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Biology of Reproduction 63, 1881-1892 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Characterization of an Androgen-Specific Response Region Within the 5' Flanking Region of the Murine Epididymal Retinoic Acid Binding Protein Gene1

Jean-Jacques Lareyre3,a, Kim Reide, Colleen Nelsone, Susan Kasperb,c, Paul S. Renniee, Marie-Claire Orgebin-Crist2,a,b,d, and Robert J. Matusikb,c

a Departments of Obstetrics and Gynecology, b Cell Biology, c Urologic Surgery, and d Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-2633 e The Prostate Centre, Jack Bell Research Centre, Vancouver, British Columbia V6H 3Z6, Canada

ABSTRACT

The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5' flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions -445/-459 and -102/-88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the -543/-88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo.

androgen receptor, gene regulation, sperm maturation

INTRODUCTION

It is now well described that mammalian spermatozoa are subjected to morphological and biochemical changes as they transit through the epididymis. This maturation process allows spermatozoa to acquire the forward motility and fertilizing ability [1]. It is believed that interactions between spermatozoa and epididymal secretory proteins are required for normal male fertility. Along the epididymis the sperm maturation process is progressive. This is likely because the epididymis displays a highly region- and cell-specific expression pattern of genes encoding ubiquitous or epididymis-specific proteins [2–4]. Little is known about the molecular mechanisms that trigger the regionalization of gene expression in the epididymis. Orchiectomy and hypophysectomy studies have demonstrated that epididymal function is primarily dependent on testicular androgens [3]. Nevertheless, unidentified sperm-associated factor [5], growth factors [6], estrogen [7], and retinoic acid [8] have also been implicated in regulating gene expression in the epididymis.

We have previously identified two peptides (minor and major form) resulting from the differential cleavage of a single precursor protein named MEP10 [9]. This protein is synthesized in the principal cells from the mid/distal caput epididymidis. It is secreted into the luminal fluid but does not tightly bind to spermatozoa. The MEP10 protein binds retinoic acid (9 cis and all trans) but not retinol [10], and was therefore renamed murine epididymal retinoic acid-binding protein (mE-RABP) [11]. The mE-RABP protein is the mouse orthologue of two other retinoic acid binding proteins identified previously in the rat epididymis and named successively proteins B/C [12], EBP1 and EBP2 [13], ERABP [14, 15], and ESPI [16]. The amino acid sequence analysis reveals that mE-RABP belongs to the lipocalin superfamily. The three-dimensional structure of the lipocalin proteins, constituted by a ß-barrel closed at one end by a short {alpha} helix, is particularly well-adapted for the binding and transport of small lipophilic molecules [17]. Therefore, mE-RABP may function as a retinoic acid carrier protein in the epididymis.

The gene encoding mE-RABP is localized in the region [A3,B] of mouse chromosome 2 that is rich in genes that encode lipocalins. These lipocalin genes have a genomic structure similar to that of the mE-RABP gene [18]. In previous studies, we have shown that mE-RABP gene expression was androgen-responsive [11]. Murine E-RABP mRNA disappeared from the epididymis of adult mice 10 days after bilateral orchiectomy and gene expression was restored upon androgen replacement.

The androgen receptor (AR) belongs to the ligand-inducible transcription factors superfamily that includes steroid, thyroid, and retinoic acid receptors [19, 20]. Once activated, AR binds to cis-DNA regulatory elements called androgen response elements (AREs). The ARE consensus sequence is similar to that of the glucocorticoids (GREs), mineralocorticoids (MREs), and progestin response elements (PREs) [21]. This consensus sequence (GRE/ARE) is an imperfect palindrome that consists of two hexamers repeated in reverse orientation and separated by three nucleotides (5'-GGTACAnnnTGTTCT-3' [21]). A cascade of direct actions or indirect interactions, or both between AR and the preinitiation complex enhances the transcription of the target gene. The molecular mechanisms that lead to a specific androgen response in vivo are not well understood. A synthetic and specific DNA binding site for the AR has been described recently [22]. However, physiologic AREs employed in transient transfection assays were activated by glucocorticoids receptors depending on the cell lines used [23–26].

