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Volume 272, Number 48, Issue of November 28, 1997
pp. 30025-30031
(Received for publication, June 6, 1997, and in revised form, August 12, 1997)
From the Division of Drug Metabolism, Faculty of Pharmaceutical
Sciences, Hokkaido University, N12W6, Kita-ku, Sapporo,
Hokkaido 060, Japan
ABSTRACT A xenobiotic-responsive element (XRE)-binding
factor(s) other than the AhR·Arnt complex was found to inhibit the
transcription of CYP1A1 gene in the liver from adult
rabbits, known to be nonresponsive to CYP1A1 inducers. The constitutive
factor(s) in liver nuclear extracts bound to the core sequence of XRE.
The binding was eliminated by the presence of an excess amount of the
AhR·Arnt complex synthesized in vitro. To identify the
constitutive factor(s), a sequence similar to rabbit XRE was sought. It
was found that the sequence of rabbit XRE overlapped with that of the
upstream stimulatory factor 1 (USF1)-binding site in the mouse
metallothionein I promoter. In fact, a super shift assay using a
specific antibody against human USF1 indicated that USF1 was capable of
binding to rabbit XRE. Additionally, the AhR·Arnt-mediated activation
of XRE-TK/Luc reporter gene in RK13 cells was blocked by the
transfection with a USF1 expression vector with the amounts of the
expression vector transfected. These results indicate that the XRE of
the rabbit CYP1A1 gene is recognized by the basic
helix-loop-helix proteins to regulate the expression of
CYP1A1 in both an agonistic (AhR·Arnt) and an antagonistic (USF1) manner.
INTRODUCTION CYP1A1 is the microsomal enzyme responsible for the bioactivation
of carcinogenic compounds such as benzo[a]pyrene (1) and
is known to be induced by halogenated aromatic hydrocarbons such as
2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD)1 and polycyclic
aromatic hydrocarbons including MC (2, 3). However, the induction of
CYP1A1 by MC occurs only in neonatal but not in adult rabbits (4-7).
To date, this mechanism seen in rabbits has not been clarified despite
its significance in toxicology.
The activation of CYP1A1 gene by aryl hydrocarbons is
mediated by a soluble protein designated as AhR (8). Prior to the binding with such an inducer, AhR exists in the cytoplasm as part of a
complex that has a molecular mass of about 280 kDa. This complex is
comprised of AhR, two molecules of the 90-kDa heat shock protein
(Hsp90), and possibly other proteins (9-12). After binding with a
ligand, AhR dissociates from the above complex and translocates to the
nucleus where it heterodimerizes with Arnt (13, 14). The heterodimer
AhR·Arnt complex binds to several copies of short sequences, termed
XREs, located within the 5 AhR and Arnt proteins have two domains necessary for the TCDD-induced
activation of CYP1A1 gene. One of the two domains is the
Per-Arnt-Sim region composed of approximately 300 amino acids that
mediates the ligand binding and interaction with Hsp90. This region is
also found in the Drosophila regulatory proteins Per and Sim
(21) and the mammalian hypoxia-inducible factor 1 The bHLH motif has been seen in common in a number of transcriptional
factors. Most transcriptional factors such as Max, USF1, MyoD, and E47
contain bHLH motif and bind as dimers to the specific DNA sequence
(CACGTG), termed E-box (25-28). Recently, it has been reported that
Arnt constitutively binds as a homodimer to the E-box motif of AdMLP
(29, 30). On the other hand, the AhR·Arnt heterodimer recognizes an
asymmetrical XRE sequence that only partially resembles the E-box. The
consensus sequence of XRE has been identified as
5 MATERIALS AND METHODS To isolate the 5 Fusion
genes with various external deletions were constructed as follows. The
pBK4.5k was treated with exonuclease III after cleavage with
SacI and BamHI. The digested plasmids were
treated with T4 DNA ligase and then introduced into Escherichia
coli TG1 to yield intermediate plasmids. These intermediate
plasmids were cleaved with KpnI and HindIII to
shorten them and to obtain the various lengths of 5 A cDNA fragment for human USF1 (37) was obtained by reverse
transcription-polymerase chain reaction from human placenta RNA. The
entire coding regions of rabbit AhR (7), rabbit Arnt (7), and human
USF1 cDNAs (37) were ligated into a mammalian expression vector
pUC-SR Nuclear extracts were
prepared from the livers of nontreated rabbits (New Zealand White,
male) at 0-4 °C as described by Schibler et al. (40),
except for 1% aprotinin (Sigma) and 0.1 mM PMSF (Wako,
Osaka, Japan) in an initial homogenization buffer. Nuclear extracts
were dialyzed against 25 mM Hepes (pH 7.9) containing 40 mM KCl, 0.5 mM PMSF, 0.1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol twice for 2 h at
4 °C. Subsequently, the extracts were snap-frozen in 0.05-ml
aliquots at 5-10 mg of protein/ml and stored in liquid nitrogen.
