Chromatin Assembly Enhances Binding to the CYP2B1 Phenobarbital-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*

Jongsook KimDagger , Gyesik MinDagger , and Byron KemperDagger §

From the Departments of Dagger  Molecular & Integrative Physiology and § Cell & Structural Biology, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, September 5, 2000, and in revised form, December 8, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phenobarbital induction of CYP2B genes 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 vivo footprinting 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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 (1, 2). 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 (3). PB is a classical inducer of cytochrome P450 genes and is representative of a large number of structurally diverse phenobarbital-like inducers (4). The most dramatic effects of PB are on the CYP2B subfamily, but members of the CYP2C and CYP3A subfamilies are also induced. In addition, PB induces the expression of other xenobiotic-metabolizing enzymes such as glutathione S-transferase and UDP- glucuronosyltransferases.

Considerable progress has been made in understanding the molecular mechanisms of PB induction of CYP2B genes. Trottier et al. (5) demonstrated that a sequence at about -2.3 kb in the CYP2B2 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 (6), and an analogous, nearly identical, sequence was identified in the mouse Cyp2b10 gene (7). 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 (7-9). The nuclear receptor, constitutive androstane receptor (CAR), binds to the two nuclear receptor sites and is enriched in nuclear extracts from PB-treated animals (10). 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 (11). Because CAR is constitutively active (12), 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 (13). 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 (8-10). 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 (14). 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 (15, 16). 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 or in situ.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Plasmid Construction-- 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 with BamHI 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 and BamHI. The reporter plasmids, PBRU2C1-luc and NF1 m12C1-luc have been described previously (9).

Antisera-- 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 coli BL21(DE3)pLysS was transformed with the resulting plasmid. Expression of CAR2 was induced by incubation with 1 mM isopropyl beta -D-thioglucopyranoside for 4 h at 37 °C. Bacteria were lysed in Ni-NTA equilibrium buffer (20 mM Tris-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 8 M 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 M guanidine-HCl and 15 mM beta -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.

Expression and Purification of CAR, RXR, and NF-1-- 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 A600 = 0.6. Expression was induced by addition of 0.5 mM isopropyl beta -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 mM EDTA, 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-- Gel mobility shift assays were carried out as described previously (17) 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 [alpha -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.

DMS and DNase I Footprinting-- Procedures for DNase I and DMS footprinting have been described previously (15, 16). The probe was prepared by digestion of pPBRU2C1-TZ with EcoRI and PvuII; incubation with E. coli DNA polymerase I, Klenow fragment, and [alpha -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 mM and 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 (15).

Assembly and Analysis of Chromatin-- Chromatin was assembled by the Drosophila embryo extract method (18). Drosophila embryo S-190 extract was prepared as described (18) and was a kind gift of S.-Y. Wu and C.-M. Chiang (Case Western Reserve University). Assembly of chromatin was as described (19, 20). Briefly, 30 µl of S-190 extract was incubated with 0.4 µg of purified Drosophila core histones in 10 mM KOH-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 mM ATP, 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 M CaCl2 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 [alpha -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.

Cell Culture and Transfection-- Human HepG2 and mouse Hepa1c1c7 cells were maintained in Dulbecco's modified Eagle's medium and alpha -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 (9), fused to the minimal CYP2C1 promoter/firefly luciferase reporter (6), 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 the Renilla luciferase values.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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CAR/RXR and NF-1 Bind Simultaneously to the CYP2B1 PBRU-- The CYP2B1 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 the CYP2B1 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 9 and 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.



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Fig. 1.   Sequence of the PBRU region of CYP2B1. The sequence of CYP2B1 from -2299 to -2137 is shown. The NF-1 site is underlined and brackets indicate the NR sites. Positions of protection of guanines in the antisense strand (cytosines in the sense strand shown) from methylation by DMS are indicated by the solid circles for CAR/RXR binding and solid triangles for NF-1 binding. The asterisks indicate positions of hypersensitivity to methylation. The solid bar over the sequence indicates the regions protected from DNase I digestion by NF-1 binding, and the hatched bars indicate regions protected by CAR/RXR binding.



