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• W1999859103 abstract "The functional mapping of the human cytochrome P4502D6 (CYP2D6) promoter in HepG2 cells revealed the presence of both positive and negative regulatory elements. One of these regulatory elements overlapped a sequence that is highly conserved in most members of the CYP2 family. This element, which consists of a degenerate AGGTCA direct repeat spaced by 1 base pair (DR1) and is known to be a target for members of the steroid receptor superfamily, was found to bind in vitro translated hepatocyte nuclear factor 4 (HNF4) in gel retardation analysis. Using HepG2 nuclear extracts, three protein-DNA complexes were formed on the DR1 element, one of which was confirmed to be dependent on the binding of HNF4. The other DR1 complexes were shown to be due to the interaction of the orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI). Experiments in COS-7 cells showed that HNF4 could activate the CYP2D6 promoter 30-fold. Surprisingly, mutation of the DR1 element produced a relatively minor 23% decrease in activity in HepG2 cells. Additionally, COUP-TFI was shown to inhibit HNF4 stimulation of the CYP2D6 promoter in COS-7 cells, suggesting that COUP-TFI could attenuate the effect of HNF4 in HepG2 cells. However, when HNF4 levels were increased in HepG2 cells by co-transfection, it resulted in the enhancement of CYP2D6 promoter activity, indicating that HNF4 could overcome the repressive effect of COUP-TFI. Therefore, the contribution of the DR1 element in controlling the transcription of the CYP2D6 gene depends on the balance between positively and negatively acting transcription factors. The functional mapping of the human cytochrome P4502D6 (CYP2D6) promoter in HepG2 cells revealed the presence of both positive and negative regulatory elements. One of these regulatory elements overlapped a sequence that is highly conserved in most members of the CYP2 family. This element, which consists of a degenerate AGGTCA direct repeat spaced by 1 base pair (DR1) and is known to be a target for members of the steroid receptor superfamily, was found to bind in vitro translated hepatocyte nuclear factor 4 (HNF4) in gel retardation analysis. Using HepG2 nuclear extracts, three protein-DNA complexes were formed on the DR1 element, one of which was confirmed to be dependent on the binding of HNF4. The other DR1 complexes were shown to be due to the interaction of the orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI). Experiments in COS-7 cells showed that HNF4 could activate the CYP2D6 promoter 30-fold. Surprisingly, mutation of the DR1 element produced a relatively minor 23% decrease in activity in HepG2 cells. Additionally, COUP-TFI was shown to inhibit HNF4 stimulation of the CYP2D6 promoter in COS-7 cells, suggesting that COUP-TFI could attenuate the effect of HNF4 in HepG2 cells. However, when HNF4 levels were increased in HepG2 cells by co-transfection, it resulted in the enhancement of CYP2D6 promoter activity, indicating that HNF4 could overcome the repressive effect of COUP-TFI. Therefore, the contribution of the DR1 element in controlling the transcription of the CYP2D6 gene depends on the balance between positively and negatively acting transcription factors. The cytochrome P450 superfamily represents a group of enzymes that are involved in the oxidative metabolism of both endogenous and foreign compound (1Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1657) Google Scholar, 2Gonzalez F.J. Pharmacol. & Ther. 1990; 45: 1-38Crossref PubMed Scopus (494) Google Scholar). The expression of P450 genes is subject to diverse regulatory controls, which display tissue-specific, sex-specific, and developmental patterns (2Gonzalez F.J. Pharmacol. & Ther. 1990; 45: 1-38Crossref PubMed Scopus (494) Google Scholar). Most foreign compound-metabolizing P450s are mainly expressed in the liver; however, some enzymes can also be detected in extrahepatic tissues such as lung, kidney, and intestine and in the brain (2Gonzalez F.J. Pharmacol. & Ther. 1990; 45: 1-38Crossref PubMed Scopus (494) Google Scholar). Certain hepatic P450s are constitutively expressed, while