequence lacks a classical GRE, fails to bind GR, but binds to a pattern of liver nuclear proteins from control or dexamethasone-treated rats that appears the same, we conclude that activation of CYP3A1 gene transcription by glucocorticoids may involve a novel indirect interaction of GR with CYP3A1.MATERIALS AND METHODSAnimals and MaterialsAdult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 190-220 g were maintained with free access to animal chow and water for 1-2 weeks prior to use. Collagenase type I was purchased from Worthington Biochemical Corp. (Freehold, NJ), and all other tissue culture reagents were of the highest grade commercially available from Sigma. Matrigel was prepared from the Engelbreth-Holm-Swarm tumor(10Kleinman H.K. McGarvey M.L. Hassell J.R. Star V.L. Cannon F.B. Laurie G.W. Martin G.R. Biochemistry. 1985; 25: 312-318Crossref Scopus (1195) Google Scholar), kindly supplied by Dr. Hynda K. Kleinman, NIDR, NIH, Bethesda, MD. Other reagents were purchased from the following: Lipofectin (Life Technologies, Inc.), CAT-ELISA kit (Boehringer Mannheim), Nycodenz (Sigma), and enzymes (Life Technologies, Inc.). Dexamethasone was purchased from Sigma and PCN was a gift from John Babcock (Upjohn). pMMTVCAT (11Cato A.C.B. Miksicek R. Schutz G. Arnemann J. Beato M. EMBO J. 1986; 5: 2237-2240Crossref PubMed Scopus (271) Google Scholar) was provided by R. Miksicek and pBLCAT2 (12Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 224Google Scholar) was provided by G. Schutz (Institute for Cell and Tumor Biology, Heidelberg, Germany).Preparation of Hepatocytes and DNA TransfectionsPrimary cultures of adult rat hepatocytes were prepared as described previously (13Li D. Schuetz E.G. Guzelian P.S. Methods Enzymol. 1991; 206: 335-344Crossref PubMed Scopus (34) Google Scholar). Hepatocytes freshly isolated by collagenase perfusion of the livers of untreated rats were incubated in a humidified atmosphere of 95% air, 5% CO2 in our standard medium, a modification of Waymouth's MB-752 medium containing insulin (0.1 μM) as the only hormone. For DNA transfection experiments, 0.4 × 106 cells were plated in 35-mm plastic culture dishes (Lux) in 1 ml of medium supplemented with 5% fetal calf serum. The cultures were maintained from 24 to 66 h in serum-free Eagle's minimal essential medium (Life Technologies, Inc.) containing 0.1 μM insulin. At 66 h each dish of hepatocytes was washed with 1 ml of Opti-MEM (Life Technologies, Inc.) and then exposed to a mixture formed by combining 12 μg of Lipofectin with 3.5 μg of DNA (as specified by Life Technologies, Inc.). After 6 h of incubation with the cells, the Lipofectin/DNA mixture was removed and the cultures were incubated with 1 ml of Waymouth's medium containing 300 μg of Matrigel and inducers. Twenty-four h later the medium was replaced with Waymouth's medium containing inducers only. Inducers were added as 1000-fold stocks in dimethyl sulfoxide.Assay of CAT ProteinAfter 48 h of treatment with inducers, cell lysates were harvested and assayed for CAT protein (CAT-ELISA assay, Boehringer Mannheim). Briefly, each dish was washed two times with phosphate buffered saline and then incubated 30 min at 4°C in 1 ml of 40 mM Tris buffer, pH 7.4, containing 5 mM EDTA and 150 mM NaCl to solubilize the Matrigel. This buffer was replaced with 350 μl of lysis buffer (Boehringer Mannheim). Following a 30-min incubation at room temperature, the lysates were centrifuged at 12,000 × g for 10 min. CAT protein was measured according to instructions provided with the kit. Total cellular protein concentration was determined using a bicinchoninic acid microassay (Sigma). Transfection efficiency in several culture preparations was in the range of 5-10%, and CAT protein measured from duplicate plates varied by less than 10%.Plasmid Constructions and Preparation of Double-stranded