Nuclear extracts from RK13 cells were prepared according to the method
of Dignam et al. (41). Twice the cell volumes of buffer A
(10 mM Hepes (pH 7.9) containinng 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol, and 0.5 mM PMSF) were added into a tube
containing cells. The cell suspension was kept on ice for 10 min. Then
the cells were homogenized (20-30 strokes) and centrifuged. The cells
were washed once with the buffer A and were resuspended with a pellet
volume of a dialysis buffer (20 mM Hepes (pH 7.9) containing 100 mM KCl, 12.5 mM
MgCl2, 0.1 mM EDTA, 17% glycerol, 2 mM dithiothreitol, and 0.5 mM PMSF), followed
by the addition of the same volume of the dialysis buffer containing
1.26 M NaCl. The solution was centrifuged at 200,000 × g for 30 min. The supernatant was dialyzed toward the
dialysis buffer. The solution was used as the RK13 nuclear extract.
Gel shift assay was performed with 10 µl
of a reaction mixture containing 25 mM Hepes (pH 7.9), 4%
Ficoll, 40 mM KCl, 0.5 mM dithiothreitol, 0.1 mM EGTA, 1 mM MgCl2, 0.5 µg of
poly(dI-dC), 1 µg of carrier DNA, 5% glycerol, 5-10 µg of nuclear
extracts, and a 32P-labeled probe DNA (5 fmol). The mixture
was incubated at 25 °C for 1 h. Oligonucleotide primers used as
probes are as follows: XRE3, 5 In vitro
transcription and translation assays were carried out using a rabbit
reticulocyte lysate system (Toyoinki, Tokyo, Japan). Briefly, 1 µg of
pBAhR/KS (7) or pBArnt/KS (7) was added to a reaction mixture (50 µl)
containing 50% of a rabbit reticulocyte lysate, 20 µM
complete amino acid mixture, 40 units of a ribonuclease inhibitor, and
20 units of T7 RNA polymerase. The reaction mixture was incubated at
30 °C for 90 min. AhR and Arnt were labeled by the addition of
[35S]methionine to this system. The products were
subjected to SDS-polyacrylamide gel electrophoresis for molecular
weight analysis. A complementary pair of synthetic oligonucleotides
containing the core sequence of XRE was annealed and end-labeled with
[ The Kd values of a
XRE-binding factor(s) were calculated from the results of a gel shift
assay with a fixed amount of nuclear extracts and the various
concentrations of the radiolabeled XRE3. After gel electrophoresis and
autoradiography, radiolabeled bands corresponding to the bound and the
free DNAs were excised. The resultant radioactivity was measured by a
LSC-300 liquid scintillation counter (Aloka, Tokyo, Japan).
Antibodies against human AhR and Arnt proteins
were prepared as described previously (30). Antibodies to human USF1
were purchased from Santa Cruz Biotechnology, Inc. (Santa, Cruz, CA). The super shift assay was performed using these antibodies as follows.
After incubation of probe DNAs with a rabbit nuclear extract as
described above, antibodies were added to the reaction mixtures and
incubated for 1 h at 4 °C. The products were then analyzed by a
gel shift assay.
