Negative Regulation of the Androgen Receptor Gene Promoter by NFI and an Adjacently Located Multiprotein-Binding Site

Chung S. Song, Myeong H. Jung1, Prakash C. Supakar2, Bandana Chatterjee and Arun K. Roy

Department of Cellular and Structural Biology (C.S.S., M.H.J., P.C.S., B.C., A.K.R.) The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284
Audie L. Murphy Memorial VA Hospital (B.C.) San Antonio, Texas 78284


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgenic steroids are critical signaling agents for the development and regulation of the male sexual phenotype (1). Similar to other steroid hormones, androgens influence gene expression by binding and activating the androgen receptor (AR) protein and by recruiting other accessory coactivators at target genes (2). Intracellular concentrations of both the receptor and the receptor-activating steroid ligand appear to play almost equally important roles in the mediation of androgen action (3). Cell-specific expression of the AR gene during development, maturation, and aging can determine the spatiotemporal control of target cell sensitivity to androgenic hormones. Both negative and positive controls of the AR promoter function are responsible for these tissue- and age-dependent differences in hormonal sensitivity. Understanding the role of various trans-acting proteins in the regulation of the AR promoter is essential to elucidate the mechanism of the spatiotemporal changes in androgen action. Studies in our laboratory and by others have helped to characterize the AR gene promoters from a number of species including mouse, rat, dog, and human (4, 5, 6, 7, 8, 9, 10, 11, 12). In addition to the extensive sequence homology among different mammalian species, the salient features of the AR gene promoter are the absence of a TATA- or CAAT-box near the initiation site, the presence of an approximately 100-bp long homopurine/homopyrimidine (pur/pyr) element immediately upstream of the Sp1-box, and a number of specific transcription factor-binding sites further upstream. A comparative analysis of the AR promoter sequences through phylogenetic footprinting (13) suggests the presence of about 20 transcription factor-binding sites between -1000 and -150 bp (3, 14). Since the AR is expressed only at a very low level in most tissues, except reproductive organs, many of these potential protein-binding sites may serve to quantitatively lower its rate of transcription in nonreproductive tissues by interacting with negative regulators. In this report we describe the identification of a strong negative regulatory element at the AR gene promoter containing NFI and at least two other nuclear factors.

NFI represents a family of enhancer binding proteins originally identified as a host initiation factor for adenoviral DNA replication (15). In vertebrate species, four closely related isoforms of NFI are coded by separate genes, NFI-A, NFI-B, NFI-C, and NFI-X. Primary transcripts of these isoform-specific genes also undergo alternate splicing, generating a family of transcription factors (16). Specific DNA binding sites for NFI and its positive regulatory role in the transcriptional control of a large number of eukaryotic genes have been established (17, 18, 19, 20). In addition to its predominantly positive regulatory function, in a number of cases negative regulatory effects of NFI have also been described. These include retinol-binding protein (21), {alpha}2(I) collagen (22), osteonectin (23), lipoprotein lipase (24), GH (25), neuron-specific peripherin in nonneuronal cells (26), von Willebrand factor (27), glutathione transferase P (28), and PIT-1 (29). Unlike its positive regulatory function, which is mediated through interactions with the basal transcriptional machinery, the mechanism of the negative regulatory function of NFI is largely unknown.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Deoxyribonuclease I (DNase I) footprinting of the rat AR promoter shows a long stretch of protected region covering -388 to -340 bp (Fig. 1Go). A transcription factor database search revealed that the upstream portion (-388 to -373) of the footprint corresponds to the binding sequence of the transcription factor NFI (18). However, the rest of the protected region (-372 to -340) does not show homology to the consensus binding motif for any known DNA-binding protein. Despite the continuous nature of the protected region, the upstream and downstream components of the footprint competed distinctively for separate binding proteins. Selective competition of these two footprint positions, either by a 33-mer oligonucleotide duplex representing the proximal segment of the footprinted DNA or a 30-mer duplex representing the high-affinity NFI enhancer element of the adenovirus major late promoter, is shown in lanes 3 and 4, respectively (Fig. 1Go).



