A Distinct Thyroid Hormone Response Element Mediates Repression of the Human Cholesterol 7
-Hydroxylase (CYP7A1) Gene Promoter
Victor A. B. Drover,
Norman C. W. Wong and
Luis B. Agellon
Canadian Institutes of Health Research Group in Molecular and Cell
Biology of Lipids and Department of Biochemistry (V.A.B.D., L.B.A.),
University of Alberta, Edmonton, Alberta, T6G 2S2 Canada; and the
Departments of Medicine and Biochemistry and Molecular Biology
(N.C.W.W.), University of Calgary, Calgary, Alberta, T2N 4N1
Canada
Address all correspondence and requests for reprints to: Dr. Luis B. Agellon, Department of Biochemistry, 327 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2 Canada. E-mail: luis.agellon{at}ualberta.ca
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ABSTRACT
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We examined the molecular basis by which T3 regulates
the human cholesterol 7
-hydroxylase gene (CYP7A1)
promoter. L-T3 decreased chloramphenicol
acetyltransferase activity in hepatoma cells cotransfected with a
plasmid encoding the T3 receptor (TR)
[NR1a1] and a
chimeric gene containing nucleotides -372 to +61 of the human
CYP7A1 gene fused to the chloramphenicol
acetyltransferase structural gene. Deoxyribonuclease I footprinting
revealed that recombinant TR
protected two regions in this segment
of the human CYP7A1 gene promoter. In EMSAs, TR
bound
to both regions. The binding was competed by oligonucleotides bearing
an idealized TR
binding motif and abolished by mutation of these
elements. In assays of promoter function, mutation of only one of the
TR
binding sites blocked repression by T3. The results
indicate that T3-dependent repression of human
CYP7A1 gene expression is mediated via a novel site in
the human CYP7A1 gene promoter.
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INTRODUCTION
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THE PRODUCTION OF bile acids is an
important pathway for the removal of cholesterol from the body.
Although two biochemical pathways are responsible for bile acid
synthesis, bile acids are produced preferentially by the neutral
pathway via the hepatic enzyme cholesterol 7
-hydroxylase (cyp7a, EC
1.14.13) (1). Posttranscriptional and posttranslational
regulation of this rate-limiting enzyme has been documented
(2, 3, 4). However, the control of cyp7a gene transcription
is the major mechanism for modulating cyp7a activity
(5, 6, 7, 8, 9).
Transcriptional regulation of the cyp7a gene promoter is mediated by
the complex interplay of multiple transcription factors, the majority
of which belong to the nuclear receptor superfamily (10, 11). The binding sites recognized by this family of
transcription factors define a related group of DNA sequences (related
to the sequence AGGTCA) collectively known as hormone response
elements. Previous studies have used sequence similarity and deletion
mapping of promoter-reporter gene chimeras to define two regions in the
cyp7a gene promoter (Sites I and II, centered at -62 nucleotides (nt)
and -138 nt, respectively, in the human promoter) that contain
multiple and overlapping hormone response element half-sites
(12, 13, 14, 15). The sequence of Site II is identical among the
rat (16), mouse (17), hamster
(18), human (19), and pig (GenBank accession
no. AF020317) cyp7a genes, suggesting that this cis-acting
element is important in the regulation of cyp7a gene transcription.
T3 plays an important role in the regulation of
cyp7a gene expression. Previous studies in rats and rat hepatocytes
have shown that T3 stimulates rat
Cyp7a1 gene expression (2, 9, 20, 21).
Surprisingly, analysis of the rat Cyp7a1 gene promoter in
HepG2 cells failed to show a response to T3 even
in the presence of TR
(6). In contrast,
T3 represses the human CYP7A1 proximal
gene promoter in HepG2 cells (5) and has a tendency to
decrease bile acid synthesis and CYP7A1 mRNA abundance in
human hepatocytes (21). The effects of
T3 on human CYP7A1 gene expression
in vivo are poorly understood. Here, we examined the
molecular basis of T3-mediated repression of the
human CYP7A1 gene promoter. We found that TR
can bind to
two sites in the human CYP7A1 gene promoter in
vitro. Functional characterization of these binding sites in
hepatoma cells revealed that only one site is capable of mediating the
repression of the human CYP7A1 gene promoter by
T3/TR
.