Rabbit kidney RK13
cells were grown in a minimal essential medium containing fetal calf
serum (10%) and nonessential amino acids (42). Reporter and expression
constructs were introduced into the cultured cells by a modification of
the calcium phosphate coprecipitation procedure (43). After exposure
for 16 h, the DNA-containing culture medium was removed. The cells
were washed with a minimal essential medium and then exposed to a
culture medium with either 1 µM MC or the equivalent
volume of Me2SO (0.1% (v/v) at final concentration as a
control). After another 38 h, the cells were washed with
phosphate-buffered saline (0.01 M sodium phosphate, 0.15 M NaCl) and then lysed. Insoluble materials were removed by
centrifugation. A luciferase activity was determined using Lumat LB9501
luminometer (Berthold, Tokyo, Japan). RESULTS To identify possible
regions responsible for the MC-induced expression of rabbit
CYP1A1 gene, 5
[View Larger Version of this Image (42K GIF file)]
We found a
constitutive factor(s) in rabbit liver nuclear extracts that bound to
XRE3 and XRE5. Thus, the amounts of the constitutive factor(s) present
in the nuclear extracts from neonatal livers were compared with those
from adult livers (Fig. 2). The binding of a liver-specific transcriptional factor HNF4 (44) was monitored as a
control. The amount of the constitutive factor(s) bound to XRE3 and
XRE5 was greater in the nuclear extracts from adult rabbit livers than
neonatal rabbit livers. No shifted bands were observed when the nuclear
extracts from neonatal and adult livers were added to XRE4 used as a
probe.
[View Larger Version of this Image (44K GIF file)]
As a first step to prove our idea that
the unknown XRE-binding factor(s) in the nuclear extracts from adult
rabbit livers bound to XRE to compete with the AhR·Arnt complex, we
examined the possibility that the constitutive factor(s) recognized the core sequence of XRE. As shown in Fig.
3A, the factor(s) did not bind
to a XRE possessing a mutation, indicating that the core sequence of
XRE was specifically recognized by the constitutive factor(s).
Additionally, the shifted band disappeared when a 300-fold molar excess
of a nucleotide with a consensus sequence (CANNTG) was added,
supporting the idea that this constitutive factor(s) belongs to the
bHLH family. These results indicate that the factor(s) bound to bHLH
sequence modulates CYP1A1 induction in adult rabbit livers
by competition with the AhR·Arnt complex to bind to XRE. To test this
possibility, we examined whether or not the competition occurred
between the factor(s) and AhR·Arnt heterodimer to bind to the XRE.
When nuclear extracts from adult rabbit livers were absent, the
AhR·Arnt complex clearly bound to XRE3 to show a shifted band (Fig.
3B). The amount of the AhR·Arnt heterodimer to bind to
XRE3 was decreased by the addition of the constitutive factor(s). Addition of a large amount of the AhR·Arnt complex was needed to
obtain an efficient binding to XRE3 in the presence of the factor(s)
(Fig. 3B). These results suggest that the constitutive factor(s) in adult rabbit livers regulates CYP1A1 induction
by competing with the AhR·Arnt complex.
[View Larger Version of this Image (43K GIF file)]
The affinities of the AhR·Arnt
complex and the constitutive factor(s) to bind to DNA were calculated
by saturation binding assays in which a fixed amount of the factor(s)
was added and by the binding assay by titration with the increasing
amounts of radiolabeled XRE3 (Fig. 4).
The data were plotted as the amount of the bound XRE3 versus
the total amount of XRE3. The dissociation constants were calculated
from Scatchard plots of the formation of the complex versus
the amounts of free XRE3. The results showed that Kd
values for the AhR·Arnt complex and the constitutive factor(s) were
2.6 and 1.8 nM, respectively, indicating that the affinities for XRE3 were similar.
[View Larger Version of this Image (21K GIF file)]
To prove that the
constitutive factor(s) did not contain AhR and Arnt, a gel shift assay
using antibodies to human AhR and human Arnt (Fig.
5) was performed. To remove AhR and Arnt
from the nuclear extracts, antibodies to human AhR or Arnt were added to the nuclear extracts from adult rabbit livers. The results showed
that a shifted band corresponding to the constitutive factor(s) appeared, indicating that a factor(s) other than the AhR·Arnt complex
recognized the core sequence of XRE. The binding of AhR·Arnt heterodimer to XRE disappeared when the antibodies against human AhR
were added. Although antibodies to human AhR inhibited the heterodimerization of AhR with Arnt, antibodies to human Arnt did not
inhibit the formation of the AhR·Arnt complex (30). Thus, a super
shifted band was detected by the presence of the antibodies against
human Arnt.
[View Larger Version of this Image (23K GIF file)]
We found that the sequence of rabbit XRE3 was similar to
that of the USF1-binding site (Fig. 6).