We have recently shown that the 5' flanking region of the mE-RABP gene confers both androgen regulation and epididymis-specific gene expression in transgenic mice [27]. In the present study, we describe the identification of a complex and androgen-specific response region (ARR) within the 5' flanking region of the mE-RABP gene.

MATERIALS AND METHODS

Chimeric Constructs

DNA fragments encompassing the mE-RABP gene promoter were generated from the pHindIII genomic clone [18] using appropriate restriction enzymes. DNA fragments were purified on a 1% (w/v) agarose gel and then ligated into the pBLCAT2 plasmid or promoterless pBLCAT3 plasmid [28] using standard methods [29]. Mutations of the AR binding sites were performed by site-directed mutagenesis as described previously [30]. Briefly, a reverse primer carrying the appropriate mutation (illustrated in bold characters) of ARBS-1 (mARBS-1 primer: 5'-GGAGTGGGCCAAAAAATCATAATCCC-3') was used with the forward primer TONTON (5'-CAGTGCTGGCTTCAGCCCGGGCATG-3') in a first polymerase chain reaction (PCR) amplification using the pCATSm plasmid as a template. The PCR product was used as the forward primer and combined to the reverse primer, SM23 (5'-CCGGATCCTGGGTTCAGCTCCCCACCAGA-3') in a second PCR amplification to generate DNA fragments encompassing the region -543/+26 of the mE-RABP gene promoter. Mutation of ARBS-0 was performed using the forward mutant primer mARBS-0 (5'-GGTTTACAGTTTGTTTCCAACCCACC-3') and reverse primer, chloroamphenicol acetyltransferase (CAT; 5'-GCTCCTGAAAATCTCGCCAAGCT-3') in the first PCR amplification. Then, the PCR product was combined with the primer TONTON in a second PCR amplification to generate the -543/+26 DNA fragment.

All final PCR products were subcloned into the pGEM-T plasmid (Promega, Madison, WI), digested with appropriate restriction enzymes, and ligated into the pBLCAT3 plasmid in front of the CAT reporter gene. All chimeric constructs were purified on CsCl gradient [29] and verified by sequence analysis. DNA sequencing was performed as described in the Thermo sequenase fluorescent-labeled primer cycle sequencing kit (Perkin Elmer, Branchburg, NJ).

Cell Transfection and CAT Assays

HeLa and PC-3 cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 2 mM glutamine, 2 µg/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% (v/v) dextran-charcoal-treated fetal bovine serum at 37°C in 5% CO2. Cells were plated at 106 cells/10 cm dish the day before the transfection, and then transfected using the calcium phosphate/DNA precipitation method. Briefly, 15 µg of the appropriate mE-RABP/CAT chimeric construct and 2 µg of the human androgen or glucocorticoid receptor expression vectors were incubated in 125 mM CaCl2, 25 mM HEPES pH 7.1, 140 mM NaCl, and 0.75 mM Na2HPO4, for 30 min at room temperature. Expression vectors encoding full-length androgen [31] and glucocorticoid [32] receptors were used during the course of the study. Cells were incubated overnight in the presence of the precipitate. The next morning, cells were washed twice with 3 ml PBS 1x, and a fresh medium containing the appropriate hormone was added. After 48 h, cells were washed once with PBS 1x and incubated for 5 min at room temperature with 2 ml TEN buffer (40 mM Tris-Cl pH 7.5, 1 mM EDTA, and 150 mM NaCl). Cells were scraped and centrifuged at 800 x g in 4°C for 5 min. The pellet was washed with 2 ml TEN buffer and centrifuged again. Finally, cells were resuspended in 0.1 M Tris-Cl pH 7.8, 0.1% (v/v) Triton X100, and incubated for 15 min on ice. Cells were lysed by three cycles of freezing in liquid nitrogen and thawing at 37°C. Insoluble materials were removed by centrifugation at 14 500 x g for 15 min at 4°C. The CAT activity in the cell extract was determined by the two-phase fluor diffusion method as described previously [33]. Average induction and standard deviations were calculated from three independent transfections of one representative experiment. All experiments were corroborated two other times.