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Figure 1. DNase I Footprint Pattern of the Rat AR Gene Promoter Spanning -486 to -324 bp Positions

Lanes 1 and 5, Naked DNA digested with DNase I; lane 2, DNA treated with rat liver nuclear extract before digestion with DNase I; lane 3, same as 2 except with a 300-fold molar excess of a competitor oligonucleotide duplex corresponding to -372 to -340 positions of the rat AR gene; lane 4, same as 2 except with a 300-fold molar excess of a competitor oligonucleotide duplex corresponding to the NFI element of the adenovirus major late (AdML) promoter. Numbers on the left show base positions within the promoter sequence. The nucleotide sequences of the footprint, the putative NFI element, and the MBS are shown on the right.

 
Identity of the -388/-373 footprint site as the NFI element was substantiated by cross-competition between the AR promoter sequence and the adenoviral NFI element in electrophoretic mobility shift assay (EMSA) (Fig. 2Go). The labeled oligonucleotide duplex corresponding to the -388/-373 footprint produced a retarded complex containing closely migrating bands characteristic of the EMSA pattern produced by the multiple forms of the NFI protein. These closely migrating bands can be almost completely competed out with either a 50-fold molar excess of the unlabeled homologous oligonucleotide (lane 4), or 10-fold molar excess of a 30-mer duplex corresponding to the NFI-binding site of the adenovirus major late (AdML) promoter (lane 5). However, even 100-fold molar excess of another closely related cis-element cognate to CCAAT/enhancer-binding protein (C/EBP) (30, 31) failed to cause any significant reduction of the band intensity (lane 8).



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Figure 2. Electrophoretic Mobility Shift Pattern of the Rat AR -388/-373 Element

32P-labeled oligonucleotide duplex corresponding to the -388/-373 rat AR element was used as the probe in mobility shift assay. Competition of the specific protein binding by different oligonucleotide duplexes and cis-elements at indicated fold molar excesses are shown on the top.

 
Identity of the bound proteins as NFI isoforms was further authenticated by their immunoreactivity with the polyclonal antibody to NFI (Fig. 3Go, lane 6). The adenovirus NFI element contains two CCAAT-like boxes (box-A and -B) that are present in an inverted orientation, while in the AR promoter these two boxes occur in a direct orientation (Table 1Go). Point mutations at three of the five bases within the B-box of the -388/-373 footprinted site of the AR promoter prevented it from functioning as an effective competitor for the wild-type AR element (Fig. 3Go, lane 4). From all of these results we can conclude that the AR -388/-373 footprint site contains an authentic NFI element, but its binding affinity is lower than that of the adenovirus NFI site.



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Figure 3. Mutational Inactivation and Immunoreactivity of the Rat AR NFI Binding Site

Lanes 1–3 show band patterns without any competitor oligonucleotide, with 100-fold molar excesses of the homologous rat AR -388/-373 oligo, and the adenovirus major late promoter NFI oligo, respectively. Lane 4 shows lack of competition with the mutant -388/-373 oligo containing three- point mutations (described in Materials and Methods). Lane 5 shows lack of competition with the related cis-element for C/EBP. Lanes 6 and 7 show that polyclonal antibody to NFI, but not the preimmune IgG, eliminates specific protein binding.

 

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Table 1. Sequence Comparison between -388/-373 Site and the NF1-Binding Site of the Adenovirus Major Late Promoter (AdMLP)

 
The proximal part (-372/-340) of the footprinted site appears to contain binding sites for at least two uncharacterized DNA-binding proteins. EMSA with a labeled probe corresponding to this footprinted site and the rat liver nuclear extract suggested multiple protein binding with a distinct slower-migrating upper complex and a faster-migrating lower complex (Fig. 4Go). Although protein binding to both of these complexes can be competed out with a 100-fold excess of unlabeled homologous oligonucleotide duplex (lane 2), oligonucleotides corresponding to the consensus binding elements of two other transcription factors, C/EBP and NF{kappa}B, at the same molar ratio failed to compete for binding with the labeled probe (lanes 3 and 4). These results and our failure to find any match of sequence homology from the transcription factor database have led us to conclude that the rat AR -372/-340 site specifically binds to at least two yet-to-be-characterized sequence-specific DNA-binding proteins.