The USF1-binding site was originally identified as the E-box motif
(CACGTG) of AdMLP (45). This site was also found in human growth
hormone (46), mouse metallothionein I (47), rat
[View Larger Version of this Image (24K GIF file)]
To
determine if USF1 could bind to XRE3, we performed a competition assay
using an E-box motif of AdMLP as a competitor. When nuclear extracts
from adult rabbit livers were added, the formation of the complex with
XRE3 was abolished by the presence of a 50-fold molar excess of the
unlabeled AdMLP (Fig. 7A).
Super shift bands were detectable with antibodies against USF1,
although the amounts of super shift bands varied depending on the probe
(Fig. 7B). To further prove the specific binding of USF1
with XRE3, USF1 was expressed in RK13 cells by transfection of the
expression plasmid USF-SR
[View Larger Version of this Image (38K GIF file)]
To
determine the functional consequences of the interaction of USF1 with
XRE3 of rabbit CYP1A1 gene, the expression plasmid carrying
USF1 was cotransfected to RK13 cells with the expression vectors for
AhR and Arnt in addition to the XRE-TK/Luc or TK/Luc. Relative to
pUC-SR
[View Larger Version of this Image (24K GIF file)]
DISCUSSION The induction of CYP1A1 by MC occurs only in neonatal and not in
adult rabbits, probably resulting in low susceptibility to carcinogens
(4-7). However, the mechanism(s) involved in the nonresponsive nature
of rabbits to such an inducer in rabbits has not been clarified despite
its significance in toxicology. Our hypothesis to account for this
phenomenon is that bHLH proteins in addition to the AhR·Arnt complex
are capable of recognizing the core sequence of XRE and that a
factor(s) that binds to XRE inhibits the induction of CYP1A1 by MC in
adult rabbits. To examine this idea, we performed a gel shift
assay using nuclear extracts from adult rabbit livers as a competitor
to the binding of the AhR·Arnt complex with XRE3 and found a
constitutive factor(s) that inhibited the binding as was expected.
Scatchard plots showed that the binding affinity of the factor(s) for
XRE3 was similar to that of the AhR·Arnt complex (Fig. 4). In fact,
the binding of the AhR·Arnt complex to XRE3 was not observed even
after treatment of adult rabbits with MC. Alternatively, the
constitutive factor(s) bound to XRE3 (data not shown). Recently, we
reported that there was a lesser amount of Arnt mRNA in rabbit livers than in extrahepatic tissues (7). Thus, it is possible that the
amount of Arnt limits the amount of the AhR·Arnt complex, leading to
the low level of induction of CYP1A1. These results indicate that the
interaction of a constitutive factor(s) with XRE3 occurs predominantly
in the livers from MC-treated rabbits, accounting for the known fact
that the induction by MC does not occur in adult rabbits.
A computer search was performed to find a sequence(s) similar to XRE3
in the rabbit CYP1A1 gene. The sequence of rabbit XRE3 was
highly homologous to that of USF1-binding sites (Fig. 6). The presence
of the conserved sequences has been reported in AdMLP (45), human
growth hormone (46), mouse metallothionein I (47), rat Our super shift assay indicated that one of the constitutive factor(s)
was USF1, a bHLH protein (Fig. 5). However, the E-box motif is reported
to be recognized by a number of bHLH proteins such as USF2, Max, MyoD,
and E47 (25-28, 53, 54). This suggests that a factor(s) in addition to
USF1 also binds to the core sequence of XRE3. In fact, only some of the
bands were super shifted by antibodies to USF1 when XRE3 and AdMLP were
used as probes. Similar to the binding shown in the present study, the
factor(s) constitutively bound to XRE has been found in human
keratinocytes (55) and human fibroblasts (56). It has also been
reported that an additional XRE-binding factor was detectable only in
the presence of TCDD (17). Because these findings were obtained with
XREs that did not contain the binding site of USF1 within the core
sequence, the constitutive factor (USF1) found in the present study was distinct from the above factors. Recently, it has been reported that
AhR2 negatively regulates the MC-induced expression of
CYP1A1 gene by blocking the heterodimerization of AhR with
Arnt or competing with the AhR·Arnt complex on the same binding site
(57). Thus, a factor such as AhR2 may also contribute to the negative
regulation of the induction of CYP1A1 in rabbits.