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Fig. 2.   Binding of NF-1 and CAR/RXR to the rat CYP2B1 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 contained CYP2B1 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 the left of the figures as are the complexes with slower mobility after addition of antisera (supershifts).

NF-1, NR-1, and NR-2 Sites Are Occupied Simultaneously and Binding to NR-2 Is Enhanced by NF-1 Binding-- 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 or vice 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.



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Fig. 3.   DNase I footprinting of the CYP2B1 PBRU region by NF-1 and CAR/RXR. A 540-bp EcoRI/PvuII CYP2B1 fragment, which was labeled with 32P at the EcoRI site, was incubated with recombinant proteins as indicated, and the DNA was partially digested with DNase I and fractionated by electrophoresis on 6% denaturing polyacrylamide gels. The triangles indicate increasing amounts of CAR (10, 30, and 90 ng), RXR (5, 15, and 45 ng), or NF-1 (10, 20, and 30 ng). The "+" corresponds to the lowest of these concentrations. The positions of the regions protected by CAR/RXR (NR-1, NR-2, NR-3) and NF-1 (NF-1) are indicated on the left.

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 the 3'-end of the NF-1 motif when NF-1-bound. A similar hypersensitive site was observed in in vivo DMS footprinting of the PBRU in hepatic chromatin (16), which indicates that NF-1 is bound to the PBRU in vivo. All the guanines protected from methylation by the factors individually were protected to the same degree when both CAR/RXR and NF-1 were added to the binding reaction, which suggests that little or no interaction occurs among the factors. These DNase I and DMS footprinting data show that CAR/RXR and NF-1 can bind simultaneously to their sites in the core of the PBRU. In addition, the binding of the proteins is largely independent, although binding of CAR/RXR to the NR-2 site may be increased by NF-1 binding.



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Fig. 4.   DMS footprinting of the CYP2B1 PBRU region by NF-1 and CAR/RXR. A 540-bp EcoRI/PvuI CYP2B1 fragment, which was labeled with 32P at the EcoRI site, was incubated with recombinant proteins as indicated, and the DNA was partially reacted with 20 mM DMS for 3 min at room temperature. The DNA was cleaved at methylated guanines with piperidine and fractionated by polyacrylamide gel electrophoresis. The amounts of CAR, RXR, and NF-1, were 30, 15, and 30 ng, respectively. The positions of the sequence corresponding to the NR-1, NR-2, NR-3, and NF-1 sites are indicated on the left. The sequences of the three NR sites and the NF-1 site are shown below. Guanine residues in the 3'-end-labeled antisense strand (corresponding to C's in the sense strand sequence shown) that were protected from methylation are indicated by dots, and the residues hypersensitive to methylation are indicated by asterisks along the sides of the autoradiogram and over the sequences at the bottom.

The DNase I and DMS footprinting studies with purified CAR/RXR both revealed that these proteins bound to a third site within the PBRU region, designated NR-3 (Figs. 3 and 4). The NR-3 sequence (-2274) 5'-TGCACTtcagTGACCT-3' (-2259) is very similar to the NR-1 site (-2215) 5'-TGTACTttccTGACCT-3' (-2200), and the apparent affinity of CAR/RXR is about the same for NR-1 and NR-3 and greater than that for NR-2. NR-3 is about 60 bp 5' of NR-1, and its functional significance is not clear, but deletion of this region resulted in a modest decrease in PB responsiveness (21). Interestingly, if the PBRU is present in a nucleosome, the NR-3 site on one loop of DNA would be adjacent to the NF-1 site in the NR-1·NF-1·NR-2 core on the other loop of DNA in the nucleosome so that interactions between proteins binding to these sites might be possible.

