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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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
-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
-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
-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 [
-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 [
-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 [
-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
-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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RESULTS |
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).
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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DISCUSSION |
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.