To date, the mechanism responsible for the suppression of the induction
of CYP1A1 has been widely discussed. A negative regulatory element has
been found in the 5 In the present study, we showed that USF1 bound to XRE3 to compete with
the AhR·Arnt complex in the rabbit CYP1A1 gene. Because the sequence of XRE3 in the rabbit, to which USF1 bound, was not present in the 5 FOOTNOTES REFERENCES
Inhibition of the Transcription of CYP1A1 Gene by the
Upstream Stimulatory Factor 1 in Rabbits
COMPETITIVE BINDING OF USF1 WITH AhR·Arnt COMPLEX*
,
-flanking region of CYP1A1 gene
to stimulate the synthesis of CYP1A1 protein and several other proteins
involved in xenobiotic metabolism (15-18). Thus, the induction of
CYP1A1 is regulated exclusively at the transcriptional level (19,
20).
(22). The other is
a bHLH motif essential to the DNA binding and the dimerization of AhR
and Arnt toward their amino termini (23, 24).
-(T/G)NGCGTG(A/C)(G/C)(A/T)-3 (31-33). Four core nucleotides
(5-CGTG-3) within XRE are absolutely required for the binding with
AhR·Arnt heterodimer. Arnt binds to the thymidine in the 5-CGTG-3
core identical to an E-box half site (GTG), whereas AhR binds to 5
proximal to the 5-CGTG-3 core in the XRE (34). In the present study,
we found USF1 to be an additional factor capable of binding to the
rabbit XRE to inhibit the interaction of the AhR·Arnt complex with
XRE in rabbits.
-Flanking Region of Rabbit CYP1A1
Gene
-flanking region of rabbit
CYP1A1 gene (35), a gene library prepared from the rabbit
DNA cleaved by EcoRI and cloned into 
ZAPII vector
(Stratagene, La Jolla, CA) was screened with a 32P-labeled
CYP1A1 cDNA as a probe (36). By screening about 1.1 × 105 plaques, we obtained a genomic clone (pBK4.5k)
containing the 5-flanking region of rabbit CYP1A1 gene and
mapped for further studies.
-flanking
sequences. The fragments were inserted into the unique
KpnI-HindIII site of the basic vector 2 (Stratagene).
(38). The resultant expression plasmids, designated
AhR-SR, Arnt-SR, and USF-SR, were used for the transfection. To ascertain the contribution of USF1 in rabbit kidney RK13 cells, reporter plasmids XRE-TK/Luc and TK/Luc were constructed. The double-stranded XRE was subcloned into the basic vector 2 by a blunt
end ligation. The basal TK promoter was then inserted into the
NheI-HindIII site of the plasmid. The copy number
and the direction of the oligonucleotides inserted into the reporter
plasmids were confirmed by sequence analysis (39).
-TCCCCCCAGTTCGCGTGACTGCGCCGGGA-3 and
3-AGGGGGGTCAAGCGCACTGACGCGGCCCT-5; mutant XRE3,
5-TCCCCCCAGTTCGATTGACTGCGCCGGGA-3 and
3-AGGGGGGTCAAGCTAACTGACGCGGCCCT-5; XRE4,
5-CGCCCAGGAGTTGCGTGAGAAGGGCTTGGA-3 and
3-GCGGGTCCTCAACGCACTCTTCCCGAACCT-5; XRE5,
5-GAGTCCCTGCTCGCGTGAGAAGCTCAGCGACC-3 and
3-CTCAGGGACGAGCGCACTCTTCGAGTCGCTGG-5; AdMLP,
5-GATCCGTAGGCCACGTGACCGGG-3 and 3-CTAGGCATCCGGTGCACTGGCCC-5.
-32P]dATP. A nonspecific competitor, poly(dI-dC) (4.5 ng), was mixed with rabbit AhR and Arnt proteins, expressed in
vitro, and incubated for 20 min at 25 °C. The radiolabeled
probe (1 × 105 cpm) was then added and incubated for
20 min at 25 °C, followed by a nondenaturing gel electrophoresis and
autoradiography.
