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• W2014233887 abstract "Phenobarbital induction of CYP2Bgenes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivofootprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR. Phenobarbital induction of CYP2Bgenes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivofootprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR. cytochrome P450 cytochrome P450 gene phenobarbital PB-responsive unit nuclear receptor nuclear factor-1 constitutive androstane receptor retinoid X receptor 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene dithiothreitol dimethyl sulfate micrococcal nuclease murine mammary tumor virus kilobase(s) base pair(s) nickel-nitrilotriacetic acid Cytochromes P450 (P450s)1 play an important role in the metabolism of xenobiotics and in the biosynthesis of endogenous compounds. A characteristic of the xenobiotic-metabolizing forms is that subsets of the P450s are induced by a variety of chemicals (1Waxman D.J. Arch. Biochem. Biophys. 1999; 369: 11-23Crossref PubMed Scopus (670) Google Scholar, 2Savas Ü. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1999; 56: 851-857Crossref PubMed Scopus (98) Google Scholar). This induction may alter the turnover of a drug that is given chronically and is the basis of drug interactions, because individual P450s are able to metabolize many substrates. The major human P450, P450 3A4, for example is responsible for the metabolism of nearly 50% of all therapeutic drugs (3Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 1-17Crossref PubMed Scopus (1070) Google Scholar). PB is a classical inducer of cytochrome P450 genes and is representative of a large number of structurally diverse phenobarbital-like inducers (4Waxman D.J. Azaroff L. Biochem. J. 1992; 281: 577-592Crossref PubMed Scopus (536) Google Scholar). The most dramatic effects of PB are on the CYP2B subfamily, but members of theCYP2C and CYP3A subfamilies are also induced. In addition, PB induces the expression of other xenobiotic-metabolizing enzymes such as glutathioneS-transferase and UDP- glucuronosyltransferases. Considerable progress has been made in understanding the molecular mechanisms of PB induction of CYP2B genes. Trottier et al. (5Trottier E. Belzil A. Stoltz C. Anderson A. Gene. 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar) demonstrated that a sequence at about −2.3 kb in theCYP2B2 gene had the properties of a PB-responsive enhancer either in its normal context or fused to a heterologous gene when assayed by transient transfection in primary cultures of rat hepatocytes. This sequence was also shown to mediate PB induction with heterologous promoters in rat hepatocytes in situ when DNA was directly injected into the liver (6Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and an analogous, nearly identical, sequence was identified in the mouse Cyp2b10 gene (7Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Mutational analysis established that the PB-responsive enhancer was a complex enhancer that contained multiple redundant regulatory binding sites, including two nuclear receptor binding sites, NR-1 and NR-2, and an NF-1 site (7Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 8Stoltz C. Vachon M.-H. Trottier E. Dubois S. Paquet Y. Anderson A. J. Biol. Chem. 1998; 273: 8528-8536Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Liu S. Park Y. Rivera-Rivera I. Li H. Kemper B. DNA Cell Biol. 1998; 17: 461-470Crossref PubMed Scopus (27) Google Scholar). The nuclear receptor, constitutive androstane receptor (CAR), binds to the two nuclear receptor sites and is enriched in nuclear extracts from PB-treated animals (10Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar). In the liver, CAR is present mainly in the cytoplasm of hepatocytes, until treatment with PB-like inducers, which causes translocation to the nucleus and