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Figure 4. Multiple Protein Binding at the -372/-340 Rat AR- Regulatory Element

Electrophoretic mobility shift with the 32P-labeled -372/-340 oligonucleotide duplex was performed with the rat liver nuclear extract. Competition of the specific binding with 100-fold molar excesses of the unlabeled homologous oligo, NF{kappa}B consensus element, and C/EBP consensus element are shown in lanes 2, 3, and 4, respectively.

 
To further delineate this multiprotein binding site (MBS), EMSA with labeled probes containing truncated sequences was performed. As shown in Fig. 5Go, removal of 5 bp from the 3'-end is sufficient to cause a marked decline in the formation of both the lower and the upper complexes, and removal of 2 more base pairs from the 3'-end almost completely disrupted both of these complexes. Removal of 8 bp from the 5'-boundary of the proximal half of the footprint site did not disrupt formation of the upper complex but generated a new complex of intermediate mobility (lane 4). However, truncation of 11 bp totally eliminated the upper complex and at the same time increased the intensity of the intermediate complex (lane 5). Removal of 13 bp from the 5'-end abolished all protein binding to the DNA duplex (lane 6), and no specifically retarded bands were observed. From all of these results, we conclude that the -372/-340 segment of the footprinted site contains overlapping sites for multiple DNA-binding proteins, and that the DNA sequence encompassing -347 to -359 bp is essential for the formation of all of these DNA-protein complexes. This essential component (-347/-359) contains four G residues on the upper strand, and mutation of these G residues to T completely abolished the ability of the -372/-340 oligonucleotide duplex to bind to cognate binding proteins (Fig. 5Go, lane 7).



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Figure 5. The Overlapping Nature of Specific Protein Binding at the -372/-340 Rat AR-Regulatory Element

Each lane in the upper panel (A) shows the electrophoretic mobility shift pattern of individually labeled oligonucleotide duplexes as indicated on the top. The lower panel (B) shows the nucleotide sequences of the oligonucleotides used for EMSA in panel A. The central core region (-359/-347) essential for formation of both of the retarded complexes is indicated with a box at the top, and mutant positions are marked with underlines at the bottom.

 
The functional role of the composite protein-binding element covering the entire footprinted region (-388/-340) was examined by transfectional analysis of promoter-reporter constructs containing a promoter fragment spanning -1040 to +560 bp of the rat AR gene ligated to the firefly luciferase coding DNA. A mutated form of the proximal MBS (mMBS) of the promoter-reporter construct contained four-base substitutions (GGCCTGTG-> TTCCTTTT) within the essential -347/-359 binding region. The promoter mutated at the NFI site (mNFI) contained substitutions at sequences from -378 to -376 (TGG -> GTT). In EMSA, both of these mutations were shown to destroy specific binding activity to cognate binding proteins (Fig. 3Go, lane 4, and Fig. 5Go, lane 7). In addition to the promoter-reporter constructs that contain individual mutations at these two sites, a third double mutant (DMT) containing mutations within both of these protein-binding sites was also tested in cell transfection assay. Results presented in Fig. 6Go show that inactivation of protein binding by mutations at either the NFI site (-388/-373) or the proximal multiprotein-binding site (-372/-340) caused an approximately 10-fold increase in the promoter activity. However, mutations at both of these sites together (DMT) did not produce any additional increase in the promoter activity. These results indicate that the entire footprinted area spanning -388 to -340 functions as a negative regulatory region of the rat AR gene, and the same degree of derepression after mutational inactivation of either one or both of these sites suggests a cooperative interaction among these sequence-specific DNA-binding proteins.



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Figure 6. Relative Activities of the Wild-Type and Mutant Forms of the AR Promoter in Transfected HepG2 Cells

WT, wild type; mMBS, mutated MBS; mNFI, mutated NFI site; DMT, a double mutant containing mutations at both the MBS and the NFI site. Each point is derived from separate transfection experiments with corresponding wild-type controls.