Electrophoretic Mobility Shift Assays

DNA fragments of the mE-RABP promoter were linearized using appropriate restriction enzymes to generate a 5' overhang. Templates (0.2 µg) were incubated for 25 min at room temperature with 1x B buffer (Boehringer, Mannheim, Germany), 2 U Klenow enzyme (New England Biolabs, Beverly, MA), [{alpha}32P]dATP (100 µCi, 6000 Ci/mmol) and 1 µM each of dCTP, dTTP, and dGTP. DNA fragments of interest were excised from the cloning vector using a second restriction enzyme and purified on a nondenaturing acrylamide gel using standard procedures [29]. Double-stranded oligomers were end-labeled using the T4 polynucleotide kinase according to the manufacturer's instructions (New England Biolabs). One nanogram (20 000 disintegrations per minute [dpm]) of the probe was incubated on ice for 10 min with 0–400 ng of purified GST-AR2 fusion protein, 0.5 µg poly(dI-dC), 10 mM HEPES pH 7.9, 7.5 mM MgCl2, 50 mM KCl, 0.1 mM EDTA, 10% (v/v) glycerol, 0.5 mM phenylmethylsulfonide fluoride (PMSF) and 0.5 mM dithiothreitol (DTT). The DNA/protein complexes were separated on a 0.75-mm nondenaturing PAGE containing acrylamide/bisacrylamide (20/1) 5% (v/v), 0.5x TBE, and 10% glycerol. Gels were run at room temperature in 1x TBE and dried before being autoradiographed.

DMS Methylation Protection Assays

Labeling mE-RABP promoter fragments pBluescript SK (+) vectors (Stratagene, La Jolla, CA) containing two DNA fragments of the mE-RABP promoter (-195 to +26 base pair [bp] and -543 to -166 bp) were linearized using the restriction endonuclease HindIII (New England Biolabs). The DNA (10 µg) was then incubated at 37°C for 1 h with 1x Buffer 2 (New England Biolabs) and 10 U of calf intestinal phosphatase (New England Biolabs) followed by heat denaturing at 85°C for 15 min. The reactions were then subjected to phenol/chloroform/isoamyl alcohol (25:24:1) extraction and ethanol-precipitated. The dephosphorylated DNA was incubated at 37°C for 1 h with 1x polynucleotide kinase buffer (New England Biolabs), 60 µCi [{gamma}32P]ATP (6000 Ci/mmol) (Amersham Pharmacia Biotech, Baie d'Urfe, PQ, Canada), and 10 U polynucleotide kinase (New England Biolabs) followed by heat denaturing at 75°C for 20 min. To label the noncoding strand, the HindIII linearized plasmids containing mE-RABP promoter fragments (10 µg) were incubated at room temperature (25°C) for 15 min with 1x Buffer 2 (New England Biolabs), 200 µM each of dATP, dCTP, and dGTP (Life Technologies, Burlington, ON, Canada), 50 µCi of [{alpha}32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech) and 5 U of Klenow Fragment (New England Biolabs) followed by heat denaturing at 75°C for 20 min. Each {gamma}ATP and {alpha}dCTP-labeled sample was passed through an S-200 column (Amersham Pharmacia Biotech) to remove unincorporated radionucleotides, and ethanol-precipitated. To generate single-end labeled probes, the DNA fragments were incubated at 37°C for 1 h with 1x Buffer 2 (New England Biolabs), 100 ng/µl BSA (New England Biolabs), and 30 U of the restriction endonuclease, XbaI (fragment -543/-166 was further digested with AflII to generate single-end labeled fragment -543/-435) (New England Biolabs). The labeled DNA fragments of the mE-RABP promoter were separated from the remaining plasmid vector on a 0.75-mm nondenaturing gel containing acrylamide:bisacrylamide (29:1) 5% (v/v), and 1x TBE (0.089 M Tris base, 0.089 M boric acid, 2 mM EDTA pH 8.0. Gels were run at 350 V for 2 h at room temperature, covered in plastic film, and exposed to Biomax MR autoradiograph film (Kodak) for 2 min. Three probes were cut out (135 bp, 256 bp, and 412 bp) and eluted from the gel in 500 µl of elution buffer (0.6 M ammonium acetate, 0.1% sodium dodecyl sulfate, and 1 mM EDTA) while rotating overnight at room temperature. The probes were ethanol-precipitated and resuspended in binding buffer (20 mM Hepes pH 7.9, 5% glycerol, 100 mM KCl, and 1 mM DTT).