binding to the NR sites as a CAR/RXR heterodimer (11Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar). Because CAR is constitutively active (12Choi H.S. Chung M. Tzameli I. Simha D. Lee Y.K. Seol W. Moore D.D. J. Biol. Chem. 1997; 272: 23565-23571Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), translocation may be sufficient for activation. However, it has also been demonstrated that binding of CAR to the coactivator steroid receptor coactivator-1 is increased after incubation with the PB-like inducer, TCPOBOP, although the same could not be demonstrated for PB (13Tzameli I. Pissios P. Schuetz E.G. Moore D.D. Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (364) Google Scholar). Thus, PB induction may involve translocation of CAR to the nucleus and, for some PB-like ligands, activation of CAR as well. These experiments provide evidence for the key role of CAR in the PB induction of CYP2B genes. Consistent with these results, mutation of the NR-1 and NR-2 sites decreases the response to PB in transient transfection studies (8Stoltz C. Vachon M.-H. Trottier E. Dubois S. Paquet Y. Anderson A. J. Biol. Chem. 1998; 273: 8528-8536Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Liu S. Park Y. Rivera-Rivera I. Li H. Kemper B. DNA Cell Biol. 1998; 17: 461-470Crossref PubMed Scopus (27) Google Scholar, 10Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar). In these studies, mutation of the NF-1 site also reduced the response to PB. Contrary to the transient transfection experiments, mutation of NF-1 in transgenes containing the PBRU did not reduce the expression after PB treatment, but the basal level of expression in untreated animals was increased (14Ramsden R. Beck N.B. Sommer K.M. Omiecinski C. Gene. 1999; 228: 169-179Crossref PubMed Scopus (35) Google Scholar). The contributions of the NF-1 site to activation of the PBRU, therefore, are controversial but mutation of the NF-1 site affects the function of the PBRU in both the transgenic studies and the transient transfections. Furthermore, in vivo footprints of the PBRU in hepatic chromatin demonstrate that the NF-1 region is occupied by proteins in untreated animals (15Kim J. Kemper B. J. Biol. Chem. 1997; 272: 29423-29426Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 16Kim J. Rivera-Rivera I. Kemper B. Nucleic Acids Res. 2000; 28: 1126-1132Crossref PubMed Scopus (12) Google Scholar). This region is still protected after PB treatment, but the region of protection is expanded over the flanking NR-1 and NR-2 sites. The NR sites are adjacent to the NF-1 site, and the NR-1 site overlaps by one nucleotide with the NF-1 site. The close proximity of the binding sites raised the question of whether interactions between CAR/RXR and NF-1, which bind to these sites, might play a role in transcriptional activation mediated by the PBRU. The experiments described above provide strong evidence that the PBRU in CYP2B genes is the principle mediator of PB induction these genes. For PB induction, the PB treatment most likely causes the translocation of CAR from the cytoplasm to the nucleus, where this constitutively active receptor binds to and trans-activates the PBRU. The mechanism by which this transactivation occurs has not been elucidated but requires other accessory factors, including NF-1, for maximal activation by PB in transient transfection analyses. In vitro evidence for the role of NF-1 in PBRU function has depended on analysis of protein binding using crude nuclear extracts and mutagenesis of the NF-1 site followed by functional analysis, studies in which potential interactions between NF-1 and CAR cannot be studied. The apparent differences in the effect of mutation of the NF-1 motif in transient transfections and transgenes suggest that functional interactions between NF-1 and CAR are different in chromatin and DNA. To study potential interactions between NF-1 