 
NFI appears to be expressed ubiquitously, albeit in a cell- and tissue-specific isoform composition. We have examined the cellular distribution of NFI and the unknown nuclear factors that interact with the proximal portion of the footprinted sequence in nuclear extracts derived from cells of different tissue origins (Fig. 7Go). The isoform composition of NFI in nuclear extracts derived from the liver and kidney (as indicated by the gel shift pattern) showed a marked difference, with the kidney extract displaying a predominance of higher mobility complexes (Fig. 7AGo, lanes 1 and 2). However, predominance of NFI isoforms that yield higher mobility DNA-protein complexes does not appear to correlate with a higher level of AR expression. This is indicated by band patterns shown in the next two lanes (lanes 3 and 4), where the AR-positive LNCaP cell extract shows slower migrating bands as compared with the AR-negative PC3 cells. Nuclear extracts from the other four cell lines, i.e. CHO, HeLa, COS1, and FTO2B (lanes 5–8), show band patterns similar to those of the rat liver (lane 1). The component binding proteins for the proximal (-372/-340) portion of the regulatory region appear to be present not only in the rat liver but also in nuclear extracts derived from the kidney and from LNCaP, PC3, CHO, HeLa, COS1, and FTO2B cells (Fig. 7BGo). Although tissue and cellular distributions of the ratio of the two component bands show some variations, no correlation of band patterns between AR-expressing tissues and cell lines (liver, kidney, and LNCaP) and AR-negative cells was observed. All tissue and cell extracts that were examined contained both of these specific DNA-binding proteins. These results suggest that derepression of the AR gene from the negative control of this composite regulatory element may not be due to simple tissue-specific differences in levels of the nuclear factors that specifically bind to this promoter region, and may, in fact, be the result of complex interaction of this element with other regulatory regions in the context of nucleosomal structure and/or interactions with other proteins that can function as corepressors along with these sequence-specific enhancer-binding factors.



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Figure 7. Ubiquitous Expression of NFI and the Nuclear Proteins That Bind to the -372/-340 Site of the Rat AR Gene

Various nuclear extracts used for the EMSA are indicated at the top. A, EMSA with the labeled NFI (-388/-373) probe; B, EMSA with the adjacent multiprotein binding site (-372/-340) labeled probe.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The AR gene is expressed in all tissues of the rat, albeit at markedly different levels. A relatively high level of AR expression is found only in specific cells of the male reproductive tissues and in the adrenal cortex and kidney (3). Even where it is expressed at a low level, as in the liver, the level of expression can vary over a range of approximately 100-fold at different stages of life (7). Efficient tissue-specific expression of the AR at a low level and its regulation during maturation and aging require interactions among a number of positive (7, 8, 10, 12) and negative regulatory elements (9, 11, 12). In several earlier reports we have described the positive regulatory role of an age-dependent factor and Sp1 in rat AR gene expression and negative regulation of this receptor gene by NF{kappa}B, all of which undergo marked age-dependent changes (7, 11, 12). Evidence for the presence of a strong negative control region presented in this article further extends the regulatory mechanisms available for spatiotemporal expression of the AR gene.

The ubiquitous transcription factor NFI appears to be one of the important regulatory components of the multiprotein binding site at -388/-340 of the rat AR promoter. The NFI family of transcription factors primarily functions as a positive regulator; nevertheless, in addition to the present report, the negative regulatory role of NFI has also been described for a number of genes (21, 22, 23, 24, 25, 26, 27, 28, 29). Unlike its positive regulatory function, which is mediated by direct interaction of the enhancer-bound NFI to the general transcription factors, the mechanism(s) for the NFI-mediated negative regulation is still largely unknown. Gil et al. (19) initially proposed that the sterol-mediated feedback inhibition of HMG-CoA reductase gene expression may be mediated through disruption of the protein-protein interaction between NFI and a hypothetical downstream partner. The presence of such downstream protein-binding sites within the negative regulatory elements of both the rat AR gene and the rat GH gene provides experimental support for such a hypothetical mechanism. The structural arrangement and regulatory properties of the NFI-dependent negative regulatory region of the rat GH gene (also known as the silencer-1) are very similar to the negative regulatory region of the rat AR as described in this article. The silencer-1 region of the rat GH gene contains an NFI-binding site that is immediately followed downstream by the binding site of a yet-to-be-characterized nuclear protein called SBP2 (silencer binding protein 2) (32). Although the SBP2 site does not have any sequence homology to the MBS site of the rat AR gene, analogous to the case for the rat AR promoter, both NFI and SBP2 elements of the rat GH promoter provide independent, but not additive, repressive effects, indicating a cooperative mode of function between these two regulatory sites.