OligonucleotidesThe construction of the chimeric plasmid containing the CYP3A1 gene 5′-flanking sequences from -1525 to -56 bp upstream of the transcription start site has been described previously (5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). CYP3A1 sequences consisting of 5′ and 3′ deletions were generated by the polymerase chain reaction and sequenced (14Sanger F. Nicklen S. Coulsen A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52343) Google Scholar) to confirm the accuracy of the amplification reaction. All CYP3A1 5′-flanking sequences were cloned in the 5′ to 3′ orientation in front of the thymidine kinase (TK) promoter of the pBLCAT2 expression vector(12Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 224Google Scholar). Constructions containing these sequences inserted 5′ to the CYP3A1 promoter appeared to support dexamethasone- and PCN-mediated CAT expression when transfected transiently into cultured hepatocytes, but the values were too low to give reliable results. Complimentary oligonucleotides spanning sequences protected against DNase I digestion (FP1, FP2), containing BamHI restriction sites at each end, were synthesized (Macromolecular Resources, Fort Collins, CO), annealed and the double-stranded (ds)-oligonucleotides purified by polyacrylamide gel electrophoresis. The ds-oligonucleotides were cloned into the BamHI site of pBLCAT2 as single and multiple copies and in both orientations. The FP1 oligonucleotide corresponds to the position -148 to -115 and FP2 oligonucleotide to -116 to -69. The sequence of the upper strands are as follows: FP1, 5′-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3′; FP2, 5′-GGATCCTAGGGGAAGAGAGTACCAAAGTCCACGTGAGGCAGAGGTGATCCATCTGGATCC-3′.Treatment of Animals and Preparation of Nuclear ExtractTwenty-one-day-old male, Sprague-Dawley rats were given dexamethasone 21-acetate (Sigma) in phosphate-buffered saline plus 10% ethanol intraperitoneally at a concentration of 100 mg/kg for 3 h. After treatment, animals were sacrificed, livers removed, and nuclei prepared by centrifugation through Nycodenz(15Graham J.M. Ford T. Rickwood D. Anal. Biochem. 1990; 187: 318-323Crossref PubMed Scopus (47) Google Scholar). Purified nuclei were extracted for proteins with high salt buffer containing 20 mM HEPES (pH 7.9), 12.5% glycerol, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.4 M KCl, and the protease inhibitors Pefabloc, leupeptin, and pepstatin A (Boehringer Mannheim). Extracts were dialyzed against 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM Pefabloc, and 0.5 mM DTT, aliquoted, and stored at -80°C until use. Protein concentration was determined using a bicinchoninic acid microassay.DNase I Footprinting AssayThe 165-bp BamHI-HindIII fragment (position -220 to -56) was labeled with 32P at the BamHI site (upper strand). The labeled probe was incubated with 60 μg of crude nuclear extract. Incubations were performed as specified in the instruction manual for the HotFoot footprinting kit (Stratagene, La Jolla, CA). After incubation, a partial DNase I digest was carried out by adding 50 μl of DNase I buffer and concentrations of DNase I (Stratagene) from 0.02 to 0.1 unit. DNA was extracted with phenol-chloroform, recovered by ethanol precipitation, and analyzed on a 12% denaturing polyacrylamide gel. Sequencing of the DNA probe was performed by the Maxam-Gilbert method (16Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (8991) Google Scholar) using a sequencing kit from United States Biochemical Corp. An Escherichia coli expressed protein consisting of the DNA-binding domain of the glucocorticoid receptor (dbGR, apoT7X556) (17Freedman L.P. Luisi B.F. Korszun Z.R. Basavappa R. Sigler P.B. Yamamoto K.R. Nature. 