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RESULTS
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T3 Represses the Human CYP7A1 Promoter
In rats, T3 increases cyp7a mRNA abundance
(20) and thyroidectomy decreases cyp7a gene transcription
(2). In contrast, the activity of the human
CYP7A1 promoter is inhibited by T3
(5). To determine the effect of T3
on the activity of the human CYP7A1 proximal promoter,
phcyp7a-CAT was transfected into McArdle RH7777 rat hepatoma (RH7777)
cells in the presence and absence of T3.
Chloramphenicol acetyltransferase (CAT) activity was used as an
indicator of promoter activity. Physiological and supraphysiological
concentrations of T3 as high as 10
µM had no effect on CAT activity in RH7777
cells (data not shown). As T3 function is
mediated by TRs, we surmised that RH7777 cells do not have sufficient
amounts of this receptor to allow the regulation of
T3 responsive genes. Accordingly, immunoblot
analysis of whole-cell protein extracts from RH7777 cells did not
reveal the presence of TRs while RXRs were readily detectable (Fig. 1A
). Thus, the lack of change in promoter
activity in the presence of T3 is attributable to
the absence of TRs in RH7777 cells.
When RH7777 cells were cotransfected with a plasmid encoding TR
,
promoter activity increased approximately 20% (P <
0.05). The subsequent addition of T3 decreased
promoter activity in a concentration-dependent manner.
T3 concentrations as low as 100
nM were sufficient to reduce promoter activity by
50% (P < 0.05; Fig. 1B
). TRs are transcriptionally
active as monomers, homodimers, or as heterodimers with RXRs
(22, 23, 24). Cells cotransfected with both TR
and RXR
exhibited similar magnitudes of T3-dependent
repression of promoter activity (Fig. 1C
) as observed in cells
transfected with TR
only. Thus, repression of the human
CYP7A1 gene promoter by T3 is
dependent upon the presence of TR
.
The Human CYP7A1 Gene Contains Two Elements That Bind
TR
To determine the promoter elements required for the
T3 response, we mapped the TR
binding sites in
the CYP7A1 promoter by deoxyribonuclease I (DNAse I)
protection experiments using recombinant TR
. Two protected regions
were identified. The first region spans nt -119 to -149 (Fig. 2A
) and corresponds to Site II
(14). The second protected region spans nt -227 to -247
(Fig. 2B
) and was designated Site III. As TR
can heterodimerize with
RXR
, EMSAs were performed in the presence of both recombinant
receptors to determine whether TR
can bind Site II and Site III as a
TR
:RXR
heterodimer. TR homodimers can be distinguished from
TR:RXR heterodimers by a difference in electrophoretic mobility
(25). When both TR and RXR were incubated with an
idealized thyroid hormone response element (TRE; a direct repeat
separated by 4 nt), the shifted complex migrated more slowly than when
the receptors were incubated with either Site II or Site III (Fig. 2C
, lanes 2, 4, and 6, respectively). These results indicate that Site II
and Site III bind only TR
even in the presence of RXR
.
Analysis of TR
Binding to Site II and Site III
EMSAs were performed to characterize the receptor binding to these
sites. Radiolabeled oligonucleotides containing the Site II sequence
bound recombinant TR
(Fig. 3A
, lane
2). Addition of T3 to the binding reaction had no
effect on this interaction (data not shown). The TRE competed for TR
binding (Fig. 3A
, lanes 36), while a 100-fold excess of a different
sequence (the glucocorticoid response element, GRE) had no effect on
TR
binding (Fig. 3A
, lane 10). An antibody recognizing TRs was used
to further illustrate the specific interaction of Site II with TR
(Fig. 3B
, lane 13). Similarly, Site III bound TR
and was competed by
the TRE but not by the GRE (Fig. 3B
, lanes 2, 36, and 710,
respectively). These results demonstrate that TR
can interact
specifically with two regions in the human CYP7A1 proximal
promoter.
The Site II sequence was previously described (14) and
features two half-sites separated by 1 nt (Table 1
). To determine the nature of the
interaction between TR
and Site II, mutant oligonucleotides
containing substitutions of either of these half-sites were
synthesized. Mutagenesis of the 5'-half-site of Site II abrogated TR
binding (Site II m1; Fig. 4A
, lane 4).
Only residual binding of TR
was detected when the 3'-half-site was
altered (Site II m2; Fig. 4A
, lane 6). The requirement of both
half-sites for efficient receptor binding suggests Site II is bound by
two molecules of TR
in vitro. In addition, the mobility
of a previously characterized TR
dimer [site A of the ApoA-I
promoter (26); Fig. 4A
, lane 8] matches the mobility of
TR
bound to Site II, consistent with the presence of two molecules
of TR
at this site.