others are known to be induced by various foreign chemicals including phenobarbital, polycyclic aromatic hydrocarbons, and peroxisome proliferators (3Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). The latter two classes of chemicals act through the aryl hydrocarbon receptor (4Fujisawa-Sehara A. Yamane M. Fujii-Kuriyama Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5859-5863Crossref PubMed Scopus (165) Google Scholar) and peroxisome proliferator-activated receptor (5Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3059) Google Scholar), respectively, while the exact mechanism(s) responsible for transducing the response to phenobarbital have yet to be elucidated (3Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). In addition, the expression of some P450 enzymes can be modulated by endogenous steroid and peptide hormones (3Denison M.S. Whitlock Jr., J.P. J. Biol. Chem. 1995; 270: 18175-18178Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 6Mode A. J. Reprod. Fert. Suppl. 1993; 46: 77-86PubMed Google Scholar). In rodents for example, the sexually dimorphic expression of certain P450s is controlled by the sex-specific pattern of growth hormone secretion (6Mode A. J. Reprod. Fert. Suppl. 1993; 46: 77-86PubMed Google Scholar). Most of the regulatory effects on P450 expression are at the transcriptional level. However in some instances, such as the induction of cytochrome P4502E1 (CYP2E1) 1The abbreviations used are: CYPncytochrome P450n (eg. CYP2E1, cytochrome P4502E1)RXRretinoid X receptorCOUP-TFIchicken ovalbumin upstream promoter transcription factor IHNFhepatocyte nuclear factorCATchloramphenicol acetyltransferaseCTEcommon transcription elementARP1apoAI-regulatory protein 1EAR3v-ErbA related protein 3PPARperoxisome proliferator-activated receptorDBPD-box binding proteinC/EBPCCAAT-box/enhancer binding proteinWCEwhole cell extractsCMVcytomegalovirusDR1degenerate AGGTCA direct repeat spaced by 1 base pair. by ethanol, post-transcriptional mechanisms are also involved (7Koop D.R. Tierney D.J. BioEssays. 1990; 12: 429-435Crossref PubMed Scopus (221) Google Scholar). cytochrome P450n (eg. CYP2E1, cytochrome P4502E1) retinoid X receptor chicken ovalbumin upstream promoter transcription factor I hepatocyte nuclear factor chloramphenicol acetyltransferase common transcription element apoAI-regulatory protein 1 v-ErbA related protein 3 peroxisome proliferator-activated receptor D-box binding protein CCAAT-box/enhancer binding protein whole cell extracts cytomegalovirus degenerate AGGTCA direct repeat spaced by 1 base pair. Within the P450 superfamily, the CYP2 family is the largest and most diverse (1Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1657) Google Scholar). This family, whose members are mainly expressed in the liver, contains many of the drug-metabolizing isoforms and also some of the enzymes involved in the metabolism of endogenous substrates (for review see 8Henderson C.J. Wolf C.R. Gibson G.G. Progress in Drug Metabolism. Vol 13. Taylor & Francis, London1992: 73Google Scholar). In addition to the constitutively expressed CYP2 members, this family also contains isoforms that are regulated by phenobarbital, ethanol, and growth hormone (8Henderson C.J. Wolf C.R. Gibson G.G. Progress in Drug Metabolism. Vol 13. Taylor & Francis, London1992: 73Google Scholar). Regarding the study of CYP2 gene regulatory DNA elements and their corresponding transacting transcription factors, relatively little is known in comparison to members of the CYP1 family or some of the steroid metabolizing P450s (2Gonzalez F.J. Pharmacol. & Ther. 1990; 45: 1-38Crossref PubMed Scopus (494) Google Scholar). Research in this area has been hampered by the difficulty in maintaining the expression of, or the ability to induce, P450s in isolated hepatocytes or liver-derived cell lines. As a result, most of the data generated to date comes from studies using transient transfection of promoter constructs into various hepatoma cell lines. Nevertheless, using this approach some information has been obtained about the transcriptional control of the CYP2 genes. The transcription of the CYP2E1 gene was reported to