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Molecular Endocrinology 16 (1): 14-23
Copyright © 2002 by The Endocrine Society

A Distinct Thyroid Hormone Response Element Mediates Repression of the Human Cholesterol 7{alpha}-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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We examined the molecular basis by which T3 regulates the human cholesterol 7{alpha}-hydroxylase gene (CYP7A1) promoter. L-T3 decreased chloramphenicol acetyltransferase activity in hepatoma cells cotransfected with a plasmid encoding the T3 receptor (TR) {alpha} [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{alpha} protected two regions in this segment of the human CYP7A1 gene promoter. In EMSAs, TR{alpha} bound to both regions. The binding was competed by oligonucleotides bearing an idealized TR{alpha} binding motif and abolished by mutation of these elements. In assays of promoter function, mutation of only one of the TR{alpha} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-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{alpha} (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{alpha} 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{alpha}.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). Thus, the lack of change in promoter activity in the presence of T3 is attributable to the absence of TRs in RH7777 cells.



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  		Figure 1. Activity of the Human CYP7A1 Gene Promoter in RH7777 Cells

A, Whole-cell protein extracts from mock transfected RH7777 cells were prepared and assessed for the presence of TRs and RXRs by immunoblot analysis. Recombinant receptors produced in a cell-free transcription/translation system were used as positive controls (TNT control). The mass of cell extract used in the experiment is shown above the lanes. B, phcyp7a-CAT was cotransfected into RH7777 cells with an expression vector encoding TR{alpha} and treated with the indicated concentrations of T3 for 18 h. After treatment, the cells were harvested and assayed for CAT activity. C, phcyp7a-CAT was cotransfected into RH7777 cells with expression vectors encoding TR{alpha} and RXR{alpha}, grown for 18 h in the presence of ethanol (carrier, open bars) or 100 nM T3 (solid bars), and then assayed for CAT activity. The activity of the promoter in the absence of both T3 and TR was taken as 100%. The data shown are the mean ± SEM relative CAT activity of triplicate assays from two experiments. *, Differences were evaluated using a two-sample t test and were considered significant when P < 0.05.

 
When RH7777 cells were cotransfected with a plasmid encoding TR{alpha}, 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. 1BGo). TRs are transcriptionally active as monomers, homodimers, or as heterodimers with RXRs (22, 23, 24). Cells cotransfected with both TR{alpha} and RXR{alpha} exhibited similar magnitudes of T3-dependent repression of promoter activity (Fig. 1CGo) as observed in cells transfected with TR{alpha} only. Thus, repression of the human CYP7A1 gene promoter by T3 is dependent upon the presence of TR{alpha}.

The Human CYP7A1 Gene Contains Two Elements That Bind TR{alpha}
To determine the promoter elements required for the T3 response, we mapped the TR{alpha} binding sites in the CYP7A1 promoter by deoxyribonuclease I (DNAse I) protection experiments using recombinant TR{alpha}. Two protected regions were identified. The first region spans nt -119 to -149 (Fig. 2AGo) and corresponds to Site II (14). The second protected region spans nt -227 to -247 (Fig. 2BGo) and was designated Site III. As TR{alpha} can heterodimerize with RXR{alpha}, EMSAs were performed in the presence of both recombinant receptors to determine whether TR{alpha} can bind Site II and Site III as a TR{alpha}:RXR{alpha} 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. 2CGo, lanes 2, 4, and 6, respectively). These results indicate that Site II and Site III bind only TR{alpha} even in the presence of RXR{alpha}.



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  		Figure 2. Mapping TR{alpha} Binding Sites in the Human CYP7A1 Gene Promoter

A and B, Human CYP7A1 gene promoter fragments labeled in one strand at either the 3'- or 5'-end (panels A and B, respectively) were incubated with recombinant TR{alpha} and partially degraded with DNAse I. The products were analyzed by denaturing electrophoresis on a 6% polyacrylamide gel. Protected regions (Site II and Site III) are indicated by the vertical lines at the right. The corresponding positions in the human CYP7A1 gene promoter are indicated at left. C, Labeled double-stranded oligonucleotides corresponding to TRE, Site II, or Site III (see Table 1Go) were incubated with recombinant TR{alpha} and RXR{alpha}. Protein-DNA complexes were separated from free probe by nondenaturing electrophoresis on a 5% polyacrylamide gel. The migration of the free probe and shifted complexes are indicated at the left.

 
Analysis of TR{alpha} 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{alpha} (Fig. 3AGo, lane 2). Addition of T3 to the binding reaction had no effect on this interaction (data not shown). The TRE competed for TR{alpha} binding (Fig. 3AGo, lanes 3–6), while a 100-fold excess of a different sequence (the glucocorticoid response element, GRE) had no effect on TR{alpha} binding (Fig. 3AGo, lane 10). An antibody recognizing TRs was used to further illustrate the specific interaction of Site II with TR{alpha} (Fig. 3BGo, lane 13). Similarly, Site III bound TR{alpha} and was competed by the TRE but not by the GRE (Fig. 3BGo, lanes 2, 3–6, and 7–10, respectively). These results demonstrate that TR{alpha} can interact specifically with two regions in the human CYP7A1 proximal promoter.