1988; 334: 543-546Crossref PubMed Scopus (345) Google Scholar) was a gift from Dr. Keith Yamamoto, University of California, San Francisco. The footprinting assay using the dbGR was essentially as described for nuclear extracts, except that 600 ng of dbGR was preincubated for 30 min on ice with ZnCl (final concentration, 250 mM) prior to the binding reaction. The MMTV DNA fragment, from -222 to +104 relative to the transcriptional start site, in the 5′ long terminal repeat of the mouse mammary tumor virus was labeled and used as a positive control for GR binding. This fragment contains three regions protected to varying degrees when used in nuclease protection assays employing partially purified receptor (18Miksicek R. Heber A. Schmid W. Danesch U. Posseckert G. Beato M. Schutz G. Cell. 1986; 46: 283-290Abstract Full Text PDF PubMed Scopus (143) Google Scholar).Electrophoretic Mobility Shift AssayThe ds-oligonucleotide, FP1, corresponding to the position -148 to -115, was radiolabeled with [γ-32P]ATP and polynucleotide kinase. For this assay, 15 μg of nuclear protein were incubated at room temperature for 20 min with 3 μg of poly(dI-dC) in a binding buffer consisting of 20 mM HEPES, pH 7.9, 60 mM KCl, 5 mM MgCl2, 2 mM DTT, and 10% glycerol. The binding reaction was continued for 20 min after addition of labeled oligonucleotide to the reaction. Competitor DNA was added during the preincubation period, prior to addition of labeled oligonucleotide. Gel electrophoresis was conducted using 1 × TBE buffer (0.089 M Tris borate, 0.089 M boric acid, 0.002 M EDTA). The sequences of the oligonucleotides used in the electrophoretic mobility shift assay (upper strands) are as follows: FP1, 5′-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3′; GRE, 5′-CTAGGCTGTACAGGATGTTCTGCCTAG-3′; NF-κB, 5′-AGTTGAGGGGACTTTCCCAGG-3′.RESULTSPreliminary analysis of deletions of chimeric plasmid TKCAT-1525/-56 that contains CYP3A1 sequences from -56 to -1525 relative to the transcriptional start site, previously identified a region of the rat CYP3A1 gene between -220 and -56 that, when transiently expressed in primary cultures of adult rat hepatocytes, responded to treatment of the cells with dexamethasone or PCN and exhibited dexamethasone plus PCN synergy(5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar). We noted that -220 to -56 contains neither a classical sequence for the 15-bp GRE (19Lucas P.C. Granner D.K. Annu. Rev. Biochem. 1992; 61: 1131-1173Crossref PubMed Scopus (163) Google Scholar) nor sequences identical to the consensus sequence, TGTTCT, for "half-sites" in the classical GRE(20Strahle U. Klock G. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7871-7875Crossref PubMed Scopus (258) Google Scholar). We prepared additional deletion mutants of the chimeric CAT plasmid, TKCAT-220/-56 (Fig. 1), transiently expressed these in primary cultures of rat hepatocytes, and measured CAT expression following 48 h treatment of hepatocytes with 10 μM dexamethasone or PCN (Fig. 1). Deletion of sequences between -220 and -176 or between -71 and -56 resulted in no loss of dexamethasone or PCN induction of CAT expression, whereas a further deletion between -176 and -148 resulted in a 2-fold loss of dexamethasone induction. The deletion extending beyond position -56 to -37 bp appeared to reduce CAT expression by PCN treatment, although CAT expression by dexamethasone treatment was not altered. These data suggest the possibility that a negative element lies between -56 and -37. However, the induction by dexamethasone on both the TKCAT-176/-71 (20-fold) and TKCAT-148/-71 (10-fold) constructions was substantial. As shown previously for the TKCAT-220/-56 construction(5Burger H.-J. Schuetz J.D. Schuetz E.G. Guzelian P.