-Galactosidase activity was determined as described (43). Each luciferase
activity was corrected by -galactosidase activity.
deletion mutants were constructed as shown
in Fig. 1A. These plasmids
were transfected into rabbit kidney RK13 cells to show the inducibility
of CYP1A1 by some inducers (data not shown). The cells were then
exposed to MC. The maximal MC-induced activity was seen in the cells
transfected with Luc1.6. The activity decreased with progressive
deletions starting from 
1.6 kb of rabbit CYP1A1 gene. The
luciferase activity was significantly decreased by the deletion of the
sequences between nucleotides 1.06 and 0.95 kb, 0.95 and 0.83
kb, and 0.68 and 0.45 kb from the transcriptional start sites.
These results indicate that at least three regions containing essential
XREs are responsible for the induction of rabbit CYP1A1
gene. Deletions to 1.28 kb resulted in an approximately 55% decrease
in a constitutive expression, whereas further deletions to 0.18 kb
decreased to about 5%. Comparing the sequences of possible XREs in the
5-flanking region of rabbit CYP1A1 gene up to 1.4 kb with
the consensus sequence (Fig. 1B) reported so far, XRE3,
XRE4, and XRE5 were found to be identical with the reported consensus
sequence (31-33). However, other XREs possessed the replacement of
some nucleotides from the consensus sequence. To analyze the
specificity of AhR·Arnt to bind to the XREs, a competition analysis
was carried out (Fig. 1C). The specific binding of
AhR·Arnt disappeared when a 150-fold molar excess of an unlabeled
probe (XRE3, XRE4, or XRE5) was added. Thus, we focused on the XREs
that possibly regulate the CYP1A1 induction by MC in RK13
cells.
Fig. 1.
Regulatory DNA elements in the 5-flanking
region of rabbit CYP1A1 gene. A, structure and
transcriptional activity of 5 deletion mutants in the 5-flanking
region of CYP1A1 gene. The construction of deletion mutants
is described under "Materials and Methods." The numbers
given to deletion plasmids indicate the 5 ends of the 5-flanking
sequence of rabbit CYP1A1 gene counted negatively from the
transcriptional start site. The XRE most distant from the mRNA
start site was defined as XRE1. The extent of induction by MC is shown
as the ratio of the induced to the uninduced activity. The position of
the XRE most distant from XRE6 is shown by a vertical arrow.
All values represent the means ± S.D. with the number of
independent experiments shown in parentheses. The data are
expressed as a percentage of the basal activity obtained with Luc4.5.
B, alignment of possible XREs found in the 5-flanking
region of rabbit CYP1A1 gene. The sequence of the rabbit XRE
was compared with that of consensus sequences identified in the
CYP1A1 gene reported so far (31-33). The numbers
given to XREs indicate the position of the nucleotide counted
negatively from the transcriptional initiation site. C, competition analysis with various XREs. A synthetic XRE4 was used as a
labeled probe. Rabbit AhR and Arnt proteins produced by in vitro translation were incubated in the presence of 1 µM MC, followed by gel shift assay as described under
"Materials and Methods." The arrow indicates the
AhR·Arnt·XRE4 complex. A competitor DNA was added at 75- or
150-fold molar excess of the probe XRE4 DNA. The elimination of the
shifted band by the addition of a 150-fold molar excess of XRE3, XRE4,
or XRE5 demonstrates the specificity of the complex. Mutant XRE used as
a competitor is as follows: XRE4 mu,
5-CGCCCAGGAGTTGATTGAGAAGGGCTTGGA-3 and
3-GCGGGTCCTCAACTAACTCTTCCCGAACCT-5. To prove the specificity of
AhR·Arnt heterodimer, a 150-fold molar excess of XRE4 mutant was
added.
Fig. 2.
Comparison of the amounts of constitutive
factor(s) in neonatal and adult rabbit livers that bound to XRE.
Liver nuclear extracts from nontreated rabbits (New Zealand White,
male) were prepared according to the method of Schibler et
al. (40). A 32P-labeled double-stranded XRE3, XRE4, or
XRE5 was incubated with 10 µg of nuclear extracts from neonatal or
adult rabbit livers at 25 °C for 1 h as described under
"Materials and Methods." The arrow indicates the major
constitutive factor(s) bound to rabbit XREs. As a control experiment,
the binding activity of HNF4 was determined in neonatal and adult
livers.
Fig. 3.
