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(Received for publication, June 6, 1995; and in revised form, August 18, 1995) From the
The induction by dexamethasone of rat liver CYP3A1 differs from
classical glucocorticoid gene regulation in part because both
glucocorticoids and antiglucocorticoids such as pregnenolone
16
CYP3A1, a member of the cytochrome P450 supergene family, is
prominently induced in liver microsomes of rats or in primary cultures
of adult rat hepatocytes treated with the synthetic glucocorticoid,
dexamethasone and, paradoxically, by such antiglucocorticoids as
pregnenolone 16 A
possible explanation for our unusual findings was that glucocorticoids
and antiglucocorticoids might affect induction of CYP3A1 at different
steps in gene expression including post-transcriptional events such as
translation or degradation of CYP3A1 mRNA. To investigate the role of
CYP3A1 transcription specifically, we established a system for
transient expression of 1.5 kilobases of DNA 5`-flanking the CYP3A1
gene
Preliminary analysis of deletions of chimeric plasmid
TKCAT-1525/-56 that contains CYP3A1 sequences from
-56 to -1525 relative to the transcriptional start site,
previously identified a region of the rat CYP3A1 gene between
-220 and -56 that, when transiently expressed in primary
cultures of adult rat hepatocytes, responded to treatment of the cells
with dexamethasone or PCN and exhibited dexamethasone plus PCN
synergy(5) . We noted that -220 to -56 contains
neither a classical sequence for the 15-bp GRE (19) nor
sequences identical to the consensus sequence, TGTTCT, for
``half-sites'' in the classical GRE(20) . We prepared
additional deletion mutants of the chimeric CAT plasmid,
TKCAT-220/-56 (Fig. 1), transiently expressed these
in primary cultures of rat hepatocytes, and measured CAT expression
following 48 h treatment of hepatocytes with 10 µM dexamethasone or PCN (Fig. 1). Deletion of sequences
between -220 and -176 or between -71 and -56
resulted in no loss of dexamethasone or PCN induction of CAT
expression, whereas a further deletion between -176 and
-148 resulted in a 2-fold loss of dexamethasone induction. The
deletion extending beyond position -56 to -37 bp appeared
to reduce CAT expression by PCN treatment, although CAT expression by
dexamethasone treatment was not altered. These data suggest the
possibility that a negative element lies between -56 and
-37. However, the induction by dexamethasone on both the
TKCAT-176/-71 (20-fold) and TKCAT-148/-71
(10-fold) constructions was substantial. As shown previously for the
TKCAT-220/-56 construction(5) , dexamethasone also
produced dose-dependent increases in CAT expression for
TKCAT-148/-71 (data not shown). From these results we
concluded that the sequences between -148 and -71 are
essential for the steroid inductions, although upstream sequences
(-176 to -148) may potentiate hormone responsiveness.
Figure 1:
Expression of chimeric CYP3A1-TKCAT
plasmids in primary rat hepatocytes. Chimeric plasmids were generated
by amplification of CYP3A1 sequences using polymerase chain reaction,
and sequences cloned into the pBLCAT2 vector (TKCAT) as
described under ``Materials and Methods.'' Negative
numbers represent distances from the transcriptional start site.
Chimeric plasmids were transiently expressed in primary cultures of rat
hepatocytes. Cells were treated with dexamethasone (10 µM)
or PCN (10 µM) for 48 h, followed by analysis of CAT
expression. The -fold induction refers to the ratio of CAT protein of
induced cells to uninduced control cells. Data are the average results
(mean ± standard error of the mean) of at least three
independent transfection experiments.
To
examine whether trans-acting proteins bind to the identified
CYP3A1 dexamethasone-responsive region, we performed in vitro DNase I footprinting assays. Radioactively labeled CYP3A1 DNA
(-220/-56) was incubated with nuclear extracts from control
rat liver, and then the mixture was digested with DNase I. The results
of these ``footprinting'' studies showed two regions strongly
protected and one region weakly protected against DNase I activity (Fig. 2, lane 3). The reaction was specific because
addition of excess unlabeled DNA (-220/-56) was able to
compete for binding to both protected regions (Fig. 2, lane
5), whereas the presence of excess nonspecific DNA in the reaction
did not compete (Fig. 2, lane 6). Although
dexamethasone induction of CYP3A1 in the liver might be expected to
alter the pattern perhaps by creating additional footprints, use of
nuclear extracts from dexamethasone-treated rat liver in the same
experiment produced the identical footprints (Fig. 2, lane
4). Moreover, we found the same footprints when this experiment
was carried out with the use of nuclear extracts isolated from
untreated and dexamethasone-treated cultures of rat hepatocytes (data
not shown).