Binding of NF-1 and CAR/RXR to the PBRU in Assembled Chromatin-- Binding of proteins to naked DNA template in vitro may not accurately represent the binding affinity of the proteins in vivo when the DNA is in a chromatin structure. The binding of NF-1, for example, has been shown to be greatly decreased by nucleosome structures in the MMTV promoter (22, 23). To examine the effect of chromatin structure on the binding of NF-1 and CAR/RXR to the PBRU, plasmid DNA containing PBRU sequence was assembled into chromatin using the Drosophila S-190 embryo extract (18) as illustrated in Fig. 5A. The assembly of chromatin was monitored by partial micrococcal nuclease digestion in each experiment. An example of such analysis is shown in Fig. 5B. After digestion, a ladder of DNA fragments of about 180 bp was observed for bulk DNA by Southern blotting using total plasmid DNA as a probe (Fig. 5B) or ethidium bromide staining of agarose gels (not shown). In contrast, DNA, which had been mock assembled into chromatin was digested to small fragments by the same concentrations of micrococcal nuclease. These results demonstrate that the assembly of chromatin was successful. To examine whether the nucleosomes that were formed were translationally phased in the PBRU region, indirect end-labeling Southern analysis was used (see Fig. 5D). Again, a ladder of fragments with a repeat of about 180 bp was observed indicating that the nucleosomes were linearly phased in the PBRU region while mock assembled chromatin was completely digested (Fig. 5C). The central NR-1·NF-1·NR-2 region of the PBRU was in the core of a nucleosome toward its 3'-end (Fig. 5D), which is consistent with conclusions from micrococcal nuclease digestion of native hepatic chromatin (16).



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Fig. 5.   Micrococcal nuclease digestion of assembled chromatin using Drosophila embryo extract. Chromatin was assembled, digested with micrococcal nuclease (MNase), and analyzed by electrophoresis in agarose gels as described under "Experimental Procedures." In parallel, control samples, in which the Drosophila S-190 extract was omitted, mock chromatin (Mock), were analyzed. In A, the assembly process and protein binding assays are illustrated schematically. The results of MNase analysis are shown, and the DNase I analysis is shown in Fig. 6. In B and C, the triangles indicate increasing times of incubation with 5 units of MNase, 1, 3, and 9 min. To detect bulk DNA, radioactive plasmid DNA was used as a probe (B) for a Southern analysis. In the indirect end-labeled Southern analysis in C, the probe was a radiolabeled 21-nucleotide oligonucleotide that was complementary to the sequence starting at the EcoRI site. Radiolabeled 123-bp ladder DNA was used as a marker (M). The positions of linker regions indicated by the increased sensitivity to MNase in the assembled chromatin samples are indicated by arrows. In D, a diagram of the PBRU region from the EcoRI site is shown. The horizontal arrow indicates the oligonucleotide probe used for the indirect Southern, and the vertical arrows indicate the regions of increased sensitivity to MNase in the chromatin samples. The positions of the NR-1/NF-1/NR-2 sites are shown.

The binding of NF-1 and CAR/RXR to either assembled chromatin or to mock assembled chromatin as a control was examined, and a representative experiment is shown in Fig. 6. These results were reproducible in four different experiments. NF-1 bound with an affinity about 10 times greater to chromatin than to the mock-assembled chromatin control (compare lanes 21-25 with lanes 7-11). Although the protected region was the same in both chromatin and the control, there was a hypersensitive site present at the 3'-end of the footprint that was not observed in the mock chromatin control (compare lane 23 with lane 9) and was not present in DNase I footprints to naked DNA (Fig. 3). In addition to the differences in the hypersensitivity, the concentration of micrococcal nuclease that was required for partial digestion of the chromatin samples was about 300-fold greater than that required for the mock assembled chromatin, which provides additional evidence that the PBRU sequence is assembled into chromatin. Micrococcal nuclease digestion of the chromatin after incubation with NF-1, CAR, and RXR indicated that nucleosomal structure was not affected by the binding of the proteins (data not shown). Binding of CAR/RXR to the PBRU could also be detected in the chromatin samples, primarily because of increased hypersensitivity to DNase I at the 3'-side of NR-1 (Fig. 6, lanes 18-20). In this case the protection was less evident compared with that of mock assembled chromatin (lanes 4-6), suggesting that the nucleosomal structures inhibited binding of CAR/RXR. The assembly of the PBRU into chromatin, therefore, dramatically altered the relative affinities of NF-1 and CAR/RXR.