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  		Figure 3. Binding of Recombinant TR{alpha} to Site II and Site III

A and B, Recombinant TR{alpha} was incubated with the labeled, double-stranded oligonucleotides (see Table 1Go) corresponding to Site II or Site III (panels A and B, respectively). Unlabeled specific and nonspecific competitors (TRE and GRE, respectively) were added (where indicated) in 0.1-, 1-, 10-, and 100-fold molar excess. An antibody specific for TRs was added where indicated. Protein-DNA complexes were separated from free probe by nondenaturing electrophoresis on a 5% polyacrylamide gel. The migration of TR{alpha} homodimers and the free probe are indicated at the left, while supershifted complexes of TR{alpha} homodimers and anti-TR antibodies are indicated at the right.

 
The Site II sequence was previously described (14) and features two half-sites separated by 1 nt (Table 1Go). To determine the nature of the interaction between TR{alpha} 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{alpha} binding (Site II m1; Fig. 4AGo, lane 4). Only residual binding of TR{alpha} was detected when the 3'-half-site was altered (Site II m2; Fig. 4AGo, lane 6). The requirement of both half-sites for efficient receptor binding suggests Site II is bound by two molecules of TR{alpha} in vitro. In addition, the mobility of a previously characterized TR{alpha} dimer [site A of the ApoA-I promoter (26); Fig. 4AGo, lane 8] matches the mobility of TR{alpha} bound to Site II, consistent with the presence of two molecules of TR{alpha} at this site.


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  		Table 1. Oligonucleotides used for EMSAs

 


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  		Figure 4. Half-Site Dependence of TR{alpha} Binding to Site II and Site III

Wild-type and mutant labeled, double-stranded oligonucleotides (see Table 1Go) corresponding to Site II (panel A) or Site III (panels B, C, and D) were incubated with or without recombinant TR{alpha}. Protein-DNA complexes were separated from free probe by nondenaturing electrophoresis on a 5% polyacrylamide gel. The migration of TR{alpha} homodimers, bound to the Apo A-1 side A (26 ), and the free probe are indicated at the left. The F2-TRE (panel C) was used as a marker for TR monomers and was incubated with TR in the presence of T3 (10-7 M).

 
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 1Go). 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{alpha} to Site III entirely (Site III m1; Fig. 4BGo, lane 4). Mutation of the 3'-half-site resulted in reduced receptor binding (Site III m2; Fig. 4BGo, lane 6) and the appearance of a second complex with faster mobility (Fig. 4BGo, arrow). The mobility of this second complex was the same as that of the F2 silencer (Fig. 4CGo) 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. 4DGo, 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. 4DGo, 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{alpha} molecules in vitro.

One Half-Site in Site III Independently Mediates T3-Dependent Repression of the Human CYP7A1 Gene Promoter
Since TR{alpha} 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. 5AGo, phcyp7a-CAT), promoter activity was repressed by T3 in the presence of TR{alpha} (Fig. 5BGo). This finding indicates that Site III, the only remaining TR{alpha} 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. 5CGo).



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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{alpha}. 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.

 
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. 5DGo). Taken together, the results show that a single half-site in Site III is necessary and sufficient for the T3/TR{alpha}-mediated repression of the human CYP7A1 gene promoter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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{alpha} and TRß), which are encoded by two distinct genes, are expressed in a wide variety of tissues. Analysis of the expression of the TR{alpha} and TRß genes revealed a disparity in the abundance of their mRNAs. However, quantitative indirect immunofluorescence studies have revealed that TR{alpha} 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{alpha} and TRß are present in comparable amounts (29), suggesting that both isoforms are readily available for T3-dependent gene regulation.

Recent studies using TR{alpha}- 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{alpha} (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{alpha} 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{alpha}-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{alpha}-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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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{alpha}1 and human RXR{alpha} (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{alpha} and RXR{alpha} were prepared using the TNT Coupled Reticulocyte Lysate System (Promega Corp.) programmed with 0.5 µg of plasmid encoding either rat TR{alpha}1 (41) or human RXR{alpha} (42). Proteins were separated by SDS-PAGE (43) and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) using the manufacturer’s 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{alpha}1 and TRß1 isoforms] or RXRs [sc-774 (Santa Cruz Biotechnology, Inc.) detects RXR{alpha} and RXRß isoforms]. The primary antibody-antigen complexes were visualized with the enhanced chemiluminescence-based detection system (Amersham Pharmacia Biotech, Baie d’Urfé, Quebec, Canada) using horseradish peroxidase-conjugated antirabbit IgG as the secondary antibody.

Recombinant TR{alpha} and RXR{alpha}
Plasmids encoding rat TR{alpha}1 or human RXR{alpha} 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 [{alpha}-32P]dATP, 50 µCi of [{alpha}-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{alpha} 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 [{alpha}-32P]dATP (3,000 Ci/mmol) and 50 µCi of [{alpha}-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
 
We thank Anthony Taylor for technical advice. We also thank Daisy Sahoo, and Eric Labonté for critical reading of the manuscript.


   FOOTNOTES
 
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{alpha}-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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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