Figure 2:
DNase I footprint analysis of nuclear
protein binding to the CYP3A1 dexamethasone-responsive element. The
By alignment with the CYP3A1 DNA (-220/-56)
sequence, we determined that the first protected region (FP1) was
located from -135 to -117, while the second footprint (FP2)
was from -108 to -85 ( Fig. 2and 3A). Thus,
within the -220 to -56 DNA probe, both footprints, FP1 and
FP2, were found within the 78-bp fragment identified from deletional
analysis to be dexamethasone- and PCN-responsive (Fig. 1,
TKCAT-148/-71). Close examination of the FP1 sequence (Fig. 3B) finds 8 bp in common with a 12-bp consensus
GRE (19) and 4 of 5 nucleotides suggested as critical for GR
binding(21) . The FP1 sequence also contains a palindromic
sequence (underlined), ATGAACTTCAT, that overlaps one of two
``direct repeats'' of ATGAACT separated by 2 nucleotides (Fig. 3C). A 6-bp portion of these 7-bp repeated
sequences, TGAACT, when examined on the lower strand (AGTTCA), is a DNA
motif representing a high affinity binding site for several types of
nuclear receptors including the peroxisome proliferator activator (22) and the retinoic acid receptors(23) . The CYP3A1
gene is not regulated by peroxisome proliferators or retinoic acids,
thus emphasizing that a DNA binding site identified by sequence
analysis is not necessarily functional. In addition, we identified a
sequence at the 3` end of FP2 similar to that of an AP1 binding site (24) (Fig. 3A).
Figure 3:
Summary of the DNA-protein interactions at
the CYP3A1 dexamethasone-responsive element. A, regions
protected against DNase I digestion, referred to as FP1 and FP2, of
dexamethasone-responsive CYP3A1 (-220/-56) are indicated by solid lines. The dashed line indicates a footprint
possibly due to weak interactions. The sequence similar to an AP1
binding site (24) is boxed. B, alignment of
FP1 with the consensus GRE sequence(19) . The 5 bases critical
for glucocorticoid receptor binding are shown in bold(21) . Lines between nucleotides show
identity. C, the sequence of the ds-oligonucleotide containing
the FP1 sequence (underlined) is shown. Arrows indicate its palindromic structure and a 7-bp direct
repeat.
We prepared overlapping
ds-oligonucleotides that span the 78-bp responsive region in CYP3A1
corresponding to -148 to -115 (contains FP1) and to
-116 to -69 (contains FP2) to determine which if either of
the sequences that were protected against DNase I retains
responsiveness to inducers. When the ds-oligonucleotides were cloned
into pBLCAT2 and transiently expressed in hepatocytes, only sequences
spanning the area protected by FP1 (TKCAT-148/-115) were
responsive to dexamethasone and PCN (6- and 3-fold, respectively) (Fig. 4A). The magnitude of the response of the FP1
containing construction to dexamethasone was comparable to the
endogenous hepatic levels of CYP3A1 transcriptional activation by
dexamethasone as demonstrated by nuclear run-on transcription
assays(6, 7, 8) . When placed in the opposite
orientation, i.e. 3` to 5`, this -148/-115
ds-oligonucleotide mediates a similar level of CAT induction; however,
multiple copies of this sequence did not increase CAT induction over
and above the results with a single copy (data not shown). Thus, this
single 33-bp element is able to confer both dexamethasone and PCN
induction on CYP3A1 gene expression in cultured hepatocytes, although
sequences flanking this element may be important in the modulation of
responsiveness, as indicated by an increase in CAT induction when
sequences from -148 to -176 are included.
Figure 4:
Transient expression in cultured
hepatocytes of CYP3A1 5`-flanking sequences encompassing FP1 and FP2. A, ds-oligonucleotides corresponding to -148/-115
(contains FP1) and -116/-69 (contains FP2) were cloned 5`
to TKCAT and transiently expressed in rat hepatocytes. Hepatocytes were
treated with dexamethasone (10 µM) or PCN (10
µM). Footprint sequences are underlined. B, hepatocytes were transfected with either MMTVCAT or
TKCAT-148/-115 and treated with either dexamethasone (0.1
µM), PCN (10 µM), or dexamethasone (0.1
µM) plus PCN (10 µM) for 48 h. CAT protein
was determined as described under ``Materials and Methods.''