and CAR and the role of chromatin structure, we have examined the binding of partially purified bacterially expressed CAR, RXR, and NF-1 to the CYP2B1 PBRU either in DNA or in assembled chromatin. The results indicate that CAR/RXR and NF-1 bind independently and simultaneously to the PBRU. In assembled chromatin, nucleosomes are linearly phased in the PBRU region, and the apparent affinity of NF-1 is increased 10-fold while that of CAR/RXR is reduced. Functionally, CAR-dependent activation of the PBRU in cultured cells is decreased by mutations of the NF-1 site and coexpression of NF-1 with CAR enhances the CAR-dependent activation in HepG2 cells just as mutation of the NF-1 site inhibits PB induction in transiently transfected hepatocytes in primary culture orin situ. Mouse CAR1 and CAR2 cDNAs were isolated from a mouse liver cDNA library by polymerase chain reaction and verified by sequencing. For bacterial expression of CAR1 with a His tag at the N terminus, CAR1 cDNA digested withBamHI and EcoRI was inserted into pET28a+ (Novagen Corp.) to produce pETCAR. For expression of CAR in mammalian cells, a BamHI/EcoRI fragment containing the CAR1 cDNA isolated from pGEX2TK-CAR was inserted into pcDNA3 (Invitrogen) digested with the same enzymes to produce pcDNA3-CAR. For bacterial expression of His- and FLAG-tagged NF-1 and FLAG-tagged RXR, the vectors pf:His-CTF1 and pf:RXR, respectively, were obtained from C. Chiang (Case Western Reserve University). For expression of NF-1 in mammalian cells, an EcoRI/BglII fragment from NF1L21 (A. Nicosia, Istito di Ricerche di Biologia Molecolare, Rome, Italy), which contained the NF-1 cDNA was inserted into pCMV5 digested with EcoRI andBamHI. The reporter plasmids, PBRU2C1-luc and NF1 m12C1-luc have been described previously (9Liu S. Park Y. Rivera-Rivera I. Li H. Kemper B. DNA Cell Biol. 1998; 17: 461-470Crossref PubMed Scopus (27) Google Scholar). RXR and NF-1 antisera were obtained from Santa Cruz Biotechnology and N. Tanese (New York University Medical Center, New York), respectively. For production of CAR antisera, CAR2 cDNA was inserted into pET28a+, and Escherichia coliBL21(DE3)pLysS was transformed with the resulting plasmid. Expression of CAR2 was induced by incubation with 1 mm isopropyl β-d-thioglucopyranoside for 4 h at 37 °C. Bacteria were lysed in Ni-NTA equilibrium buffer (20 mmTris-HCl, pH 8.0; 500 mm NaCl; 10 mm imidazole; 0.2 mm phenylmethylsulfonyl fluoride; 1 mm DTT; 2 μg/ml leupeptin, pepstatin, and aprotinin; and 10 μg/ml benzamidine) by passing through a French press. CAR2, which was in inclusion bodies, was pelleted by centrifugation for 15 min at 15,000 × g. The pellet was washed twice with 8m urea and once with 6 m guanidine-HCl in Ni-NTA equilibrium buffer plus 1% Nonidet P-40 minus the protease inhibitors. The protein was solubilized in 6 mguanidine-HCl and 15 mm β-mercaptoethanol in Ni-NTA equilibration buffer, and the solubilized protein was purified by affinity chromatography on a nickel-NTA column. The sample was dialyzed against phosphate-buffered saline, which resulted in precipitation of the protein. The precipitated protein was resuspended at a concentration of 5 mg/ml in phosphate-buffered saline for injection into a rabbit for antibody production. For expression of proteins in bacteria, 1-liter cultures of E. coli BL21(DE3)pLysS in LB broth were inoculated with 1/20 volume of overnight cultures and incubated at 37 °C for about 1 h to an A 600 = 0.6. Expression was induced by addition of 0.5 mm isopropyl β-d-thioglucopyranoside, and the samples were incubated for 4 h at 37 °C. The bacteria were pelleted by centrifugation, and the pellet was resuspended in 20 ml of 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, 20% glycerol, 0.2 mmEDTA, 0.1% Nonidet P-40, 4 mm DTT, and protease inhibitors. The cells were lysed by sonication and centrifuged at 22,000 × g