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Fig. 6.   DNase I footprinting of the CYP2B1 PBRU in assembled chromatin by NF-1 and CAR/RXR. Chromatin or mock chromatin (Mock) was assembled, incubated with recombinant NF-1, CAR, and RXR, and analyzed by polyacrylamide gel electrophoresis as described under "Experimental Procedures." The addition of recombinant proteins is indicated at the top, and the triangles indicate increasing amounts of the proteins, 0.75 to 300 ng for CAR, 0.3 to 120 ng for RXR, and 0.15 to 120 ng for NF-1. The "+" indicates an amount equal to 25 and 12.5 ng for CAR and RXR, respectively. The positions of the protected regions over the NR-1, NR-2, and NF-1 sites are indicated by a line at the left side of the autoradiogram.

To determine whether the binding of NF-1 affected the binding of CAR/RXR, or vice versa, in chromatin, the binding of each in the presence of the other was examined. Binding of CAR/RXR was not detectably affected by increasing concentrations of NF-1 (Fig. 6, lanes 27-29), nor was the binding of NF-1 detectably affected by increasing concentrations of CAR/RXR (not shown). These results indicate that binding of NF-1 and CAR/RXR to chromatin is largely independent.

Effect of NF-1 on CAR-mediated Transactivation of the PBRU-- To determine whether there were functional interactions between the NF-1 binding and CAR/RXR binding, CAR-mediated transactivation of the PBRU was examined in human HepG2 and mouse Hepa1c1c7 cells. These cells were cotransfected with a CAR expression vector and a reporter plasmid, which contained a single copy of the CYP2B1 PBRU fused to the minimal CYP2C1 promoter/firefly luciferase reporter. Expression of CAR resulted in an increase in firefly luciferase activity of about 6- and 20-fold for the Hepa1c1c7 and HepG2 cells, respectively (Fig. 7). The CAR transactivation was dependent on the PBRU sequence, because no activation was observed with just the CYP2C1 promoter/firefly luciferase reporter (Fig. 7). Coexpression of RXR did not result in an increase in transcription (data not shown).



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Fig. 7.   CAR-dependent transactivation of the CYP2B1 PBRU. Either HepG2 cells or Hepa1c1c7 cells were cotransfected with pcDNA3-CAR and either PBRU2C1-luc or 2C1-luc as indicated and with pRL-SV40, which expresses Renilla luciferase. Values for firefly luciferase were normalized by dividing the firefly luciferase activity by that of Renilla luciferase. The averages of the -fold increase over activity without CAR transfection from two (HEPG2) or three (HEPA1C1C7) experiments in which triplicate samples were analyzed are shown. Standard errors are shown for the Hepa1c1c7 experiments.

Mutations in the 5' part of the bipartite motif (NF1 m1) of the CYP2B1 PBRU have been shown to eliminate NF-1 binding and reduce PB induction in hepatocytes transfected in situ (9). Binding of CAR/RXR to NR-1 and NR-2 was not detectably affected by the mutation of the NF-1 motif (Fig. 8A). To examine the role of the NF-1 site in the CAR-mediated transactivation, the NF1 m1 mutation was introduced into the PBRU fused to the CYP2C1/firefly luciferase reporter. The mutation of the NF-1 motif had little effect on basal transcriptional activity in HepG2 and Hepa1c1c7 cells that were not transfected with the CAR expression vector (Fig. 8B). However, CAR-mediated activation of the PBRU was decreased by about 80% by mutation of the NF-1 motif when 1 or 5 ng of CAR expression vector was transfected. At 25 ng, inhibition was slightly less, because transactivation with the wild type reporter had reached a plateau while that of the mutant reporter continued to increase. These results suggest that NF-1 itself has little transactivating activity, because basal expression was not affected by the mutation, but NF-1 enhances substantially the response to CAR. The trend of increased transactivation of the mutant reporter, but not the wild type reporter, up to the highest concentration of CAR tested, suggests that NF-1 binding may increase the affinity of CAR/RXR binding in the cells.