The -fold induction refers to the ratio of CAT protein of induced cells
to uninduced control cells. Data are the average results (numbers above bars) of two to three independent experiments. Open bars, MMTVCAT; solid bars,
TKCAT-148/-115. N.I., no induction; ratio of CAT
protein of induced cells to uninduced control cells <
1.0.
We also
demonstrated that the 33-bp element containing the FP1 sequence still
confers (although at an attenuated level) the synergistic effect of PCN
on CYP3A1 induction by dexamethasone demonstrated previously for the
larger CYP3A1 5`-flanking DNA segments(5) . In cultured
hepatocytes transfected with TKCAT-148/-115 (one copy of
the 33-bp element) and treated with a low dose of dexamethasone (0.1
µM), with PCN (10 µM), or with their
combination, the induction of CAT was <2-, 2-, and 10-fold,
respectively (Fig. 4B). As a control, we transfected
hepatocyte cultures with MMTVCAT, a vector containing a typical GR
regulated gene promoter (11) and found, as expected from
previous studies(5) , that dexamethasone treatment induced CAT
expression and that PCN treatment induced no CAT expression and
modestly antagonized dexamethasone induction of MMTVCAT (Fig. 4B). Inasmuch as the 33-bp element is
sufficient to confer dexamethasone responsiveness in cultured
hepatocytes and contains sequences in its FP1 segment similar to a
simple GRE (Fig. 3B), we performed in vitro DNase I footprinting to determine if the GR actually is capable of
being bound by this CYP3A1 DNA fragment. When the radioactively labeled
CYP3A1 DNA (-220/-56) containing the 33-bp element was
incubated with saturating amounts (approximately 50 pmol) of dbGR, a
bacterially expressed protein corresponding to the DNA-binding domain
of the rat GR(17) , no protection of the CYP3A1 fragment could
be detected (Fig. 5, lanes 12 and 13). In
contrast, the MMTV promoter, a DNA control for GR binding that contains
multiple GREs(18) , was bound by dbGR as shown by protected
sequences within the MMTV promoter corresponding to the GRE showing
partial dyad symmetry (-174/-185) (Fig. 5, lanes
4 and 5). These results suggest that if the GR is
directly involved in the transcriptional activation of the CYP3A1 gene,
it likely interacts with DNA-bound protein rather than with DNA itself.
Figure 5:
Test
for binding to the dexamethasone-responsive CYP3A1 element by dbGR (DNA
binding domain of the glucocorticoid receptor protein) with the in
vitro DNase I footprint assay. DNase I footprinting was carried
out after binding the dbGR to the MMTV promoter and the
CYP3A1-responsive element (-220/-56). Lane 1,
A+G sequence for the MMTV promoter; lanes 2 and 3, MMTV promoter, no protein; lanes 4 and 5,
MMTV promoter incubated with 600 ng of dbGR; lanes 6-9,
CYP3A1 promoter (-220/-56) sequence; lanes 10 and 11, CYP3A1 promoter, no protein; lanes 12 and 13, CYP3A1 promoter incubated with 600 ng dbGR. - refers
to absence of dbGR protein in the binding reaction. The GRE in the MMTV
promoter protected against DNase I digestion is bracketed.
Further evidence against the possible direct binding of the GR with
the CYP3A1 gene was developed with the use of the electrophoretic
mobility shift assay. When we incubated bacterially expressed GR (dbGR)
with the radiolabeled 33-bp ds-oligonucleotide-responsive element
(containing FP1), no change in its mobility in gel electrophoresis was
observed, whereas control incubations containing GR plus the TAT GRE
were retarded in their migration as expected (data not shown). We did
detect a protein-DNA complex when the radiolabeled 33-bp
ds-oligonucleotide was incubated with liver nuclear extracts from
untreated or from dexamethasone-treated rats (Fig. 6, lanes
2 and 3). As evidence of sequence specificity, the
binding of nuclear proteins to the 33-bp oligonucleotide could be
abolished in the presence of 50-fold molar excess of unlabeled 33-bp
oligonucleotide, but not by 150-fold molar excess of an unrelated
oligonucleotide containing a binding site for NF-
Figure 6:
Binding of rat liver nuclear proteins to
the 33-bp responsive element. The 5`-end-labeled double-stranded
oligonucleotide corresponding to the 33-bp responsive element
(containing FP1 sequence) was incubated with liver nuclear extracts
from untreated control (C) and dexamethasone-treated (DX) rats. For competition, 50-fold molar excess of unlabeled
FP1 DNA, 50-fold (lane 5) and 150-fold (lane 6)
excess of unlabeled GRE oligonucleotide, and 150-fold excess of
nonspecific DNA (NS) was incubated prior to addition of probe.