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2145-2149Crossref PubMed Scopus (89) Google Scholar), dexamethasone also produced dose-dependent increases in CAT expression for TKCAT-148/-71 (data not shown). From these results we concluded that the sequences between -148 and -71 are essential for the steroid inductions, although upstream sequences (-176 to -148) may potentiate hormone responsiveness.To examine whether trans-acting proteins bind to the identified CYP3A1 dexamethasone-responsive region, we performed in vitro DNase I footprinting assays. Radioactively labeled CYP3A1 DNA (-220/-56) was incubated with nuclear extracts from control rat liver, and then the mixture was digested with DNase I. The results of these "footprinting" studies showed two regions strongly protected and one region weakly protected against DNase I activity (Fig. 2, lane 3). The reaction was specific because addition of excess unlabeled DNA (-220/-56) was able to compete for binding to both protected regions (Fig. 2, lane 5), whereas the presence of excess nonspecific DNA in the reaction did not compete (Fig. 2, lane 6). Although dexamethasone induction of CYP3A1 in the liver might be expected to alter the pattern perhaps by creating additional footprints, use of nuclear extracts from dexamethasone-treated rat liver in the same experiment produced the identical footprints (Fig. 2, lane 4). Moreover, we found the same footprints when this experiment was carried out with the use of nuclear extracts isolated from untreated and dexamethasone-treated cultures of rat hepatocytes (data not shown).Figure 2DNase I footprint analysis of nuclear protein binding to the CYP3A1 dexamethasone-responsive element. The 32P-labeled CYP3A1 fragment from -220 to -56 (upper strand) was incubated with 60 μg of crude nuclear extract from untreated control and dexamethasone-treated animals. Lane 1, A+G sequence reaction; lane 2, CYP3A1 DNA, no nuclear extract; lane 3, control extract; lanes 4-6, induced extract. Specific binding was determined by competition reactions in which 100-fold molar excess of unlabeled CYP3A1 DNA (-220/-56) (lane 5, +) or nonspecific DNA (lane 6, +) was added prior to addition of labeled CYP3A1 DNA. Sequencing of CYP3A1 DNA (-220/-56) was performed by the Maxam-Gilbert method(16Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (8991) Google Scholar). Footprints 1 and 2 (FP1, FP2) are marked by solid lines. The dashed line indicates a footprint possibly due to weak interactions. Base pairs protected are delineated by negative numbers representing distance from the start of transcription.View Large Image Figure ViewerDownload Hi-res image Download (PPT)By alignment with the CYP3A1 DNA (-220/-56) sequence, we determined that the first protected region (FP1) was located from -135 to -117, while the second footprint (FP2) was from -108 to -85 (Fig. 2 and 3A). Thus, within the -220 to -56 DNA probe, both footprints, FP1 and FP2, were found within the 78-bp fragment identified from deletional analysis to be dexamethasone- and PCN-responsive (Fig. 1, TKCAT-148/-71). Close examination of the FP1 sequence (Fig. 3B) finds 8 bp in common with a 12-bp consensus GRE (19Lucas P.C. Granner D.K. Annu. Rev. Biochem. 1992; 61: 1131-1173Crossref PubMed Scopus (163) Google Scholar) and 4 of 5 nucleotides suggested as critical for GR binding(21Nordeen S.K. Suh B.J. Kuhnel B. Hutchison III, C.A. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar). The FP1 sequence also contains a palindromic sequence (underlined), ATGAACTTCAT, that overlaps one of two "direct repeats" of ATGAACT separated by 2 nucleotides (Fig. 3C). A 6-bp portion of these 7-bp repeated sequences, TGAACT, when examined on the lower strand (AGTTCA), is a DNA motif representing a high affinity binding site for several types of nuclear receptors including the peroxisome proliferator activator (22Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1514) Google Scholar) and the retinoic acid receptors(23Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1484) Google Scholar). The CYP3A1 gene is not regulated by peroxisome