be partly controlled by HNF1α (9Ueno T. Gonzalez F.J. Mol. Cell. Biol. 1990; 10: 4495-4505Crossref PubMed Scopus (86) Google Scholar), and that of CYP2C6 by DBP (10Yano M. Falvey E. Gonzalez F.J. Mol. Cell Biol. 1992; 12: 2847-2854Crossref PubMed Scopus (46) Google Scholar), both of these transcription factors being hepatocyte-enriched. A phenobarbital-responsive region was identified in the chicken CYP2H1 gene using transient transfection of primary chicken hepatocytes (11Hahn C.N. Hansen A.J. May B.K. J. Biol. Chem. 1991; 266: 17031-17039Abstract Full Text PDF PubMed Google Scholar), and a functional glucocorticoid response element was identified in the rat CYP2B2 gene promoter (12Jaiswal A.K. Haaparanta T. Luc P.V. Schembri J. Adesnik M. Nucleic Acids Res. 1990; 18: 4237-4242Crossref PubMed Scopus (54) Google Scholar). Analysis of the rabbit CYP2C1 and CYP2C2 promoters in HepG2 cells revealed the presence of a regulatory element, which was shown to be a target for HNF4 (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar), a member of the steroid receptor superfamily (14Sladek F.M. Zhong W. Lai E. Darnell Jr., J.E. Genes & Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (860) Google Scholar). Co-expression of HNF4 in COS-1 cells resulted in the induction of the CYP2C2 promoter (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar). Furthermore, mutation of the CYP2C2 HNF4 element resulted in a marked decrease in promoter activity in HepG2 cells (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar). This HNF4 element has been reported to be conserved in other members of the CYP2 family, and it was proposed to be of importance in the transcriptional control of other CYP2 genes (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar). Additional studies have also demonstrated a role for HNF4 in the transcriptional control of the human CYP2C9 gene (15Ibeanu G.C. Goldstein J.A. Biochemistry. 1995; 34: 8028-8036Crossref PubMed Scopus (73) Google Scholar). However, in contrast, studies of the rat CYP2C genes (CYP2C7, CYP2C11, CYP2C12, and CYP2C13) demonstrated that co-expressed HNF4 gave only a maximal 3-fold induction of promoter activity in COS-7 cells (16Strom A. Westin S. Eguchi H. Gustafsson J.-A. Mode A. J. Biol. Chem. 1995; 270: 11276-11281Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Furthermore, mutation of the HNF4 binding site in the respective promoters had no effect on the activity of the 2C7 or 2C11 promoters in HepG2 cells, while it caused decreases to 60 and 80% in the activity of CYP2C13 and CYP2C12 promoters, respectively (16Strom A. Westin S. Eguchi H. Gustafsson J.-A. Mode A. J. Biol. Chem. 1995; 270: 11276-11281Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Regarding the CYP2D subfamily, using in vitro transcription analysis and transient transfections into HepG2 cells, it was possible to identify basal and sex-specific regulatory elements in the mouse CYP2D genes (17Yoshioka H. Lang M. Wong G. Negishi M. J. Biol. Chem. 1990; 265: 14612-14617Abstract Full Text PDF PubMed Google Scholar, 18Yokomori N. Kobayashi R. Moore R. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1995; 15: 5355-5362Crossref PubMed Scopus (81) Google Scholar). Another CYP2D promoter that has been analyzed is that of the rat CYP2D5 gene, where it was reported that C/EBP and Sp1 cooperate in controlling its transcriptional activity (19Lee Y.-L. Yano M. Liu S.-Y. Matsunaga E. Johnson P.F. Gonzalez F.J. Mol. Cell. Biol. 1994; 14: 1383-1394Crossref PubMed Google Scholar). There was no evidence presented in support of a role for the HNF4 binding site in the modulation of CYP2D5 expression (19Lee Y.-L. Yano M. Liu S.-Y. Matsunaga E. Johnson P.F. Gonzalez F.J. Mol. Cell. Biol. 1994; 14: 1383-1394Crossref PubMed Google Scholar). Human CYP2D6 is known to play a major role in the metabolism of a wide range of clinically important drugs (20Daly A.K. Cholerton S. Gregory W. Idle J.R. Pharmacol. & Ther. 1993; 57: 129-160Crossref PubMed Scopus (201) Google Scholar). It is also polymorphic, with 5-10% of the Caucasian population classified as poor metabolizers of CYP2D6 substrates (21Daly A.K. Armstrong M. Monkman S.C. Idle M.E. Idle J.R. Pharmacogenetics. 