The GRE ds-oligonucleotide represents the human tyrosine
aminotransferase gene response element, and the nonspecific DNA is a
ds-oligonucleotide that contains the DNA binding site for NF-
CYP3A1 is one of a family of homologous liver cytochrome
proteins inducible by glucocorticoids in many species(27) ,
including humans (28) , where the 3A cytochromes are the
dominant forms accounting for the metabolism of numerous clinically
important drugs including nifedipine and cyclosporin as well as such
endogenous substrates as cortisol(29) . Isolation of the rat
CYP3A1 gene (30) and the development of a system for primary
culture of adult rat hepatocytes in which CYP3A1 expression and
inducibility by dexamethasone can be maintained (31) has
afforded an opportunity to investigate in detail, the unique mode of
regulation of CYP3A1 by glucocorticoids. Inhibition of protein
synthesis in hepatocyte cultures and in rat liver actually increases
CYP3A1 mRNA, making this approach unhelpful in distinguishing induction
of CYP3A1 by dexamethasone as a primary or secondary glucocorticoid
response(32) . Favoring the former is a report that CYP3A1 is
transcriptionally activated within 1 h of dexamethasone treatment to
rats(8) . Attempting to penetrate this problem directly, we
established previously that a 165-bp fragment contained within 1.5
kilobases of DNA 5`-flanking the CYP3A1 gene transiently expressed in
hepatocyte cultures mirrored changes in accumulation of endogenous
CYP3A1 mRNA in being transcriptionally activated by treatment of the
cells with dexamethasone by a process that is slightly antagonized
(RU486) or was actually enhanced synergistically by antiglucocorticoids
(PCN)(5) . We now show that transcriptional responses to
dexamethasone and PCN and to the synergistic effect of PCN are mediated
by a single 33-bp element located at -135 in the CYP3A1 gene.
Failure to find within these 33-bp a consensus GRE sequence
(5`-AGAACAnnnTGTTCT-3`), multiple GRE half-sites (TGTTCT) (reviewed in (19) and (33) ), or sequences associated with a
delayed, secondary glucocorticoid response element (34) is
consistent with our proposal from pharmacologic studies that
glucocorticoid induction of CYP3A1 proceeds by a non-classical
pathway(3, 4) . While it remains to be proven
unequivocally that the GR is obligatory for dexamethasone induction of
CYP3A1 in primary cultures of adult rat hepatocytes (or in rat liver),
there is strong pharmacologic evidence in such cultures to support this
idea (only glucocorticoids to the exclusion of androgens, estrogens and
progestins induce CYP3A protein; (3) ). Sequence comparisons of
the dexamethasone-responsive CYP3A1 33-bp segment
(-148/-115) to mouse proliferin (25) and
phosphoenolpyruvate carboxykinase (35) genes, previously
demonstrated to bind the GR, suggested sufficient homology to a 12-bp
consensus element (19) and critical GR binding nucleotides (21) to test whether the GR can bind to the functional CYP3A1
33-bp segment. However, assays of the binding of dbGR protein with the
use of in vitro DNase I footprinting as well as
electrophoretic mobility shift assay ruled out an interaction of GR
with CYP3A1 at least under conditions in which binding occurs with the
classical MMTV GRE (Fig. 5) or the TAT GRE (data not shown),
respectively. Furthermore, neither of these techniques disclosed the
appearance of an associated protein (such as the GR) that binds to the
functional CYP3A1 33-bp segment when it undergoes dexamethasone
induction, as would be expected from similar studies of classical
GRE-controlled genes(9, 36) . It is a formal but
unlikely possibility that proteins bound to the 3A1 enhancer sequence
in the basal, steady state are ``switched'' with other
dexamethasone induction-specific proteins that have the same
characteristics in the two assay systems we employed. We conclude that
the glucocorticoids and antiglucocorticoids activate expression of the
CYP3A1 gene hormone-responsive element by enhancing transcription
through a mechanism that may involve a protein or proteins already
bound to the CYP3A1 enhancer in the basal, steady state. Fresh
insight into the control of CYP3A1 arose from DNase I footprinting
studies, which disclosed the same pattern of factor(s) capable of
binding to this gene in the basal, steady state and following
dexamethasone treatment (Fig. 2). One of these footprint areas,
FP1, was associated with a 33-bp segment that, by a mechanism that