Analysis of the Site III sequence revealed three putative
response elements: a 5'-half-site, a 3'-half-site, and another
half-site that overlaps with the 5'-half-site (Table 1
). The
5'-half-site and 3'-half-site are separated by 4 nt whereas the
overlapping half-site and the 3'-half-site are separated by 1 nt.
Mutation of the 5'-half-site abrogated binding of TR
to Site III
entirely (Site III m1; Fig. 4B
, lane 4). Mutation of the 3'-half-site
resulted in reduced receptor binding (Site III m2; Fig. 4B
, lane 6) and
the appearance of a second complex with faster mobility (Fig. 4B
, arrow). The mobility of this second complex was the same as
that of the F2 silencer (Fig. 4C
) from the chicken lysozyme gene, which
is known to bind TR as a monomer (27).
As the initial mutation of the 5'-half-site also disrupted the
overlapping half-site, we synthesized oligonucleotides containing more
subtle mutations to delineate which half-site was required for TR
binding. Substitutions that preserved the 5'-half-site (Site III m3)
abrogated receptor binding (Fig. 4D
, lane 4). Oligonucleotides
containing substitutions that preserved the overlapping half-site (Site
III m4) displayed an electrophoretic mobility comparable to wild-type
Site III (Fig. 4D
, lane 6). These data show that the overlapping and
3'-half-sites of Site III (apparently arranged as an everted repeat
spaced by 1 nt) direct the binding of two TR
molecules in
vitro.
One Half-Site in Site III Independently Mediates
T3-Dependent Repression of the Human CYP7A1
Gene Promoter
Since TR
is able to bind two sites in the human
CYP7A1 gene promoter, gene chimeras containing mutations at
these sites were characterized to define their functional significance.
A gene chimera containing a substitution at the 5'-half-site of Site II
(pM1.CAT) was created. Like the wild-type chimera (Fig. 5A
, phcyp7a-CAT), promoter activity was
repressed by T3 in the presence of TR
(Fig. 5B
). This finding indicates that Site III, the only remaining TR
binding site in pM1.CAT, is sufficient to mediate
T3-dependent repression of promoter activity. A
second mutant gene chimera (pM16.CAT) was created in which the
3'-half-site of Site III was altered. Surprisingly, the addition of
T3 still resulted in repression of promoter
activity (Fig. 5C
).

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Figure 5. Functional Analysis of Gene Chimeras Containing
Mutant Site II and Site III Sequences
RH7777 cells were cotransfected with parental (phcyp7a-CAT; panel A) or
mutant (pM1. CAT, pM16. CAT, pM3. CAT; panels B, C, and D,
respectively) gene chimeras and an expression vector encoding TR .
Transfected cells were then grown for 18 h in the presence of
ethanol (carrier, open bars) or 100 nM
T3 (solid bars) and assayed for CAT
activity. The activities of the promoters in the absence of both
T3 and TR were taken as 100%. The data shown are the
mean ± SEM relative CAT activity of triplicate assays
from two experiments. The specific activities
(pmol·min-1·mg-1 total cell protein)
are: phcyp7-CAT, 1.68; pM1. CAT, 1.41; pM16. CAT, 1.06; pM3. CAT, 0.6).
*, Differences were evaluated using a two-sample t test
and were considered significant when P < 0.05. The
locations of Site II and Site III are shown schematically
above each panel. Open boxes indicate the
location of TR-binding half-sites at Site II and Site III. The boxes
designated with an X represent mutagenized half-sites.
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We hypothesized that the overlapping half-site of Site III, which was
still intact in both pM1.CAT and pM16.CAT gene chimeras, might be
sufficient to mediate the T3 response. To test
this hypothesis, we created another gene chimera (pM3.CAT) in which
only the overlapping half-site of Site III was altered. This
substitution rendered the promoter unresponsive to
T3 (Fig. 5D
). Taken together, the results show
that a single half-site in Site III is necessary and sufficient for the
T3/TR
-mediated repression of the human
CYP7A1 gene promoter.