1991; 1: 33-41Crossref PubMed Scopus (114) Google Scholar). This is caused by mutations within the gene resulting in the absence of CYP2D6 protein (22Gough A.C. Miles J.S. Spurr N.K. Moss J.E. Gaedigk A. Eichelbaum M. Wolf C.R. Nature. 1990; 347: 773-776Crossref PubMed Scopus (325) Google Scholar). This polymorphism was subsequently reported to be associated with the incidence of various forms of cancer (23Wolf C.R. Smith C.A.D. Gough A.C. Moss J.E. Vallis K.A. Howard G. Carey F.J. Mills K. McNee W. Carmichael J. Spurr N.K. Carcinogenesis. 1992; 13: 1035-1038Crossref PubMed Scopus (139) Google Scholar) and the susceptibility to Parkinson's disease (24Smith C.A.D. Gough A.C. Leigh P.N. Summers B.A. Harding A.E. Maranganore D.M. Sturman S.G. Schapira A.H.V. Williams A.C. Spurr N.K. Wolf C.R. Lancet. 1992; 339: 1375-1377Abstract PubMed Scopus (428) Google Scholar). The CYP2D6 protein was also reported to be absent until the first week after birth (25Treluyer J.-M. Jacqz-Aigrain E. Alvarez F. Cresteil T. Eur. J. Biochem. 1991; 202: 583-588Crossref PubMed Scopus (188) Google Scholar), suggesting that its expression might be repressed by maternal hormones. Given that the levels of CYP2D6 expression may be critical in the responsiveness to certain clinically used drugs and in disease susceptibility, it is important to understand how CYP2D6 expression is controlled at the transcriptional level. Therefore, in this paper, we have performed the functional analysis of the CYP2D6 promoter and investigated what role the HNF4 binding site plays in controlling transcription of the CYP2D6 gene. First, the results indicate that both positive and negative regulatory elements contribute toward promoter activity. Second, although the HNF4 binding site alone appears to play a relatively minor role in HepG2 cells, the findings indicate that the balance between HNF4 and negatively acting transcription factors is an important factor. HepG2 and COS-7 cells were grown in monolayer and cultured in Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin (all from Life Technologies, Inc.) at 37°C in 5% CO2. DNA transfections were carried out by the calcium phosphate method (26Parker B.A. Stark G.R. J. Virol. 1979; 31: 360-369Crossref PubMed Google Scholar) as described by Gorman (27Gorman C. Glover D.M. DNA Cloning: A Practical Approach. Vol 3. IRL Press, Oxford1986: 143Google Scholar) with the exception that the glycerol step was omitted. Cells were harvested 24-36 h after transfection, extracts were prepared, and chloramphenicol acetyltransferase (CAT) activity was assayed as described by Gorman et al. (28Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). Cell extracts were assayed for protein content (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217507) Google Scholar). All CAT assays were performed such that the rate of acetylation was in the linear range. In all experiments, the values given represent the mean ± S.E. of at least three experiments. A minimum of two plasmid preparations were used for each construct. Cells were also co-transfected with pSVgal (Promega) to assay for β-galactosidase activity as a control for transfection efficiency. Using this technique, transfection efficiency was found to vary by less than 10%. The expression plasmid pCMVHNF4 was kindly provided by Prof. B. Kemper (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar), and the pMTHNF4 and pMTEAR3 constructs were gifts from Dr. J. Ladias (30Ladias J.A.A. Hadzopoulou-Cladaras M. Kardassis D. Cardot P. Cheng J. Zannis V. Cladaras C. J. Biol. Chem. 1992; 267: 15849-15860Abstract Full Text PDF PubMed Google Scholar). For the expression of HNF4 and COUP-TFI in COS-7 cells, 75-cm2 tissue culture flasks were transfected with 10 μg of pMTHNF4 or pMTEAR3, respectively, as described above. After 36 h, cells were harvested, and whole cell extracts were prepared by three cycles of freeze-thawing in 0.4 mM KCl, 20 mM Tris-HCl, pH 8, 2 mM dithiothreitol, and 20% (w/v) glycerol. Extracts were centrifuged at 10,000 x g for 15 min at 4°C to remove debris, and phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM. The