remains to be explained, was able to confer both dexamethasone and PCN
induction and PCN synergy to a heterologous promoter when transiently
expressed in rat hepatocytes. The possibility that dexamethasone and
PCN exerted individual effects at separate sites on the CYP3A1 gene now
seems less likely. However, inasmuch as DNA-binding factors, such as
steroid receptors, bind as dimers to their cognate DNA binding sites,
the presence of a palindrome and a 6-bp direct repeat, similar to
synthetic and natural DNA response elements for some steroid receptors,
within the 33-bp responsive element supports the conclusion that
multiple DNA-binding proteins, possibly dimers, may contact sequences
within this element. This direct repeat sequence is also found in the
rat CYP3A2 gene and was recently reported to reside in a segment of the
gene required for basal expression of a transfected CAT reporter gene
and to bind proteins from rat liver nuclear extracts (37) . Wen
and Locker (38) recently described a DNA binding site for a
novel hepatocyte transcription factor within rat
Volume 270,
Number 48,
Issue of December 1, 1995 pp. 28917-28923
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-carbonitrile (PCN) induce CYP3A1 through transcriptional gene
activation. In the present study, we transiently expressed in primary
cultures of rat hepatocytes plasmids consisting of CYP3A1 5`-flanking
sequences fused to a chloramphenicol acetyltransferase reporter
plasmid. Deletional analysis identified a 78-base pair (bp) element
located approximately 135 bp upstream of the transcriptional start site
that was inducible by treatment of the cultures with dexamethasone or
PCN and was induced synergistically by dexamethasone plus PCN. Nuclear
extract from control rat liver protected two regions within the 78-bp
sequence against digestion with DNase I. The same two regions were
protected when nuclear extracts from dexamethasone-treated animals were
used. Analysis of both of the ``footprints'' (FP1 and FP2)
failed to reveal a classical sequence for the glucocorticoid-responsive
element. A 33-bp element that includes FP1 sequences inserted into the
chloramphenicol acetyltransferase reporter plasmid and transiently
expressed in rat hepatocytes conferred a profile of dexamethasone and
PCN induction similar to that of the 78-bp element. However, an Escherichia coli expressed glucocorticoid receptor protein
failed to protect sequences within FP1 in DNase I footprinting
experiments and failed to change its mobility in gel shift assays.
Moreover, as judged by the gel shift assay, the specific protein
binding to this fragment was the same whether nuclear extracts from the
liver of untreated or dexamethasone-treated rats were used. We conclude
that the activation of CYP3A1 gene transcription by glucocorticoids may
involve proteins already bound to the controlling element in the CYP3A1
gene through a mechanism in which GR in the presence of hormone does
not bind directly to CYP3A1 DNA.
-carbonitrile
(PCN)
(1, 2, 3, 4, 5) .
Studies using isolated nuclei from rats treated with dexamethasone have
demonstrated that transcriptional activation of the CYP3A1 gene is the
primary mechanism underlying the induction
process(6, 7, 8) . However, detailed
pharmacologic analysis of these phenomena in culture disclosed that the
time course of induction and the rank order and dose response of
synthetic steroid inducers was different for CYP3A1 expression as
compared to that for a typical glucocorticoid-responsive gene, tyrosine
aminotransferase (TAT)(3, 4, 5) . Moreover,
tests of agonist-antagonist relationships demonstrated that rates of de novo synthesis of immunoreactive CYP3A1 protein and
accumulation of CYP3A1 mRNA were stimulated synergistically when
hepatocyte cultures were incubated in the presence of dexamethasone
plus PCN, even though the expression of TAT was inhibited by this same
protocol(3, 4, 5) . Glucocorticoids
up-regulate TAT by entering the hepatocyte and forming a complex with
the glucocorticoid receptor (GR). This is followed by binding of the GR
to two glucocorticoid-responsive elements (GRE) located in the
5`-flanking region of the TAT gene, an event that activates TAT gene
transcription in a cooperative manner(9) . Based on our initial
studies, we suggested that the regulation of CYP3A1 by glucocorticoids
may proceed by a mechanism that differs from classical GR binding to a
GRE that might be located upstream of the CYP3A1 gene(4) .