In an attempt to identify the presence of any specific repression domain within the NFI protein sequence, Osada et al. (33) have tested the effects of various N-terminal truncated forms of NFIA (the predominant NFI isoform of the rat liver) ligated to the heterologous yeast GAL4 DNA-binding domain on the negative regulation of the rat glutathione transferase P gene promoter. From these studies they concluded that the minimum repression domain is contained within the 318–427 amino acid region that is conserved in all four major isoforms of the NFI protein. However, glutathione-S-transferase pull-down assay failed to identify interaction of this domain with any one of the general transcription factor components of the preinitiation complex. Despite these results showing a lack of interaction between the negative regulatory domain of NFI with the known members of the preinitiation complex, the possibility that non-DNA-binding corepressors may contribute to the repressor function of NFI still remains to be examined. Recently, Gao and Kunos (34) have reported that overexpression of NFI in transfected Hep3B cells results in the activation of the {alpha}1B adrenergic receptor gene middle promoter, whereas in another cell type DDT MF-2, the same NFI isoform acts as a negative regulator of promoter function. Results of these studies also suggest that a cell type-specific expression of a non-DNA- binding nuclear protein may modulate the repressor function of NFI-X (the specific isoform used in their study) through protein-protein interaction at a region located between 243 and 416 amino acid positions of NFI-X. Although in our case we did not observe any major tissue-specific differences in the level of the two adjacently positioned DNA-binding proteins, all of these published results point to more than one mechanisms for the negative regulatory function of the NFI proteins.

With the exception of the major androgen targets such as the prostate, seminal vesicle, testis, adrenal cortex, and kidney, in most tissues the AR mRNA is expressed at a low level, but in a highly controlled fashion (3). Negative regulatory mechanisms appear to play major roles in such muted expression of the AR gene, specifically in nonreproductive tissues. In addition to its AR-mediated autoregulation (35, 36, 37, 38, 39, 40), which may have a greater importance in tissues where AR is expressed at a relatively abundant level, several negatively acting cis-elements have been described. These include two negative regulatory regions in the mouse AR with yet-to-be-characterized binding proteins (9, 41), the NF-{kappa}B element (11), and the single-strand pyrimidine-specific protein-binding site in the rat AR promoter (12). Among all of these regulatory elements, the NFI composite element described in this paper appears to provide the strongest repressor function in transient transfections. Unlike the marked increase in NF-{kappa}B during the age-dependent down-regulation of AR in the rat liver, we have not observed any correlation of the hepatic levels of AR mRNAs at different ages with the nuclear levels of NFI or the adjacently located DNA-binding proteins. Although the results presented in this article do not show any correlation between the isoform-dependent gel shift patterns of NFI and AR expression in different cell types, a significant downward shift in the electrophoretic mobility of NFI-DNA complexes in the nuclear extracts derived from old rats has been reported (42). Whether such differences in the NFI isoform composition, generated either by alternate splicing of the primary transcript (31) or by O-linked glycosylation of the protein (43), play any significant role in the age-dependent down-regulation of AR in the rat liver remains uncertain. However, altered expression of C/EBP isoforms during aging has been implicated in the changes in hepatic gene expression (44).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Nuclear Extracts
Fischer 344 male rats (3–4 months of age) were obtained from Charles River Laboratories, Inc., Wilmington, MA. All animal experiments were conducted in accordance with NIH standards of humane care, and all protocols were approved by the Institutional Animal Care Committee. Nuclear extracts from rat liver and kidney were prepared according to the method described by Hattori et al. (45). Nuclear extracts from HeLa, FTO2B, PC3, LNCaP, CHO, and COS1 cells were prepared by the method of Dignam et al. (46). All buffers contained 2 µg/ml each of aprotinin, leupeptin, bestatin, 0.1 mM phenylmethyl sulfonyl fluoride, and 1 mM dithiothreitol (DTT). Protein concentrations were determined using the Bradford assay.