for 20 min. The supernatant was mixed with the appropriate affinity resin, M2 agarose (Sigma Chemical Co.) for FLAG-NF-1 and FLAG-RXR and nickel-NTA slurry (Qiagen) for 6HIS-CAR and incubated at 4 °C overnight. After washing by centrifugation and resuspension three to five times with 20 mm Tris-HCl, pH 7.9, 300 mm NaCl, 20% glycerol, 0.2 mm EDTA, and protease inhibitors, proteins were eluted at 4 °C by resuspension and incubation for 20 min in 0.2–0.5 ml of 20 mm Tris-HCl, pH 7.9, 100 mm NaCl, 20% glycerol, 0.2 mm EDTA, which contained either 150 mm imidazole or 0.5 μg/ml of FLAG peptide for the nickel-NTA or M2-agarose resins, respectively. This elution procedure was repeated twice more. The identities of the proteins were established by Western analysis, and the purity and concentration of the proteins were estimated by Coomassie Blue staining of sodium lauryl sulfate-polyacrylamide gels using bovine serum albumin as a standard. Aliquots of the samples were stored at −80 °C. Gel mobility shift assays were carried out as described previously (17Kim J. deHaan G. Nardulli A.M. Shapiro D.J. Mol. Cell. Biol. 1997; 17: 3173-3180Crossref PubMed Scopus (20) Google Scholar) with some modifications. Binding reactions contained 50 mm KCl, 50 ng poly(dI·dC), 4 mm DTT, 10% glycerol, 5,000–10,000 cpm of 32P-labeled oligonucleotide probe, and recombinant proteins in a volume of 20 μl. The probe was a 74-bp oligonucleotide, containing CYP2B1 sequence (−2222 to −2154) plus 5 T's, and was labeled by incubation with E. coli DNA polymerase I, Klenow fragment, and [α-32P]dATP. Unincorporated nucleotide was removed with a G-25 spin column. For the binding assay, samples were incubated on ice for 10 min and then at room temperature for 15 min. Competitive oligonucleotides (100× excess) and antisera to RXR, CAR, and NF-1 were added during the binding reaction. Procedures for DNase I and DMS footprinting have been described previously (15Kim J. Kemper B. J. Biol. Chem. 1997; 272: 29423-29426Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 16Kim J. Rivera-Rivera I. Kemper B. Nucleic Acids Res. 2000; 28: 1126-1132Crossref PubMed Scopus (12) Google Scholar). The probe was prepared by digestion of pPBRU2C1-TZ with EcoRI andPvuII; incubation with E. coli DNA polymerase I, Klenow fragment, and [α-32P]dATP; and isolation by polyacrylamide gel electrophoresis of the resulting 540-bp fragment, which contained the PBRU sequence and was labeled at the 3′-end of the antisense strand. About 50,000–100,000 cpm of probe was added to the binding reactions. Purified recombinant proteins were added to the reaction as indicated in the figures. The samples were incubated for 15 min at room temperature, and either DMS was added to 20 mmand the incubation was continued for an additional 3 min at room temperature, or DNase I was added to a concentration of 2 μg/ml and the incubation was continued for 2–5 min on ice. The DMS-treated samples were incubated with piperidine to cleave at the methylated G's. The DNA fragments were analyzed by electrophoresis in 6% denaturing polyacrylamide gels as described (15Kim J. Kemper B. J. Biol. Chem. 1997; 272: 29423-29426Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Chromatin was assembled by the Drosophila embryo extract method (18Kamakaka R.T. Bulger M. Kadonaga J.T. Genes Dev. 1993; 7: 1779-1795Crossref PubMed Scopus (161) Google Scholar).Drosophila embryo S-190 extract was prepared as described (18Kamakaka R.T. Bulger M. Kadonaga J.T. Genes Dev. 1993; 7: 1779-1795Crossref PubMed Scopus (161) Google Scholar) and was a kind gift of S.-Y. Wu and C.-M. Chiang (Case Western Reserve University). Assembly of chromatin was as described (19Wu S.-Y. Thomas M.C. Hou S.Y. Likhite V. Chiang C.