fused to a chloramphenicol acetyltransferase (CAT)
reporter plasmid in primary cultures of adult rat hepatocytes that
maintain synergistic inducibility of hepatocellular CYP3A1 mRNA by
dexamethasone plus PCN(5) . We found that CAT expression from
this transfected CYP3A1 chimeric plasmid was induced by treatment of
the cultures with dexamethasone or with PCN and was induced
synergistically by treatment with dexamethasone plus PCN while
expression of a transfected control plasmid containing the classical
glucocorticoid inducible MMTV gene was induced by dexamethasone, was
not induced by PCN, and was only slightly induced by dexamethasone plus
PCN(5) . These results establish that the unique features of
CYP3A1 regulation by glucocorticoids largely involve effects on CYP3A1
gene transcription. Preliminary analysis of deletions of the 1.5
kilobases of CYP3A1 DNA defined a 165-base pair (bp) fragment
approximately 200 bp upstream of the transcriptional start site that
maintains dexamethasone and PCN inducibility and PCN synergy when
transiently expressed in cultured hepatocytes(5) . In the
present study, we have exploited the advantages of this culture system
to closely define the functional glucocorticoid controlling DNA that
flanks the CYP3A1 gene. Finding that the sequence lacks a classical
GRE, fails to bind GR, but binds to a pattern of liver nuclear proteins
from control or dexamethasone-treated rats that appears the same, we
conclude that activation of CYP3A1 gene transcription by
glucocorticoids may involve a novel indirect interaction of GR with
CYP3A1.
Animals and Materials
Adult male Sprague-Dawley
rats (Harlan, Indianapolis, IN) weighing 190-220 g were
maintained with free access to animal chow and water for 1-2
weeks prior to use. Collagenase type I was purchased from Worthington
Biochemical Corp. (Freehold, NJ), and all other tissue culture reagents
were of the highest grade commercially available from Sigma. Matrigel
was prepared from the Engelbreth-Holm-Swarm tumor(10) , kindly
supplied by Dr. Hynda K. Kleinman, NIDR, NIH, Bethesda, MD. Other
reagents were purchased from the following: Lipofectin (Life
Technologies, Inc.), CAT-ELISA kit (Boehringer Mannheim), Nycodenz
(Sigma), and enzymes (Life Technologies, Inc.). Dexamethasone was
purchased from Sigma and PCN was a gift from John Babcock (Upjohn).
pMMTVCAT (11) was provided by R. Miksicek and pBLCAT2 (12) was provided by G. Schutz (Institute for Cell and Tumor
Biology, Heidelberg, Germany).Preparation of Hepatocytes and DNA
Transfections
Primary cultures of adult rat hepatocytes were
prepared as described previously (13) . Hepatocytes freshly
isolated by collagenase perfusion of the livers of untreated rats were
incubated in a humidified atmosphere of 95% air, 5% CO in
our standard medium, a modification of Waymouth's MB-752 medium
containing insulin (0.1 µM) as the only hormone. For DNA
transfection experiments, 0.4 
10
cells were plated
in 35-mm plastic culture dishes (Lux) in 1 ml of medium supplemented
with 5% fetal calf serum. The cultures were maintained from 24 to 66 h
in serum-free Eagle's minimal essential medium (Life
Technologies, Inc.) containing 0.1 µM insulin. At 66 h
each dish of hepatocytes was washed with 1 ml of Opti-MEM (Life
Technologies, Inc.) and then exposed to a mixture formed by combining
12 µg of Lipofectin with 3.5 µg of DNA (as specified by Life
Technologies, Inc.). After 6 h of incubation with the cells, the
Lipofectin/DNA mixture was removed and the cultures were incubated with
1 ml of Waymouth's medium containing 300 µg of Matrigel and
inducers. Twenty-four h later the medium was replaced with
Waymouth's medium containing inducers only. Inducers were added
as 1000-fold stocks in dimethyl sulfoxide.Assay of CAT Protein
After 48 h of treatment with
inducers, cell lysates were harvested and assayed for CAT protein
(CAT-ELISA assay, Boehringer Mannheim). Briefly, each dish was washed
two times with phosphate buffered saline and then incubated 30 min at 4
°C in 1 ml of 40 mM Tris buffer, pH 7.4, containing 5
mM EDTA and 150 mM NaCl to solubilize the Matrigel.