CYP2D6 promoter was isolated by polymerase chain reaction from human genomic DNA using the upstream oligonucleotide 5ʹ-CAGATAAGCTTGCTGAAGGTCACTCT-3ʹ (with HindIII site) and downstream oligonucleotide 5ʹ-GGGCTCCTCTAGACACACCTCCCACC-3ʹ (with XbaI site). The resulting promoter fragment (-392 to +56) was subcloned between the HindIII and XbaI sites in pCATbasic (Promega) and checked by sequencing. Deletion fragments of the CYP2D6 promoter were generated using the Erase-a-Base system following the manufacturer's protocol (Promega). After confirming the sequence of the various promoter fragments, they were subcloned into the HindIII and XbaI sites of pCATbasic (Promega) after the addition of a HindIII linker at the 5ʹ end. The -69CATMUT construct was prepared by polymerase chain reaction using the 5ʹ-mutated oligonucleotide with HindIII linker 5ʹ-TTGGAAGCTTTTCACTCACAGCAGCTTTACACTTAATCATCAGCTCCC-3ʹ and the 3ʹ oligonucleotide with XbaI linker 5ʹ-AACCTCTAGACACACCTGGCACCCCCACCC-3ʹ. After sequencing, the mutated fragment was subcloned into the HindIII and XbaI sites of pCATbasic (Promega). Nuclear extracts from HepG2 cells were prepared as described by Dignam et al. (31Dignam S.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Radiolabeled probe for DNA-binding reactions was prepared by isolating the promoter fragment spanning -69 to +56 from -69CAT, dephosphorylating the DNA with alkaline phosphatase (Boehringer Mannheim), and phosphorylating with [γ-32P]ATP (Amersham Corp.; >5000 Ci/mmol) and T4 polynucleotide kinase (Promega). Binding reactions of 20 μl were carried out in buffer containing 10 mM Hepes, pH 7.5, 2.5 mM MgCl2, 10% (w/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 2 μg of poly(dI-dC) (Pharmacia Biotech Inc.), 50 mM KCl, 0.1-0.3 ng of radiolabeled probe, and the indicated amount of protein. Reactions were incubated at 20°C for 20 min. Free and protein-bound DNA were separated on 4% nondenaturing polyacrylamide (acrylamide:bisacrylamide, 37.5:1 (v/v)) gels, which were run at 4°C and a constant voltage of 200 V in 0.25 x TBE (22 mM Tris borate, 0.5 mM EDTA). Where indicated, competitor oligonucleotides or specific antiserum was included in the DNA-binding reactions. The CTE oligonucleotide corresponds to the sequence spanning -69 to -28 of the CYP2D6 promoter, which includes the degenerate AGGTCA direct repeat separated by one base pair (DR1) element 5ʹ-TTCACTCACAGCAGAGGGCAAAGGCCATCATCAGCTCCCTTT-3ʹ. The Sp1 oligonucleotide corresponds to the Sp1 consensus sequence 5ʹ-ATTCGATCGGGGCGGGGCGAGC-3ʹ from the SV40 early promoter (32Kadonaga J.T. Jones K.A. Tjian R. Trends Biochem. Sci. 1986; 11: 20-23Abstract Full Text PDF Scopus (878) Google Scholar). Anti-HNF4 antiserum was kindly provided by Dr. Francis Sladek (14Sladek F.M. Zhong W. Lai E. Darnell Jr., J.E. Genes & Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (860) Google Scholar). Anti-COUP antiserum was kindly provided by Dr. Ming-Jer Tsai (33Wang L.H. Tsai S.Y. Cook R.G. Beattie W.G. Tsai M.-J. O'Malley B.W. Nature. 1989; 340: 163-166Crossref PubMed Scopus (388) Google Scholar). Gel retardation analysis with COS-7 whole cell extracts was carried out under the conditions described above. The plasmid pSG5HNF4 (gift from Dr. Francis Sladek (14Sladek F.M. Zhong W. Lai E. Darnell Jr., J.E. Genes & Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (860) Google Scholar)), was linearized with BglII. T7 RNA polymerase was then used to generate HNF4 transcript, which was then translated using rabbit reticulocyte lysate. Reactions were carried out in a final volume of 25 μl containing 17.5 μl of rabbit reticulocyte lysate (Promega), 20 μM amino acids (including methionine), 20 units of RNasin (Promega), and 0.5 μg of HNF4 transcript. Reactions were incubated for 90 min at 30°C. In order to determine potential regulatory elements in the CYP2D6 promoter, progressive 5ʹ deletions were generated. The various promoter fragments were then fused to the CAT gene in pCATbasic and transiently transfected into HepG2 cells to assay for promoter activity. As can be seen in Fig. 1, the fusion of CYP2D6 promoter sequences from -392 to +56 upstream of the