-M. J. Biol. Chem. 1999; 274: 23480-23490Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20Armstrong J.A. Emerson B.M. Mol. Cell. Biol. 1996; 16: 5634-5644Crossref PubMed Google Scholar). Briefly, 30 μl of S-190 extract was incubated with 0.4 μg of purified Drosophila core histones in 10 mmKOH-Hepes, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm EGTA, and 10% glycerol on ice for 30 min. Then 400 ng of pPBRU2C1-TZ DNA was added, and the volume was adjusted to 50 μl with a final concentration of 3 mmATP, 30 mm creatine phosphate, 1 μg/ml creatine kinase, 4.1 mm MgCl2, 5 mm DTT, 20 mm Tris-HCl, pH 7.9, 50 mm NaCl, 20% glycerol, and 0.2 mm EDTA. The samples were incubated at 27 °C for 4.5 h. Mock chromatin assembly reactions were carried out as above except that the S-190 extract was omitted. To analyze protein binding to the chromatin, purified recombinant NF-1, RXR, and CAR were added to 25 ng of assembled chromatin and incubations were continued for 30 min at 27 °C. To assess the formation of chromatin, 5 units of micrococcal nuclease (Roche Molecular Biochemicals) and 1.5 μl of 0.1 mCaCl2 were added to 250 ng of DNA of the assembled chromatin in 50 μl, and 17-μl aliquots were removed after 1, 3, and 9 min of incubation at room temperature. The aliquots were added to 4 μl of 0.5% Sarkosyl, 100 mm EDTA to inhibit the nuclease, and 0.2 μg/μl RNase A was added. After further incubation at 37 °C for 10 min, 0.8 μl of 5% sodium lauryl sulfate and 2 μl of proteinase K (10 mg/ml) were added, and the reaction was heated to 55 °C for 15 min. 20 μg of glycogen was added as a carrier, and the samples were extracted with phenol/chloroform. The DNA fragments were separated by agarose gel electrophoresis, transferred to nylon membranes, and cross-linked to the membrane by ultraviolet radiation. Bulk DNA was analyzed by Southern analysis with total plasmid DNA as the probe, which was labeled with [α-32P]dATP by random hexamer priming. Hybridization was at 68 °C for 5 h, and the membranes were washed several times with the final wash at 68 °C in 20 mm sodium phosphate, pH 7.2, 0.1% sodium lauryl sulfate. The position of nucleosomes relative to the PBRU was assessed by indirect end-labeled Southern analysis. After micrococcal nuclease treatment, the DNA was digested with EcoRI and transferred to Nylon membranes as above, The probe for the hybridization was an oligonucleotide of 21 nucleotides beginning at the EcoRI site, which was labeled with 32P at the 5′-end by polynucleotide kinase. Hybridization was at 55 °C for 5 h, and the membranes were washed several times as described above. For DNase I footprinting, after incubation with NF-1, CAR, and RXR, 25 ng of DNA of chromatin was digested for 3 min at room temperature with 0.9 unit of DNase I. For mock assembled chromatin, 0.003 unit of DNase I was used. DNase I digestion was stopped by addition of 20 mm EDTA, 0.2 m NaCl, 0.1% sodium lauryl sulfate, 130 μg/ml proteinase K, and 60 μg/ml tRNA. After incubation at 55 °C for 15 min, DNA was extracted with phenol/chloroform and precipitated with ethanol. DNA was labeled by linear amplification for 15 cycles in a polymerase chain reaction machine using a sense oligonucleotide, 5′-GAATTCGAGCTCGGTACCCGG-3′, from the multiple cloning site of the plasmid as a primer. The primer was labeled with 32P at the 5′-end by polynucleotide kinase. The fragments generated by polymerase chain reaction were separated by electrophoresis on a 6% denaturing polyacrylamide gel and were detected by autoradiography. Human HepG2 and mouse Hepa1c1c7 cells were maintained in Dulbecco's modified Eagle's medium and α-minimal essential medium, respectively, with 10% charcoal dextran-stripped fetal calf serum, 100 units/ml penicillin, and 0.01% streptomycin. For transfections, cells were transfected with 1–2 μg of plasmids containing either the wild-type PBRU or the PBRU with the NF-1 site mutated, NF1 m1 (9Liu S. Park Y. Rivera-Rivera I. Li H. Kemper B. DNA Cell Biol. 1998; 17: 461-470Crossref PubMed Scopus (27) Google Scholar), fused to the minimal CYP2C1promoter/firefly luciferase reporter (6Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), 2–6 ng of pRL-SV40, containing the SV40 promoter and Renilla luciferase reporter as an internal standard, and 2 μl of LipofectAMINE 2000 (Life Technologies) as described by the manufacturer. Expression plasmids for CAR, pcDNA3CAR1, and NF-1, pCMV5-NF1, were cotransfected as indicated in the figures. 