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Fig. 8.   Inhibition of CAR-dependent transactivation of the CYP2B1 PBRU by mutations of the NF-1 motif. In A, DNase I footprinting analysis of binding of CAR/RXR to NR-1 and NR-2 is shown. Samples were analyzed as described in Fig. 3. The DNA probe was either wild type PBRU sequence or NF1 m1, which is mutated in the 5' part of the bipartite NF-1 site and does not bind to NF-1. As indicated by the triangles, increasing amounts of CAR and RXR were added to the reactions. The positions of the NR-1- and NR-2-protected regions are indicated by the line at the left side. In B, the activation of luciferase reporters with the wild type PBRU, or PBRU sequence in which the NF-1 site was mutated (NF1 m1), fused to the CYP2C1 promoter was analyzed. The ratio of the firefly to Renilla luciferase activity was determined and multiplied by 500 or 104 for HepG2 or Hepa1c1c7 cells, respectively. Standard errors are shown (n = 3).

To further examine the effect of NF-1 expression on CAR-mediated transactivation, an NF-1 expression vector was cotransfected with the CAR expression vector. In HepG2 cells NF-1 increased CAR-mediated transactivation of the PBRU-CYP2C1/firefly luciferase reporter by about 2-fold when 1, 5, or 25 ng of CAR expression vector and 100 or 500 ng of NF-1 expression vector were cotransfected (Fig. 9A). To control for effects of NF-1 expression that might indirectly affect CAR activation and not require DNA binding of NF-1, the effects of NF-1 expression on CAR-mediated transactivation of a reporter plasmid containing four copies of the NR-1 site rather than the PBRU was examined. The transactivation of (NR-1)42C1-luc by CAR was not affected by cotransfection of NF-1 (Fig. 9B). This latter result suggests that the activation of the PBRU-containing reporter is dependent on NF-1 binding to the NF-1 site. The inhibition of CAR activation by NF-1 mutants and the enhancement by overexpression of NF-1 demonstrate that NF-1 contributes to the maximal response to CAR in transfected cells and presumably to PB induction of CYP2B1 in vivo.



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Fig. 9.   Enhancement of CAR-mediated transactivation of the CYP2B1 PBRU by NF-1. HepG2 cells were cotransfected as indicated in the figure with pcDNA3-CAR and pCMV5-NF-1 expression vectors. Reporters with either the PBRU sequence (A) or four copies of the NR-1 sequence (NR1)4 (B) fused to the CYP2C1 promoter were also transfected into the cells. Values for firefly luciferase were normalized by dividing the firefly luciferase activity by that of Renilla luciferase. The averages of four samples (duplicates in two experiments) for the PBRU enhancer, the averages of three samples from two experiments for the (NR1)4 enhancer, and standard errors are shown.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prior to these studies, in vitro protein binding studies with crude extracts and functional analyses of mutated CYP2B promoters by transient transfection had established the PBRU as a complex enhancer with multiple regulatory sites (7-11). NF-1 and CAR/RXR were shown to bind to an NF-1 site and two flanking NR sites, respectively, in the core of the PRBU. The central role of CAR has been confirmed recently by the loss of PB inducibility of CYP2B genes in mice in which the CAR gene was disrupted (24). Mutagenesis of the NF-1 site reduced PB trans-activation in transient transfections, but did not reduce PB-induced expression of a CYP2B1 promoter/reporter transgene (14) so that the role of NF-1 is controversial. PB treatment caused the translocation of CAR from the cytoplasm to the nucleus (11), which may be sufficient for trans-activation of the CYP2B genes, because CAR is constitutively active. The activation of the PBRU by CAR binding, therefore, is central to the mechanism of PB induction of these genes. To gain additional insight into the activation of the PBRU, the present studies examined whether NF-1 and CAR/RXR bind independently or cooperatively, using partially purified recombinant NF-1 and CAR/RXR, and whether chromatin structure might influence the binding of these proteins in a way that would explain the differences between the NF-1 site mutations in transient transfection and transgenic experiments.