This buffer was replaced with 350 µl of lysis buffer (Boehringer
Mannheim). Following a 30-min incubation at room temperature, the
lysates were centrifuged at 12,000 g for 10 min. CAT
protein was measured according to instructions provided with the kit.
Total cellular protein concentration was determined using a
bicinchoninic acid microassay (Sigma). Transfection efficiency in
several culture preparations was in the range of 5-10%, and CAT
protein measured from duplicate plates varied by less than 10% .Plasmid Constructions and Preparation of Double-stranded
Oligonucleotides
The construction of the chimeric plasmid
containing the CYP3A1 gene 5`-flanking sequences from -1525 to
-56 bp upstream of the transcription start site has been
described previously (5) . CYP3A1 sequences consisting of 5`
and 3` deletions were generated by the polymerase chain reaction and
sequenced (14) to confirm the accuracy of the amplification
reaction. All CYP3A1 5`-flanking sequences were cloned in the 5` to 3`
orientation in front of the thymidine kinase (TK) promoter of the
pBLCAT2 expression vector(12) . Constructions containing these
sequences inserted 5` to the CYP3A1 promoter appeared to support
dexamethasone- and PCN-mediated CAT expression when transfected
transiently into cultured hepatocytes, but the values were too low to
give reliable results. Complimentary oligonucleotides spanning
sequences protected against DNase I digestion (FP1, FP2), containing BamHI restriction sites at each end, were synthesized
(Macromolecular Resources, Fort Collins, CO), annealed and the
double-stranded (ds)-oligonucleotides purified by polyacrylamide gel
electrophoresis. The ds-oligonucleotides were cloned into the BamHI site of pBLCAT2 as single and multiple copies and in
both orientations. The FP1 oligonucleotide corresponds to the position
-148 to -115 and FP2 oligonucleotide to -116 to
-69. The sequence of the upper strands are as follows: FP1,
5`-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3`; FP2,
5`-GGATCCTAGGGGAAGAGAGTACCAAAGTCCACGTGAGGCAGAGGTGATCCATCTGGATCC-3`.Treatment of Animals and Preparation of Nuclear
Extract
Twenty-one-day-old male, Sprague-Dawley rats were given
dexamethasone 21-acetate (Sigma) in phosphate-buffered saline plus 10%
ethanol intraperitoneally at a concentration of 100 mg/kg for 3 h.
After treatment, animals were sacrificed, livers removed, and nuclei
prepared by centrifugation through Nycodenz(15) . Purified
nuclei were extracted for proteins with high salt buffer containing 20
mM HEPES (pH 7.9), 12.5% glycerol, 1.5 mM MgCl, 0.1 mM EDTA, 0.5 mM
dithiothreitol (DTT), 0.4 M KCl, and the protease inhibitors
Pefabloc, leupeptin, and pepstatin A (Boehringer Mannheim). Extracts
were dialyzed against 20 mM HEPES (pH 7.9), 20% glycerol, 100
mM KCl, 0.2 mM EDTA, 0.2 mM Pefabloc, and
0.5 mM DTT, aliquoted, and stored at -80 °C until
use. Protein concentration was determined using a bicinchoninic acid
microassay.DNase I Footprinting Assay
The 165-bp BamHI-HindIII fragment (position -220 to
-56) was labeled with P at the BamHI site
(upper strand). The labeled probe was incubated with 60 µg of crude
nuclear extract. Incubations were performed as specified in the
instruction manual for the HotFoot footprinting kit (Stratagene, La
Jolla, CA). After incubation, a partial DNase I digest was carried out
by adding 50 µl of DNase I buffer and concentrations of DNase I
(Stratagene) from 0.02 to 0.1 unit. DNA was extracted with
phenol-chloroform, recovered by ethanol precipitation, and analyzed on
a 12% denaturing polyacrylamide gel. Sequencing of the DNA probe was
performed by the Maxam-Gilbert method (16) using a sequencing
kit from United States Biochemical Corp. An Escherichia coli expressed protein consisting of the DNA-binding domain of the
glucocorticoid receptor (dbGR, apoT7X556) (17) was a gift from
Dr. Keith Yamamoto, University of California, San Francisco. The
footprinting assay using the dbGR was essentially as described for
nuclear extracts, except that 600 ng of dbGR was preincubated for 30
min on ice with ZnCl (final concentration, 250 mM) prior to
the binding reaction. The MMTV DNA fragment, from -222 to
+104 relative to the transcriptional start site, in the 5` long
terminal repeat of the mouse mammary tumor virus was labeled and used
as a positive control for GR binding. This fragment contains three
regions protected to varying degrees when used in nuclease protection
assays employing partially purified receptor (18) .