EMSA
Oligonucleotide probes were labeled with 32P using T4 polynucleotide kinase and [{gamma}-32P]-ATP. Five micrograms of nuclear extracts were preincubated with 2 µg of poly(dl-dC) for 5 min in 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol at room temperature with or without the unlabeled competitor DNA. After preincubation, the labeled probe (10–20 fmol) was added to the reaction mixture and incubated for 20 min. Protein-DNA complexes were resolved by electrophoresis on 5% polyacrylamide gels. After electrophoresis, gels were dried and autoradiographed. The antibody supershift experiments were carried out using 5 µl polyclonal rabbit antiserum to NF1 containing 5 µg of IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The antibody was preincubated with the nuclear extract for 45 min at 22 C. The reaction mixture was then incubated with poly(dl-dC) for 5 min before the addition of the radiolabeled probe, and incubation continued for an additional 20 min. For the control experiment, IgG from the preimmune rabbit serum was used in place of the NF1 antibody. DNA-protein complexes were analyzed by EMSA.

DNase I Footprinting
DNase I footprinting of the rat AR promoter was performed according to the procedure described previously (47). Briefly, the 3' end-radiolabeled DNA fragment was generated by PCR using 32P-radiolabeled antisense primer (-280/-300) and unlabeled sense primer (-665/-645). The radiolabeled probe (50,000 cpm, 10 fmol) was incubated with 50 µg of nuclear extract in a 50 µl reaction mixture at 10 mM HEPES, pH 7.6, 60 mM KCl, 5% (vol/vol) glycerol, 0.5 mM DTT, 0.5 mM EDTA, and 2 µg of poly(dI-dC) double-stranded DNA. After preincubation of the reaction mixture (10 min, room temperature) without the DNA probe, the labeled DNA was added and incubation continued on ice for 30 min. The reaction mixture was then brought to 1 mM CaCl2 and MgCl2 and incubated at room temperature for 1 min, and then the protein-bound DNA was digested with 0.02–0.1 µg of DNase I (30 sec to 2 min, room temperature) under standard buffer conditions (47). For incubations with BSA, 10-fold less DNase I was used. Digested DNA fragments were extracted with phenol-chloroform (1:1), resolved on an 8% sequencing gel, and visualized by autoradiography.

Construction of Wild-Type and Mutant Plasmids
The wild-type plasmid (pAR 1.6 kb-Luc) used in this study contains the wild-type rat AR promoter from -1040 to +560 bp inserted into the luciferase vector pGL2 Basic (Promega Corp.). The four-point mutant at the proximal MBS (mMBS) contains four-base substitutions (GGCCTGTG -> TTCCTTTT) within the core sequence (-347/-359). The three-point mutant at the distal NF1 binding site contains three-base substitutions within the footprinted site spanning -378 to -376 (TGG -> GTT). The site-specific mutations were introduced into DNA fragments generated by PCR of the wild-type pAR 1.6-kb Luc plasmid template, in the presence of the mutant oligonucleotide primer containing the appropriate base substitutions and the vector-specific primer. The mutant DNA fragments generated by PCR were digested with restriction enzymes and purified by gel electrophoresis. DNA fragments containing the desired mutations were then reinserted into the wild-type plasmid sequence and authenticated by DNA sequencing.

Analysis of Promoter Function in Transfected Cells
The strength of the AR promoter to direct luciferase expression was measured in transfected HepG2 (human hepatoma) cells as described earlier (11, 12). Briefly, approximately 106 cells were seeded in the T25 flask. After overnight culture in DMEM-Hank’s F12 medium (1:1) containing 10% FBS, cells were transfected with the plasmid DNA following the calcium phosphate coprecipitation method. After transfection, cells were washed with PBS (pH 7.5), subjected to glycerol shock (10%, 3 min), and cultured for an additional 48 h before harvesting. Cytoplasmic extracts were assayed for both luciferase activity (48) and protein concentration, and the enzyme activities in different samples were normalized to the constant amount of the total protein.


    ACKNOWLEDGMENTS
 
We thank Tina Hassan, Sang Kim, and Gilbert Torralva for dedicated technical assistance and Lita Chambers for secretarial help. Mutant promoter-reporter constructs used in this study were prepared by Sun-Jin Choi.


    FOOTNOTES
 
Address requests for reprints to: Dr. Arun K. Roy, Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7762.

This work was supported by NIH Grants, AG-10486 and DK-14744. B.C. is a career scientist with the Department of Veterans Affairs.

1 Current address: National Institute of Health, Division of Cancer Research, Seoul 122–020, Korea. Back

2 Current address: Institute of Life Sciences, Bhubaneswar 751007, India. Back

Received for publication April 12, 1999. Revision received June 11, 1999. Accepted for publication June 21, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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