proliferators or retinoic acids, thus emphasizing that a DNA binding site identified by sequence analysis is not necessarily functional. In addition, we identified a sequence at the 3′ end of FP2 similar to that of an AP1 binding site (24Fisher R.C. Thorley-Lawson D.A. Mol. Cell. Biol. 1991; 11: 1614-1623Crossref PubMed Scopus (18) Google Scholar) (Fig. 3A).Figure 3Summary of the DNA-protein interactions at the CYP3A1 dexamethasone-responsive element. A, regions protected against DNase I digestion, referred to as FP1 and FP2, of dexamethasone-responsive CYP3A1 (-220/-56) are indicated by solid lines. The dashed line indicates a footprint possibly due to weak interactions. The sequence similar to an AP1 binding site (24Fisher R.C. Thorley-Lawson D.A. Mol. Cell. Biol. 1991; 11: 1614-1623Crossref PubMed Scopus (18) Google Scholar) is boxed. B, alignment of FP1 with the consensus GRE sequence(19Lucas P.C. Granner D.K. Annu. Rev. Biochem. 1992; 61: 1131-1173Crossref PubMed Scopus (163) Google Scholar). The 5 bases critical for glucocorticoid receptor binding are shown in bold(21Nordeen S.K. Suh B.J. Kuhnel B. Hutchison III, C.A. Mol. Endocrinol. 1990; 4: 1866-1873Crossref PubMed Scopus (169) Google Scholar). Lines between nucleotides show identity. C, the sequence of the ds-oligonucleotide containing the FP1 sequence (underlined) is shown. Arrows indicate its palindromic structure and a 7-bp direct repeat.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We prepared overlapping ds-oligonucleotides that span the 78-bp responsive region in CYP3A1 corresponding to -148 to -115 (contains FP1) and to -116 to -69 (contains FP2) to determine which if either of the sequences that were protected against DNase I retains responsiveness to inducers. When the ds-oligonucleotides were cloned into pBLCAT2 and transiently expressed in hepatocytes, only sequences spanning the area protected by FP1 (TKCAT-148/-115) were responsive to dexamethasone and PCN (6- and 3-fold, respectively) (Fig. 4A). The magnitude of the response of the FP1 containing construction to dexamethasone was comparable to the endogenous hepatic levels of CYP3A1 transcriptional activation by dexamethasone as demonstrated by nuclear run-on transcription assays(6Telhada M.B. Pereira T.M. Lechner M.C. Arch. Biochem. Biophys. 1992; 298: 715-725Crossref PubMed Scopus (51) Google Scholar, 7Simmons D.L. McQuiddy P. Kasper C.B. J. Biol. Chem. 1987; 262: 326-332Abstract Full Text PDF PubMed Google Scholar, 8Pereira T.M. Lechner M.C. Eur. J. Biochem. 1995; 229: 171-177Crossref PubMed Scopus (16) Google Scholar). When placed in the opposite orientation, i.e. 3′ to 5′, this -148/-115 ds-oligonucleotide mediates a similar level of CAT induction; however, multiple copies of this sequence did not increase CAT induction over and above the results with a single copy (data not shown). Thus, this single 33-bp element is able to confer both dexamethasone and PCN induction on CYP3A1 gene expression in cultured hepatocytes, although sequences flanking this element may be important in the modulation of responsiveness, as indicated by an increase in CAT induction when sequences from -148 to -176 are included.Figure 4Transient expression in cultured hepatocytes of CYP3A1 5′-flanking sequences encompassing FP1 and FP2. A, ds-oligonucleotides corresponding to -148/-115 (contains FP1) and -116/-69 (contains FP2) were cloned 5′ to TKCAT and transiently expressed in rat hepatocytes. Hepatocytes were treated with dexamethasone (10 μM) or PCN (10 μM). Footprint sequences are underlined. B, hepatocytes were transfected with either MMTVCAT or TKCAT-148/-115 and treated with either dexamethasone (0.1 μM), PCN (10 μM), or dexamethasone (0.1 μM) plus PCN (10 μM) for 48 h. CAT protein was determined as described under "Materials and Methods." The -fol
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