Binding of histidine Tag-AR-DBD to labeled mE-RABP promoter fragments Joining the N-terminus of the rat AR DNA binding domain and hinge region (AR-DBD:amino acids 524–648) to a histidine Tag moiety results in the histidine Tag-AR-DBD fusion protein. Histidine Tag-AR-DBD fusion protein was prepared according to the Ni-NTA Spin column handbook instructions (Qiagen, Mississauga, ON, Canada) except for the following modification: protein was eluted off the nickel column with 20 mM Hepes pH 7.9, 100 mM KCl, 20% glycerol, 1 mM DTT, and 250 mM imidazole. Protein concentrations were determined by the Bradford method [34]. Histidine Tag-AR-DBD (7.2 µg; 13.6 µM) was incubated at room temperature for 15 min with 2 µg poly(dI-dC) (Amersham Pharmacia Biotech) and binding buffer (20 mM Hepes pH 7.9, 5% glycerol, 100 mM KCl, and 1 mM DTT). To each binding reaction 350 000 dpm (26.5 fmole) of labeled mE-RABP probe was added to a final volume of 30 µl.

Methylation of DNA by dimethylsulfate After 10 min of incubation of this reaction at room temperature, 3 µl of 2% (210 mM) dimethylsulfate (DMS; Fisher Scientific, Nepean, ON, Canada), freshly diluted in binding buffer, was added to the binding reactions for exactly 2 min and promptly loaded onto a prerun 0.75-mm nondenaturing gel containing acrylamide:bisacrylamide (29:1) 5% (v/v), and 0.5x TBE. In addition, labeled DNA without histidine Tag-AR-DBD fusion protein was subjected to 0.18% (19 mM) DMS for 2 min and similarly loaded onto the gel for comparison. The gel was immediately run at 300 V for 2.5 h at room temperature, covered in plastic film, and exposed to Biomax MR autoradiograph film (Kodak, Rochester, NY) for 2 h. The bands indicating protein-bound and protein-free methylated probes were excised and the DNA eluted as described earlier. The methylated probes were ethanol-precipitated, resuspended in 9 µl of water, and then incubated at 90°C for 30 min with 10% (1 M) piperidine (Fisher Scientific). Samples were snap-frozen in liquid nitrogen and lyophilized in a vacuum for 1.5 h. Each sample was washed twice with 100 µl of water, snap-frozen, and lyophilized for 1.5 h after each wash. The lyophilized pellets were finally resuspended in 6 µl of loading dye (95% [v/v] formamide, 10 mM EDTA pH 8.0, 0.1% [w/v] bromophenol blue and 0.1% [w/v] xylene cyanol). Using a scintillation counter (Beckman LS 6500, Beckman Coulter Inc., Fullerton, CA), 1 µl of each sample was counted, then each sample was diluted to 2000 dpm/µl. The cleaved probes were denatured at 90°C for 5 min, cooled on ice, and 4 µl of each denatured sample was loaded onto a prerun 50°C (31 cm x 38.5 cm x 0.4 mm) denaturing gel containing acrylamide:bisacrylamide (29:1) 6%, 1x TBE, and 8.3 M Urea (Fisher Scientific). Gels were run at a constant 65 W for 2 h, transferred to Whatman 3MM paper, vacuum-dried, and exposed to Biomax MS autoradiograph film (Kodak) using a Kodak Biomax MS intensifying screen overnight at -80°C. Dried gels were also visualized using a PhosphoImager (Bio-Rad, Hercules, CA and Kodak) and the images annotated using the PhotoShop software program (Adobe, San Jose, CA).