Within the core of the CYP2B1/2 PBRU, the binding site for NF-1 is flanked by CAR/RXR binding sites, NR-1, which overlaps the NF-1 by 1 bp, and NR-2, which is separated by 3 bp from the NF-1 site. The close proximity of these sites raised the question of whether steric hindrance might impede simultaneous binding of NF-1 and CAR/RXR. Supporting this possibility, recombinant NF-1 DNase I footprints overlapped the NR-1 and NR-2 sites and CAR/RXR footprints overlapped the NF-1 site. However, recombinant NF-1 and CAR/RXR can bind simultaneously and largely independently to the NF-1, NR-1, and NR-2 sites. Binding contacts, identified by decreased methylation of guanines, were five nucleotides apart in NR-1 and NF-1, which suggests that binding to these overlapping sites occurs on opposite faces of the DNA. These studies indicate that NF-1 and CAR/RXR bind independently to the PBRU so that the cooperative increase in trans-activation of the PBRU observed in transient transfections is not the result of either negative or positive cooperativity in binding of the factors to the PBRU.

Mutation of the NF-1 site decreases induction by PB of transfected CYP2B genes in hepatocytes (8-10). In the present study, CAR-mediated trans-activation of the PBRU was decreased by mutation of the NF-1 site and increased by overexpression of NF-1 but mutation of the site or overexpression of NF-1 had little effect in the absence of CAR expression. The similar effects of mutation of the NF-1 site in the PBRU on PB induction in hepatocytes and CAR-activated expression in continuously cultured cells provide additional supporting evidence that CAR mediates the PB response and that the increase mediated by NF-1 is an enhancement of the trans-activation by CAR.

Differences in the effects of mutations of the NF-1 site in transgenes (14) compared with transient transfections might be explained if assembly of the PBRU into chromatin, as in transgenes, alters the binding of NF-1. However, as with naked DNA, the NR-1, NR-2, and NF-1 sites were occupied simultaneously and largely independently in reassembled chromatin so that this explanation is not correct. In addition to the transient transfection data, a role for NF-1 is supported by in vivo footprints in which the NF-1 site is occupied in both untreated and PB-treated animals (15, 16). The occupation of the NF-1 site in the untreated animals, in which the gene is silent, would be consistent with NF-1 enhancing trans-activation only after CAR/RXR binding. The transgenic data may be misleading if, for example, mutation of the NF-1 motif inadvertently introduced a new regulatory motif, the copy number or chromosome positions of the transgenes affected the level of expression, or the absence of NF-1 binding reduced the affinity of the CAR/RXR binding, which might not be detected with the high concentrations of PB used in the transgenic mice. Further studies will be required to resolve this issue.

A major surprise in these experiments was the observation that assembly of the PBRU into chromatin increased the apparent binding affinity for NF-1. In the MMTV promoter, NF-1 is precluded from binding if the enhancer is assembled into nucleosomes using the Drosophila extract method (25). In assembled chromatin, the binding of the activated progesterone receptor to its sites in the MMTV promoter alters chromatin structure such that NF-1 binding affinity is greatly increased (26). Similarly, the binding of dioxin to its responsive elements in the Cyp1a1 gene at a distal site alters chromatin structure and facilitates the binding of NF-1 to its site in the proximal promoter (27). On the basis of these previous studies, the stimulus and initial hypothesis driving the present experiments were that CAR/RXR binding might alter the chromatin structure and facilitate the binding of NF-1 to the chromatin. However, protection of the NF-1 site in assembled chromatin compared with mock assembled chromatin requires only about one-tenth the amount of NF-1 in the absence of CAR/RXR, and CAR/RXR binding has little effect on NF-1 binding. In contrast, protection of the NR sites by CAR/RXR required more protein in the reassembled chromatin. Several lines of evidence indicate that binding to assembled chromatin is being assessed in these experiments. First, MNase digestion indicated that chromatin assembly was successful, second, the relative affinities of NF-1 and CAR/RXR were altered, third, the amounts of DNase I required for the partial digestion of the chromatin was about 300-fold higher than for the mock assembled chromatin, and fourth, a DNase I-hypersensitive site was observed at the 3'-end of the NF-1 footprint in the chromatin that was not observed with mock assembled or naked DNA. Furthermore, the nucleosomal structure was maintained after binding of NF-1 and CAR/RXR to the PBRU. These results suggest that the effect of nucleosomal structure on the binding of NF-1 to its site is context-dependent, because its binding to MMTV promoter chromatin is inhibited and binding to PBRU chromatin is enhanced. Some indication that sequence context is important was provided in the MMTV system as well. Deletion of the hormone receptor binding sites resulted in partially restored NF-1 binding in chromatin in the absence of hormone so that the sequence flanking the NF-1 site affected its binding to DNA in chromatin (22). These results indicate that NF-1 binds to the PBRU in a nucleosomal structure in the absence of CAR/RXR binding and that binding of NF-1 has little or no effect on binding of CAR/RXR to NR-1 and NR-2.