24–36 h after transfection, cells were lysed and luciferase activities were determined by the Dual luciferase reporter assay system (Promega Biotech). For each sample, the background of extracts from untransfected cells was subtracted and the firefly luciferase values were normalized by dividing by theRenilla luciferase values. TheCYP2B1 PBRU is a complex enhancer containing multiple regulatory elements, including an NF-1 motif flanked by two NR sites. As shown in Fig. 1, the NF-1 site and the NR-1 site overlap by one nucleotide and the NF-1 and NR-2 sites are separated by only three nucleotides. This proximity raises the possibility that steric hindrance might prevent binding of NF-1 and CAR/RXR at the same time or that binding among the factors might be cooperative. To examine whether all the proteins could bind at the same time, binding of partially purified recombinant CAR/RXR and NF-1 to theCYP2B1 PBRU was examined by gel mobility shift assay (Fig.2). CAR alone did not bind to the PBRU (lane 2), but RXR alone bound weakly presumably as a homodimer (lane 3). Two complexes were formed when CAR and RXR were added together, and the ratio of the slower to more rapidly migrating complexes increased as the concentration of the proteins was increased (lanes 4–6). These results are consistent with CAR/RXR binding primarily to one of the NR sites at lower concentrations and to both NR-1 and NR-2 at the higher concentrations. NF-1 alone also bound to the PBRU, forming several specific complexes (lane 7), which were competed by unlabeled oligonucleotide containing the NF-1 motif (not shown). The reason that multiple complexes are formed is not known, but may result from partial degradation of NF-1. Addition of both NF-1 and CAR/RXR resulted in complexes corresponding to the two CAR/RXR complexes and a more slowly migrating complex (lane 8, NF-1·NR-1·NR-2). Competition with oligonucleotides containing either the NR-1 or NF-1 motif resulted in the loss of the most slowly migrating complex (lanes 9and 10), suggesting that this complex contained both NF-1 and CAR/RXR. This was shown more directly by addition of antisera to CAR, RXR, and NF-1, each of which supershifted the more slowly moving complex (lanes 11, 13, and 14), whereas addition of preimmune serum (lane 12) had no effect. These results demonstrate that CAR/RXR and NF-1 bind simultaneously to the CYP2B1 PBRU.Figure 2Binding of NF-1 and CAR/RXR to the ratCYP2B1 PBRU. NF-1, RXR, and CAR were expressed in bacteria and purified as described under “Experimental Procedures.” The probe was a double-stranded oligonucleotide that containedCYP2B1 sequence −2222 to − 2154, which contains the NF-1, NR-1, and NR-2 binding sites in the PBRU. After incubation with the indicated recombinant proteins, the samples were analyzed by polyacrylamide gel electrophoresis in 6% nondenaturing gels, and radioactivity was detected by autoradiography. Approximately 5 ng of NF-1 and 5 ng (+), 10 ng (++), or 20 ng (+++) of CAR and 2.5 ng (+), 5 ng (++), or 10 ng (+++) of RXR were added to the reactions. Competitor oligonucleotides containing either the NR-1 or NF-1 site in 100× excess or antisera to CAR, RXR, and NF-1 or preimmune serum (Pre.S.) were added during the incubations as indicated. The DNA sites occupied by proteins in the complexes are indicated at theleft of the figures as are the complexes with slower mobility after addition of antisera (supershifts).