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DISCUSSION
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Thyroid hormones play important roles in development and energy
homeostasis by regulating the transcription of a variety of target
genes via TRs. The two major isoforms of the TRs (TR
and TRß),
which are encoded by two distinct genes, are expressed in a wide
variety of tissues. Analysis of the expression of the TR
and TRß
genes revealed a disparity in the abundance of their mRNAs. However,
quantitative indirect immunofluorescence studies have revealed that
TR
and TRß proteins are present in similar quantities in the
nuclei of rat hepatocytes (28). Additionally, an
examination of TR abundance in human liver indicated that TR
and
TRß are present in comparable amounts (29), suggesting
that both isoforms are readily available for
T3-dependent gene regulation.
Recent studies using TR
- and TRß-deficient mice have implicated
TRß in the T3-dependent stimulation of
Cyp7a1 gene expression (30). A direct
interaction of TR with the murine Cyp7a1 gene promoter,
however, has not been demonstrated, and the failure of this gene to be
induced by T3 in TRß-/-
mice could be an indirect effect of the global changes in lipid
metabolism observed in this mouse strain. In contrast, we demonstrate
that TR
(and TRß, Drover, V. A. B., and L. B.
Agellon, unpublished results) binds directly to the human
CYP7A1 gene promoter and that this binding is required for
T3-dependent repression of promoter activity in
RH7777 cells.
Sites II and III were identified by DNAse I footprinting, and EMSAs
confirmed that two molecules of TR
can bind each site. The invariant
nature of the Site II sequence among the five species examined to date
suggested that this site would be important in the transcriptional
regulation of the human CYP7A1 gene promoter by
T3/TR. Surprisingly, functional analysis revealed
that T3/TR
-dependent down-regulation of the
human CYP7A1 gene promoter does not involve this Site. Site
II has been previously characterized as a focal point for the binding
of several transcription factors to the human CYP7A1, and
mouse and rat Cyp7a1 gene promoters (12, 14, 15, 31, 32, 33), and therefore it may not be available for binding of
and regulation by T3/TR.
Mutation of the 5'-half-site of Site II did not have a dramatic
effect on the basal activity of the human CYP7A1 promoter as
assessed in RH7777 cells. However, mutagenesis of both the 5'-half-site
of Site II and the 3'-half-site of Site III resulted in the
near-complete loss of promoter function (Drover, V. A. B.,
and L. B. Agellon, unpublished results), suggesting that factors
required for the maintenance of promoter activity require these
half-sites. The interaction between Site II and Site III and the
importance of the 3'-half-site of Site III in maintaining basal
activity of the human CYP7A1 gene promoter requires further
investigation.
Site III is a novel regulatory region in the human CYP7A1
gene promoter. It consists of multiple, overlapping half-sites that are
putative binding sites for members of the nuclear receptor superfamily.
Unlike Site II, the sequence of Site III is not conserved among
different species, and this may explain the divergent responses of the
human CYP7A1 and rodent Cyp7a1 genes to
T3 (2, 9, 20, 21, 30). The
stimulation of the rodent genes by T3 may be
directly related to the sequence differences at Site III or,
alternatively, rodent Cyp7a1 genes may be induced by
T3 via promoter elements specific to these
species.
A number of studies have examined the role of T3
in bile acid metabolism in humans. In hyperthyroid patients, Kosuge
et al. (34) found that the most prominent bile
acid in bile shifted from deoxycholic acid to chenodeoxycholic acid,
and Pauletzki et al. (35) observed a 47%
decrease in cholic acid pool size. This may be attributable to the
inhibition of sterol 12
-hydroxylase as observed in thyroid
hormone-treated rats (36, 37). However, Pauletzki et
al. (35) also documented a 20% decrease in total
primary bile acid synthesis. Consistent with this and other in
vitro studies (5, 21), we found that the human
CYP7A1 gene promoter was inhibited by
T3. In vitro experiments suggest that
repression of gene expression by TR homodimers involves the recruitment
of the silencing mediator of retinoid and thyroid hormone
receptors corepressor (38). Our data indicate that
the molecular basis for the repression of the human CYP7A1
gene promoter in response to T3 involves the
interaction of monomeric TR with a novel element localized in its
proximal region. Further studies are necessary to elucidate the
mechanism for this effect.
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MATERIALS AND METHODS
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Chemical Reagents
T3 and heparin agarose were
purchased from Sigma-Aldrich Corp. Canada Ltd.
(Oakville, Ontario, Canada), and poly(dI-dC) was purchased from
Roche Molecular Biochemicals (Laval, Quebec, Canada).
Tissue culture reagents and restriction and DNA-modifying enzymes were
purchased from Life Technologies, Inc. (Gaithersburg, MD).