CAT gene resulted in the 30-fold induction of CAT activity when compared with pCATbasic. No activity was observed when the same construct was transfected into HeLa cells (data not shown). The deletion of sequences from -392 to -308 resulted in a 28% drop in activity in HepG2 cells, and further deletion to -242 had no additional effect. However, removal of sequences down to -156 produced an additional 2-fold decrease in activity. The presence of a negative regulatory element between -128 and -90 was indicated by the approximately 2-fold increase in CAT activity. Further deletions to -69 and -18 produced additional 2.5- and 3.5-fold decreases in activity, respectively. Therefore, the deletion analysis revealed the presence of four positively acting regulatory regions (-392/-308, -242/-156, -90/-69, and -69/-18), and one negative element (-128/-90). It is noteworthy that none of the deletions produced a difference in activity in excess of 2-3-fold, despite that fact that overall promoter activity was 20-30-fold higher than pCATbasic. This would suggest that these regulatory elements work together to control the activity of the CYP2D6 promoter. The deletion of sequences between -69 and -18 produced the largest change in CAT activity (Fig. 1). This region contains, in addition to the TATA-box, a sequence that is highly conserved within the CYP2 family (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar) (see Fig. 2A). This element consists of a DR1. In addition to the high degree of conservation, perhaps the most striking feature is the fact that the nucleotide at the fourth position in both half-sites of every element is nonconserved with respect to the consensus DR1. Whether this characteristic has any functional significance is unclear at present. The DR1 elements of other CYP2 genes have been reported to bind HNF4 (13Chen D. Lepar G. Kemper B. J. Biol. Chem. 1994; 269: 5420-5427Abstract Full Text PDF PubMed Google Scholar, 15Ibeanu G.C. Goldstein J.A. Biochemistry. 1995; 34: 8028-8036Crossref PubMed Scopus (73) Google Scholar, 16Strom A. Westin S. Eguchi H. Gustafsson J.-A. Mode A. J. Biol. Chem. 1995; 270: 11276-11281Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Therefore, we examined if the CYP2D6 DR1 was a target for in vitro translated HNF4 in gel retardation analysis. The addition of in vitro translated HNF4 to the radiolabeled -69/+56 promoter fragment containing the DR1 element resulted in the formation of one major (complex A) and one minor (complex b) protein-DNA complex (Fig. 2B, lane 2). Complex A was shown to be specific, since it was competed out by the addition of an oligonucleotide (CTE) spanning the DR1 site (lane 3) but not by an unrelated oligonucleotide (Sp1) corresponding to the Sp1 consensus sequence from the SV40 early promoter (lane 4). Complex b was observed to be nonspecific, since its formation was not abolished by the addition of the CTE oligonucleotide (lane 3) or the unlabeled -69/+56 fragment (data not shown). Indeed, this complex was observed to be due to the reticulocyte lysate itself, since it was also seen using nonprogrammed lysate (lane 5). The identity of complex A was confirmed as being due to HNF4 by the addition of anti-HNF4 antiserum, which produced a supershift of the protein-DNA complex (lane 7), which was not seen with the nonimmune serum (lane 8). In order to test which nuclear proteins from HepG2 cells could bind to the CYP2D6 DR1 element, gel retardation analysis was performed using nuclear extracts. As shown in Fig. 2C (lane 1), the addition of HepG2 nuclear protein resulted in the formation of three protein-DNA complexes (complexes 1-3) on the -69/+56 promoter fragment. All of these complexes were observed to bind specifically to the DR1 sequence, since their formation was abolished by the addition of CTE oligonucleotide (lane 2) but not by Sp1 oligonucleotide (lane 3). Since HNF4 was previously shown to be capable of binding to this DR1 element Fig. 2B), we examined if any of the complexes were due to the interaction of HNF4. Subsequently, complex 2 was shown to be dependent on the interaction of HNF4 as it was supershifted by the inclusion of anti-HNF4 