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The gel mobility shift assays established that NF-1 and CAR/RXR bind simultaneously to the PBRU. To examine the relative affinity of CAR/RXR binding to the NR-1 and NR-2 sites and to determine directly whether both NR sites were occupied when the NF-1 site was occupied, DNase I and DMS footprinting assays of binding of the purified proteins to the PBRU were examined. Addition of increasing amounts of CAR/RXR resulted in protection from DNase I over the NR-1 site initially and both the NR-1 and NR-2 sites at higher concentrations. On the basis of these results, the complex observed in the gel shift assays with low concentrations of CAR/RXR represents binding to the NR-1 site and the more slowly migrating complex present at higher concentrations represents binding to both the NR-1 and NR-2 sites. The regions protected by CAR/RXR from DNase I cleavage extend into the NF-1 site from both the NR-1 and NR-2 sites, and binding to the NR-1 site is associated with the appearance of hypersensitive sites within the NF-1 site. Binding of NF-1 to the PBRU results in a footprint of about 30 bp over the NF-1 site, which also overlaps with the NR-1 and NR-2 sites. NF-1 binding is also associated with hypersensitivity in the NR-1 site. The overlapping footprints and the hypersensitivity induced in the binding site of the adjacent factor by binding of one factor to its site suggested that binding of NF-1 might affect binding of CAR/RXR orvice versa. However, the occupation of the NF-1 site by NF-1 has little influence on the binding of increasing concentrations of CAR/RXR to NR-1, and CAR/RXR binding has little influence on the binding of increasing concentrations of NF-1 (Fig.3). In contrast, the binding of CAR/RXR to NR-2 is modestly increased by the binding of NF-1 to its site, suggesting some interaction exists between the proteins at these sites. DMS footprinting was used to map the binding of the factors at higher resolution (Fig. 4). Protection by CAR/RXR against methylation of guanines was observed in the NR-1 site at nucleotides −2211, −2202, and −2201 and within NR-2 site at −2179 and −2170. None of the guanines on the antisense strand within the NF-1 site was protected by CAR/RXR. Likewise, protection from methylation by NF-1 was at nucleotides −2196, −2189, and −2188 within the NF-1 bipartite motif. A strong hypersensitive site was observed at" @default.
• W2014233887 created "2016-06-24" @default.
• W2014233887 creator A5012838178 @default.
• W2014233887 creator A5026464156 @default.
• W2014233887 creator A5036629320 @default.
• W2014233887 date "2001-03-01" @default.
• W2014233887 modified "2023-10-09" @default.
• W2014233887 title "Chromatin Assembly Enhances Binding to the CYP2B1Phenobarbital-responsive Unit (PBRU) of Nuclear Factor-1, Which Binds Simultaneously with Constitutive Androstane Receptor (CAR)/Retinoid X Receptor (RXR) and Enhances CAR/RXR-mediated Activation of the PBRU" @default.
• W2014233887 cites W1485938486 @default.
• W2014233887 cites W1964106638 @default.
• W2014233887 cites W1990330754 @default.
• W2014233887 cites W1990958396 @default.
• W2014233887 cites W2004352153 @default.
• W2014233887 cites W2007140214 @default.
• W2014233887 cites W2017573875 @default.
• W2014233887 cites W2027294913 @default.
• W2014233887 cites W2028273026 @default.
• W2014233887 cites W2043888927 @default.
• W2014233887 cites W2049970457 @default.
• W2014233887 cites W2056458409 @default.
• W2014233887 cites W2087770202 @default.
• W2014233887 cites W2090528164 @default.
• W2014233887 cites W2099172455 @default.
• W2014233887 cites W2123159704 @default.
• W2014233887 cites W2127059331 @default.
• W2014233887 cites W2129185784 @default.
• W2014233887 cites W2132777822 @default.
• W2014233887 cites W2135248587 @default.
• W2014233887 cites W2154088862 @default.
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