Although the efficient binding of NF-1 to chromatin was unexpected, based on the studies with the MMTV promoter and Cyp1a1 genes, it is consistent with previous in vivo footprinting analyses of protein binding to the CYP2B1 PBRU in native chromatin (15, 16). In those experiments, the NF-1 site was protected in untreated animals, in which CAR is cytoplasmic (11) and CAR/RXR presumably are not bound to the NR-1 and NR-2 sites. After PB treatment, the NF-1 region was still protected, but the protected region was extended over the NR sites that would be consistent with CAR/RXR binding. Although the in vivo footprinting experiments do not allow identification of the proteins that are bound to the sites, the region protected in native chromatin was the same size as that protected by recombinant NF-1 in vitro, and more importantly, a DMS-hypersensitive site observed at the 3'-side of the NF-1 site with recombinant NF-1 in vitro (this study) was also present in DMS footprints of native chromatin (16). These results indicate that NF-1 binds to the PBRU in vivo in both untreated and PB-treated animals. This result, combined with micrococcal nuclease digestion of native chromatin demonstrating that the PBRU was in a nucleosomal structure (16), indicates that NF-1 can bind to the PBRU in a nucleosomal structure in vivo as was observed in the binding of NF-1 to assembled chromatin in vitro.

The protein binding studies with recombinant CAR/RXR revealed a third NR site that was present about 60 bp to the 5'-side of NR-1. One of the DR-4 half sites is the same as one half site of a putative ER-7 site noted previously (8). The NR-3 site alone does not seem to contribute greatly to PB responsiveness in transient transfections, because deletion of this region either modestly reduced responsiveness by 25% or had little effect (8, 21). In these deletions both the NR-1 and NR-2 sites are present, so it is possible that in the context of these two sites the role of NR-3 is not detectable. The binding affinity of CAR/RXR for NR-3 is similar to that for NR-1 and greater than for NR-2. Furthermore, the center of NR-3 is 85 bp from the center of the NF-1 site in the PBRU core so that, in a nucleosomal structure, the NR-3 and the NF-1 sites would be nearly adjacent on the two loops of DNA in the nucleosome allowing the possibility of interactions between the two.

The present data are consistent with a combinatorial mechanism for the activation of the PBRU, which includes CAR/RXR and NF-1 and possibly other proteins that bind to the region but have not been characterized. For CAR/RXR and NF-1, CAR/RXR primarily mediate the transactivation and NF-1 appears to enhance the CAR/RXR activity. Such enhancement could be mediated by cooperative recruitment of coactivators to the PBRU, which in turn influence the basal transcriptional factors. Identification and characterization of all the proteins in the complex that interact with the PBRU and proximal promoter of the CYP2B genes will be required to completely understand the mechanism of PB induction of these genes.


    ACKNOWLEDGEMENTS

We thank Drs. Cheng-Ming Chiang and Shwu-Yuan Wu for providing the S-190 Drosophila extract and the core histones and for helpful discussions about the methods for chromatin assembly. We thank Dr. Alfredo Nicosia for the cDNA clone of rat NF-1.


    FOOTNOTES

* This work was supported by U.S. Public Health Services Grant GM39360.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1146; Fax: 217-333-1133; E-mail: byronkem@life.uiuc.edu.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008090200


    ABBREVIATIONS

The abbreviations used are: P450, cytochrome P450; CYP, cytochrome P450 gene; PB, phenobarbital; PBRU, PB-responsive unit; NR, nuclear receptor; NF-1, nuclear factor-1; CAR, constitutive androstane receptor; RXR, retinoid X receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; DTT, dithiothreitol; DMS, dimethyl sulfate; MNase, micrococcal nuclease; MMTV, murine mammary tumor virus; kb, kilobase(s); bp, base pair(s); Ni-NTA, nickel-nitrilotriacetic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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