Electrophoretic Mobility Shift Assay
The
ds-oligonucleotide, FP1, corresponding to the position -148 to
-115, was radiolabeled with [-
P]ATP
and polynucleotide kinase. For this assay, 15 µg of nuclear protein
were incubated at room temperature for 20 min with 3 µg of
poly(dI-dC) in a binding buffer consisting of 20 mM HEPES, pH
7.9, 60 mM KCl, 5 mM MgCl
, 2 mM DTT, and 10% glycerol. The binding reaction was continued for 20
min after addition of labeled oligonucleotide to the reaction.
Competitor DNA was added during the preincubation period, prior to
addition of labeled oligonucleotide. Gel electrophoresis was conducted
using 1 TBE buffer (0.089 M Tris borate, 0.089 M boric acid, 0.002 M EDTA). The sequences of the
oligonucleotides used in the electrophoretic mobility shift assay
(upper strands) are as follows: FP1,
5`-GGATCCAATAGGCTGTAGATGAACTTCATGAACTGTCTAGGATCC-3`; GRE,
5`-CTAGGCTGTACAGGATGTTCTGCCTAG-3`; NF-
B,
5`-AGTTGAGGGGACTTTCCCAGG-3`.
P-labeled CYP3A1 fragment from -220 to -56
(upper strand) was incubated with 60 µg of crude nuclear extract
from untreated control and dexamethasone-treated animals. Lane
1, A+G sequence reaction; lane 2, CYP3A1 DNA, no
nuclear extract; lane 3, control extract; lanes
4-6, induced extract. Specific binding was determined by
competition reactions in which 100-fold molar excess of unlabeled
CYP3A1 DNA (-220/-56) (lane 5, +) or
nonspecific DNA (lane 6, +) was added prior to addition
of labeled CYP3A1 DNA. Sequencing of CYP3A1 DNA (-220/-56)
was performed by the Maxam-Gilbert method(16) . Footprints 1
and 2 (FP1, FP2) are marked by solid lines. The dashed
line indicates a footprint possibly due to weak interactions. Base
pairs protected are delineated by negative numbers representing distance from the start of
transcription.
B (Fig. 6, lanes 4 and 7). The specific protein-DNA complex
appeared to migrate with the same mobility in the gel regardless of
whether nuclear extracts from untreated or dexamethasone-treated rat
liver were used in the binding reaction (Fig. 6, upper
arrow), even though the latter extract alone contained sufficient
GR protein to be detected by Western blot analysis (data not shown).
Also, addition of excess GRE oligonucleotide (50- and 150-fold) to the
binding reaction did not result in competition for the complex,
indicating that the GR was not part of the protein-DNA complex formed
at the 33-bp element (Fig. 6, lanes 5 and 6).
Failure of the GR-containing nuclear extracts from
dexamethasone-treated rats to enhance retardation of the DNA
probe's mobility does not exclude protein-protein interactions
involving the GR because such complexes are well known to be subject to
disruption under electrophoretic conditions in
vitro(25, 26) .
B.
The upper arrow indicates the position of the specific
DNA-protein complex and the lower arrow the position of the
free DNA probe.
-fetoprotein, a
gene repressed in the developing liver by glucocorticoids(39) .
This element, termed promoter-linked coupling element, that may compete
for GR binding as the mechanism for glucocorticoid
repression(38) , is similar to CYP3A1 FP1 in its sequence (8 of
12 bp in common), and in overlapping a GRE-like sequence. It remains to
be determined whether it is this protein or members of the nuclear
receptor family that recognize the CYP3A1 FP1 sequence and whether the
GR, if it is involved at all, transcriptionally activates CYP3A1 gene
expression through protein-protein interactions. New models associate
the GR with DNA-bound constitutive factor without actual binding to DNA
resulting in either down-regulation (40) or up-regulation of
gene expression (41) .
)-carbonitrile; CAT,
chloramphenicol acetyltransferase; TK, thymidine kinase; GR,
glucocorticoid receptor; dbGR, DNA binding domain of the glucocorticoid
receptor protein; GRE, glucocorticoid-responsive element; TAT, tyrosine
aminotransferase; bp, base pair(s); ELISA, enzyme-linked immunosorbent
assay; ds, double-stranded; DTT, dithiothreitol; MMTV, mouse mammary
tumor virus.)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