antiserum in the DNA-binding reaction (Fig. 2C, lane 5). This supershift was observed to be specific, since the antiserum had no effect on the mobility of the other two complexes, and the addition of nonimmune serum to the DNA-binding reactions had no effect (lane 6). It was noted that when HNF4 was effectively removed from the DNA-binding reaction by its antiserum, the intensity of complex 1 increased (lane 5), suggesting that HNF4 and the factor responsible for the formation of complex 1 may bind to the DR1 element in a mutually exclusive manner. It was previously demonstrated that DR1 elements from other genes could be recognized by not only HNF4, but also by other members of the steroid receptor family, including COUP-TFI (EAR3), ARP1 (COUP-TFII), EAR2, peroxisome proliferator-activated receptor (PPAR) and retinoid X receptor (RXR), (30Ladias J.A.A. Hadzopoulou-Cladaras M. Kardassis D. Cardot P. Cheng J. Zannis V. Cladaras C. J. Biol. Chem. 1992; 267: 15849-15860Abstract Full Text PDF PubMed Google Scholar, 34Palmer C.N.A. Hsu M.-H. Griffen K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 35Nakshatri H. Chambon P. J. Biol. Chem. 1994; 269: 890-902Abstract Full Text PDF PubMed Google Scholar). Therefore, we tested if any of the unidentified complexes formed by HepG2 nuclear extracts on the CYP2D6 DR1 element were due to the interaction of other members of the steroid receptor superfamily. Based on what was already reported in the literature, the most obvious candidates were COUP-TFI/ARP1. These two proteins are highly homologous members of the steroid receptor family (36Ladias J.A.A. Karathanasis S.K. Science. 1991; 251: 561-565Crossref PubMed Scopus (308) Google Scholar), and ARP1 has also been termed COUP-TFII. As Fig. 2D demonstrates, the addition of HepG2 nuclear extracts to radiolabeled -69/+56 fragment resulted in the formation of the same three complexes (lane 2). The inclusion of anti-COUP antiserum (which recognizes both COUP-TFI and ARP1 (37Mietus-Snyder M. Sladek F.M. Ginsburg G.S. Kuo C.F. Ladias J.A.A. Darnell Jr., J.E. Karathanasis S.K. Mol. Cell. Biol. 1992; 12: 1708-1718Crossref PubMed Scopus (231) Google Scholar)) in the DNA-binding reactions, resulted in the inhibition of complex 1 and the disappearance of complex 3, with the concomitant appearance of supershifted complexes (lane 3). None of the complexes were affected by the addition of nonimmune serum (lane 4). The identification of COUP-TFI/ARP1 as being responsible for the formation of complex 1 was in agreement with the observed relative mobilities of HNF4 and COUP-TFI/ARP1 reported by other groups (37Mietus-Snyder M. Sladek F.M. Ginsburg G.S. Kuo C.F. Ladias J.A.A. Darnell Jr., J.E. Karathanasis S.K. Mol. Cell. Biol. 1992; 12: 1708-1718Crossref PubMed Scopus (231) Google Scholar). The observation that complex 3 was also supershifted by anti-COUP antiserum suggests that its formation was also dependent on COUP-TFI/ARP1. Alternatively, the factor(s) responsible could be antigenically related to COUP-TFs. However, results from gel retardation analysis using whole cell extracts from COS-7 cells transfected with a COUP-TFI expression vector support the former (see Fig. 5A), where the addition of COUP-TFI resulted in the formation of two protein-DNA complexes similar in mobility to complexes 1 and 3. In order to investigate if HNF4 was capable of activating the CYP2D6 promoter, co-transfection experiments were performed in COS-7 cells (see Fig. 3). In contrast to the results obtained from HepG2 cells, the transfection of -392CAT into COS-7 cells did not result in any enhancement of CAT activity above that observed with pCATbasic. Co-transfection with the mammalian HNF4 expression" @default.
• W1999859103 created "2016-06-24" @default.
• W1999859103 creator A5035792835 @default.
• W1999859103 creator A5040646059 @default.
• W1999859103 creator A5068393331 @default.
• W1999859103 creator A5086807788 @default.
• W1999859103 date "1996-10-01" @default.
• W1999859103 modified "2023-10-18" @default.
• W1999859103 title "Characterization of the Human Cytochrome P4502D6 Promoter" @default.
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