RH7777 cells were obtained from ATCC (Manassas, VA). All
other reagents were of analytical grade.
Construction of Recombinant Plasmids
The parental gene chimera (phcyp7a-CAT) (14)
contained the proximal promoter region of the human CYP7A1
gene (nt -372 to +61) fused to the CAT structural gene sequence in
pCAT-basic (Promega Corp., Madison, WI). Mutant gene
chimeras were produced from phcyp7a-CAT in which the 5'-half-site of
Site II (nt -144 to -139) or the 3'-half-site of Site III (nt -238
to -233) was mutated to the sequence 5'-CTCGAG-3' (recognition
sequence of XhoI). Mutagenic sense and antisense primers
were used to amplify the entire phcyp7a-CAT plasmid. The linear 4.7-kb
product was digested with XhoI and ligated after protein
removal. Clones obtained after transformation were screened by
restriction enzyme analysis. The gene chimera pM1.CAT carries the
mutation at Site II and was produced using the primers hTRE-1 m1a
(5'-tacctgCTCGAGtagttcaaggccag) and hTRE-1 m1b2
(5'-taCTCGAGcaggtatcagaagtgg). The gene chimera pM16.CAT carries the
mutation at Site III and was produced using the primers
hTRE-2 m2a2 (5'-ccCTCGAGgaatgttaagtcaac) and hTRE-2 m2b
(5'-attcCTCGAGggggacaacagc). Another mutant, pM3.CAT, containing a
5'-AAA substitution of nt -134 to -136, was produced using mutagenic
sense (hTRE-2 m3A, 5'-tagctgttgtAAAcaggtccga) and antisense (hTRE-2
m3B, 5'-attcggacctgTTTacaacag) primers and phcyp7a-CAT as the template
DNA. The 4.7-kb product was phosphorylated with T4 polynucleotide
kinase and ligated after protein removal. The primary structure of all
mutant gene chimeras was confirmed by DNA sequencing.
Cell Culture and Transfection Assays
RH7777 cells were cultured in complete media (DMEM containing
10% carbon-stripped calf serum, 10% carbon-stripped FBS) at 37 C/5%
CO2 in 100-mm culture dishes. Cells from
confluent dishes were plated to 60-mm culture dishes and grown to 50%
confluence (18 h). The culture medium was aspirated and 20 µg of DNA
was transfected using the calcium phosphate coprecipitation method. The
DNA mixture consisted of 5 µg of promoter-reporter gene, 5 µg of a
plasmid encoding ß-galactosidase under the control of the
cytomegalovirus promoter, and 5 µg of each plasmid encoding rat
TR
1 and human RXR
(where stated). The total mass of DNA was
adjusted to 20 µg using sheared salmon sperm DNA. Transfected cells
were incubated overnight in DMEM containing 2.5% carbon-stripped calf
serum and 2.5% carbon-stripped FBS as described above. The cells were
washed with PBS and supplied with complete media containing
T3 (10-9 to
10-5 M) and ethanol (0.33%,
vol/vol; carrier) or ethanol (0.33%, vol/vol) alone. After 18 h
of treatment, cells were washed with PBS. Cell lysates were prepared by
three freeze-thaw cycles and assayed for CAT (39) and
ß-galactosidase (40) activities. The CAT activity
measured in transfected cell lysates, which was typically 8- to
30-fold above background, was normalized to ß-galactosidase
activity.
Detection of Nuclear Hormone Receptors in RH7777 Cell
Extracts
Cell lysates were prepared from RH7777 cells after a mock
transfection with sheared salmon sperm DNA as described above.
Recombinant TR
and RXR
were prepared using the TNT Coupled
Reticulocyte Lysate System (Promega Corp.) programmed with
0.5 µg of plasmid encoding either rat TR
1 (41) or
human RXR
(42). Proteins were separated by SDS-PAGE
(43) and transferred to a polyvinylidene difluoride
membrane (Millipore Corp., Bedford, MA) using the
manufacturers protocol. TRs and RXRs were detected using rabbit
polyclonal antibodies raised against the TRs [sc-772 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) detects TR
1 and TRß1
isoforms] or RXRs [sc-774 (Santa Cruz Biotechnology, Inc.) detects RXR
and RXRß isoforms]. The primary
antibody-antigen complexes were visualized with the enhanced
chemiluminescence-based detection system (Amersham Pharmacia Biotech, Baie dUrfé, Quebec, Canada) using horseradish
peroxidase-conjugated antirabbit IgG as the secondary antibody.