Constitutive factor(s) recognizing the core
sequence of XRE3. A, specificity of a constitutive binding
factor(s) to bind to XRE3. A 32P-labeled double-stranded
XRE3 was incubated with 10 µg of nuclear extracts from adult rabbit
livers at 25 °C for 1 h as described. Mutant XRE3 as described
under "Materials and Methods" was used as a probe to demonstrate
the specificity of the constitutive complex. Competitor DNAs were also
used at 100- (XRE3), 200-, or 300-fold (bHLH consensus (25-28)) molar
excess of the probe DNA. B, the competitive binding of a
constitutive factor(s) with AhR·Arnt complex. The DNA binding of the
AhR·Arnt complex and the constitutive factor(s) were examined by a
competitive binding assay in which various amounts (1-7 µl) of
AhR·Arnt complex synthesized in vitro were added. Rabbit
AhR and Arnt proteins were incubated in the presence of 1 µM MC, followed by gel shift assay as described under
"Materials and Methods."
Fig. 4.
Kinetic analysis of a constitutive factor(s)
(A) and AhR·Arnt complex to XRE3 (B).
The Kd values were calculated by Scatchard plots.
After gel electrophoresis and autoradiography, the radiolabeled bands
corresponding to the bound and free XREs were excised, and the
radioactivity was measured by the scintillation counter. The left
panels show the autographic images of the gel shift assay. The
middle panels are saturation curves in which the amount of
bound XRE3 versus total XRE3 was plotted. The right panels show Scatchard plots.
Fig. 5.
The constitutive binding factor(s) did not
contain the AhR and Arnt. A 32P-labeled
double-stranded XRE3 was incubated with 10 µg of nuclear extracts
prepared from adult rabbit livers in the absence or the presence of
anti-AhR or anti-Arnt antibodies as described under "Materials and
Methods." As a control experiment, AhR·Arnt synthesized in
vitro was incubated in the absence or the presence of the
antibodies.
-fibrinogen (48),
and Xenopus TFIIIA genes (49), although some nucleotides in
the binding site of USF1 identified in a variety of cellular and viral
promoters differed from those in the E-box motif (50). It is of
particular interest to note that the sequence of rabbit XRE3 is
identical to that of the USF1-binding site (CGCGTGAC) in the mouse
metallothionein I promoter.
Fig. 6.
Alignment of the binding sites for USF1.
The sequences are aligned with 8-base pair domain CANNTGAC, which is a
consensus binding motif for USF1 (50). The core sequence of XRE3 was
identical to that of the USF1-binding site in mouse metallothionein I
promoter (47).
into the cells. Nuclear extracts prepared
from the cells were applied to a gel shift assay. Consequently, a
specific band appeared only when nuclear extracts from USF1-transfected cells were applied. Antibodies against USF1 recognized the super shifted band. This antibody-bound complex exhibited a further retarded
mobility in the polyacrylamide gel (Fig. 7C).
Fig. 7.
Binding of USF1 to XRE3. A,
competition between a constitutive factor(s) and AdMLP to bind to XRE3.
A 32P-labeled double-stranded XRE3 fragment was incubated
with 10 µg of nuclear extracts from adult rabbit livers in the
presence of a 50-, 100-, and 150-fold molar excess amount of AdMLP at
25 °C for 1 h. The arrow indicates the complex of a
constitutive factor(s) with XRE3. B, super shift assay using
antibodies to USF1. A 32P-labeled double-stranded XRE3 or
AdMLP was incubated with nuclear extracts from adult rabbit livers in
the absence or the presence of antibody against human USF1 as described
under "Materials and Methods." The arrow indicates the
super shifted band of USF1. C, binding of human USF1
expressed in RK13 cells to XRE3. Nuclear extracts from RK13 cells
transfected with USF1-SR were prepared essentially as described by
Dignam et al. (41). A 32P-labeled
double-stranded XRE3 was incubated with 10 µg of the nuclear extracts
from RK13 cells at 25 °C for 1 h. The specificity of the
binding of USF1 was determined by super shift assay as described under
"Materials and Methods." The arrow indicates the complex
of human USF1 with XRE3.