Recombinant TR
and RXR
Plasmids encoding rat TR
1 or human RXR
were grown in
Escherichia coli strain BL21 (DE3), and the recombinant
receptor was partially purified as described earlier (44)
with the following modifications: the receptors were eluted from
heparin-agarose with GTETD375 (15% glycerol, 25
mM Tris HCl, pH 7.8, 0.5 mM
EDTA, 0.05% glycerol, 1 mM dithiothreitol, 375
mM KCl) and concentrated using Ultrafree-15
centrifugal filter device (Millipore Corp., Bedford,
MA).
DNAse I Footprinting
phcyp7a-CAT (10 µg) was digested with either
HindIII or SalI in a total reaction volume of 200
µl. The linear DNA was mixed with 6 U of the Klenow fragment of DNA
polymerase I, 50 µCi of [
-32P]dATP, 50
µCi of [
-32P]dTTP (3,000 Ci/mmol
each), and dCTP/dGTP, each to a final concentration of 181
µM, in a total volume of 220 µl. After
labeling, the DNA polymerase was heat inactivated and removed by
centrifugation of the reaction mixture through a Ultrafree Probind spin
column (Millipore Corp.). The labeled DNA was digested
with 100 U of either SalI (after HindIII) or
HindIII (after SalI) to produce fragments with
only one labeled strand. The labeled promoter fragments were separated
from the plasmid vector by agarose gel electrophoresis and purified by
elution from crushed agarose gel plugs (43). The
radiospecific activity of the labeled probe was determined by liquid
scintillation spectrometry.
TR
binding sites in the human CYP7A1 gene promoter were
mapped by DNAse I footprinting (45) in 1x binding buffer
(100 mM NaCl, 10 mM Tris,
pH 7.5, 10 mM EDTA). The locations of the
protected regions were deduced using a ladder generated by
purine-specific chemical cleavage (43) of the radiolabeled
probe.
EMSAs
Complementary synthetic oligonucleotides were annealed in
1x annealing buffer (100 mM NaCl, 10 mM
Tris-HCl, pH 7.5, 10 mM EDTA) at a final concentration of
10 pmol/µl. Double-stranded oligonucleotides (20 pmol) were
end-filled in 1x Klenow buffer (10 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 7.5
mM dithiothreitol) with 2 U of the Klenow fragment of DNA
polymerase I, 667 µM each of dCTP and dGTP, and 50 µCi
each of [
-32P]dATP (3,000 Ci/mmol) and 50
µCi of [
-32P]dTTP (3,000 Ci/mmol), in a
final reaction volume of 30 µl. After labeling, the free nt were
removed from the reaction mixture by spun-column chromatography
(43) through a 2.0-ml bed of Sephadex G-25. The
radiospecific activity of the labeled probe was determined by liquid
scintillation spectrometry.
The labeled oligonucleotide was incubated with bacterially
expressed, recombinant nuclear receptors and 1 µg of
poly(dI-dC)·poly(dI-dC) in 1x binding buffer at room temperature for
20 min. Where indicated, 1 µl of an antibody specific for TRs was
added to the reaction and incubated for an additional 30 min.
Protein-DNA complexes were separated from free probe by nondenaturing
electrophoresis on a 5% polyacrylamide gel and visualized by
autoradiography.
 |
ACKNOWLEDGMENTS
|
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We thank Anthony Taylor for technical advice. We also thank
Daisy Sahoo, and Eric Labonté for critical reading of the
manuscript.
 |
FOOTNOTES
|
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This work was supported by Grant MOP-14812 from the Canadian Institutes
of Health Research. V.A.B.D. was supported by a Studentship from the
Alberta Heritage Foundation for Medical Research. N.C.W.W. is a Senior
Investigator of the Canadian Institutes of Health Research and Senior
Scientist of the Alberta Heritage Foundation for Medical Research.
L.B.A. is a Senior Medical Scholar of the Alberta Heritage Foundation
for Medical Research.
Abbreviations: CAT, Chloramphenicol acetyltransferase; cyp7a,
cholesterol 7
-hydroxylase; DNase, deoxyribonuclease; GRE,
glucocorticoid response element; nt, nucleotides; RH7777, McArdle
RH7777 rat hepatoma cells; TRE, idealized thyroid hormone response
element.
Received for publication September 20, 2000.
Accepted for publication September 17, 2001.
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