-transfected or nontransfected cells, the transfection of AhR-
and Arnt-SR together with XRE-TK/Luc resulted in a 2-fold increase
in basal expression in the absence of MC. USF1 inhibited the
transactivation of XRE-TK/Luc reporter gene by the AhR·Arnt complex
and MC (Fig. 8B). Relative to
the maximum induction observed with the absence of USF-SR (16 ± 1-fold), the down-regulation observed with USF-SR (2 µg)
(9.5 ± 0.3-fold) and USF-SR (4 µg) (6.1 ± 0.2-fold)
was significant with p values all equal to <0.0001 but not
with USF-SR (1 µg) (14 ± 1-fold) with p value of
>0.05. The increasing amounts of the USF-SR expression vector did
not affect basal expression. However, the transfection of USF-SR (>5 µg) enhanced the basal activity of XRE-TK/Luc gene (data not shown). The co-transfection of RK13 cells with the USF1 expression vector together with Luc 1.28 reporter plasmid inhibited the induction by MC by 39% (data not shown). These results indicate that USF1 depresses the induction of CYP1A1 by MC in rabbit livers by
competing XRE3 with the AhR·Arnt complex.
Fig. 8.
Inhibition by USF1 of AhR·Arnt-mediated
transactivation of XRE3 to express CYP1A1. A, the schematic
illustration of reporter plasmids. Reporter plasmids XRE-TK/Luc and
TK/Luc were constructed as described under "Materials and Methods."
B, co-transfection to assess the contribution of USF1.
AhR-SR (3 µg), Arnt-SR (3 µg), USF-SR (0-4 µg), and
pUC-SR (3 µg) were co-transfected to RK13 cells in combination
with XRE-TK/Luc (2 µg) or TK/Luc (2 µg). An appropriate amount of
pUC-SR was added to adjust the total amount of DNA transfected to
the cells. The medium was removed 16 h after the initiation of the
transcription. The cells were then washed and exposed to a medium
supplemented with either dimethyl sulfoxide or 1 µM MC
for 38 h. All values represent the means ± S.D.
(n = 3). The mean value obtained with XRE-TK/Luc in the absence of USF-SR and in the presence of AhR-SR, Arnt-SR, and MC was defined as 100%.
-fibrinogen
(48), and Xenopus TFIIIA genes (49). Particularly, the
sequences of XRE3 and XRE5 were identical to the USF1-binding site of
mouse metallothionein I promoter (CGCGTG) (Fig. 6). The existence of
the XRE core sequence (CGCGTG) allowed us to predict that rabbit
CYP1A1 gene is regulated in a positive (AhR·Arnt) and a
negative (USF1) manner through the overlapped XRE sequence. Interestingly, the portion of XRE sequence identical with that of USF1
sequence was conserved in humans but not in rats and mice known to be
highly responsive to aromatic hydrocarbons (34, 51, 52). Unlike the
rabbit CYP1A1 gene, the binding site of USF1 was not found
in the possible XRE sequences in the 5-flanking region of rabbit
CYP1A2 gene (35). CYP1A2 is known to be induced by TCDD in
both neonatal and adult rabbit livers (35).
-flanking region of human CYP1A1 gene
(58-61). An unknown factor(s) in HepG2 cells bound to a 21-base pair
palindrome in the human CYP1A1 gene, resulting in the
negative regulation of the enhancement of the transcription of
CYP1A1 gene by TCDD. Because the negative regulatory element
was not present in the upstream region of rabbit CYP1A1
gene, the incapability of adult rabbits to induce CYP1A1 must not be
caused by the negative regulatory element-binding factor(s).
-upstream region of rat and mouse CYP1A1
gene, the inhibition of the induction by USF1 may not occur in the rat and mouse. This regulation of CYP1A1 expression may account, at least
in part, for the species differences in chemical carcinogenesis and
cytotoxicity.
Present address: Div. of Cellular Physiology, Faculty of
Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka
565, Japan.
§
Present address: Dept. of Chemistry, Faculty of Science, Tohoku
University, Sendai 980-77, Japan.
¶
To whom correspondence should be addressed. Tel. and Fax:
81-11-706-4978; E-mail: kamataki{at}pharm.hokudai.ac.jp.
1
The abbreviations used are as follows: TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin; MC,
3-methylcholanthrene; AhR, aryl hydrocarbon receptor; Arnt, Ah receptor
nuclear translocator; XRE, xenobiotic-responsive element; bHLH, basic
helix-loop-helix; USF1, upstream stimulatory factor 1; AdMLP,
adenovirus major late promoter; TK, thymidine kinase; PMSF,
phenylmethanesulfonyl fluoride; kb, kilobase(s).
Volume 272, Number 48,
Issue of November 28, 1997
pp. 30025-30031
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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