RESULTS

Identification of an Androgen-Specific Response Region Within the 5' Flanking Region of the mE-RABP Gene

We have previously shown that mE-RABP gene expression was specifically androgen-regulated in vivo [11, 27]. To investigate whether androgens could modulate the transcription of the mE-RABP gene in vitro, a set of chimeric constructs containing different lengths of the mE-RABP promoter driving the CAT reporter gene, were designed. Because there are no appropriate epididymal cell lines, the chimeric constructs were transiently cotransfected into HeLa cells with either the AR or the glucocorticoid receptor (GR) expression vector and incubated with the appropriate hormone (10-7 M dihydrotestosterone [DHT] or dexamethasone [DEX], respectively) for 48 h. The biopotency of DEX is 12 times greater than corticosterone [35]. Therefore, the resulting biopotency of 10-7 M DEX is on the order of 10-6 M corticosterone, which is higher than the circulating concentrations of corticosterone in mice. The construct, pMMTV-CAT, containing the long terminal repeat of the mouse mammary tumor virus driving the CAT reporter gene, was used as a positive control because it contained the well-described steroid hormone response element that is inducible by both glucocorticoids and androgens. The results of these experiments are shown in Figure 1. In our experimental conditions, addition of DHT and DEX led to a 26.1-fold and 74.6-fold increase of the CAT reporter gene expression when the pMMTV-CAT construct was tested. In the absence of hormone, basal CAT activity of all the chimeric constructs carrying DNA fragments of the mE-RABP promoter was low. In the presence of hormone, no significant increase of the CAT activity was observed with constructs containing promoter fragments smaller than 450 bp (constructs -195/+26 and -442/+26). However, the additional 100-bp DNA fragment, included in construct -543/+26 and containing a GRE/ARE-like sequence, led to a 24.1-fold increase of the CAT activity in the presence of 10-7 M DHT only. Surprisingly, DEX failed to activate the CAT gene. This result was not due to a defective glucocorticoid receptor (GR) because in the same experimental conditions, GR was more efficient than AR in activating the hormone response element located within the long terminal repeat of the MMTV (74.6 ± 9.2 versus 26.1 ± 5.0). Although a slight decrease of the basal expression of the construct -1101/+26 was noticed, no significant difference was found in the induction level between constructs -1101/+26, -3009/+26, and -543/+26 (17.9 ± 4.1-, 18.5 ± 4.5-, 24.1 ± 5.8-fold induction, respectively). A higher DHT-mediated induction of the CAT reporter gene expression was noticed when the construct -3806/+26 was tested (50.5 ± 8.5- versus 24.1 ± 5.8-fold induction). Finally, a decrease of CAT expression (21.5 ± 4.8-fold induction) was obtained in the presence of 10-7 M DHT when a 5 kb DNA 5' flanking fragment of the mE-RABP gene was tested with the CAT reporter gene (construct -5120/+26).



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  		FIG. 1. HeLa cells were cotransfected by the calcium phosphate/DNA precipitation method with chimeric constructs (15 µg) carrying different length of mE-RABP gene promoter ligated to the CAT reporter gene, and either AR and GR expression vector (2 µg). The pMMTV-CAT construct (5 µg) containing a well-characterized hormone response element was used as a positive control. Cells were incubated with the appropriate hormone (10-7 M DHT or DEX) for 48 h. CAT assays were performed as described in Materials and Methods. One representative experiment, which was corroborated two other times, is presented. The CAT activity of three independent transfections (n = 3) was measured for each experiment and is expressed as the mean + SEM (dpm/min per mg protein). Note that a few SEMs lie within the top line of the histogram bar. The induction factor was determined by calculating the ratio between the CAT activity measured in DHT- or DEX-treated and untreated cells

The specificity for androgens was also observed when the chimeric constructs -543/+26 and -5120/+26 were transiently cotransfected in PC-3 cells (Fig. 2). Therefore, the specificity to androgens was not HeLa cell-specific.



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  		FIG. 2. PC-3 cells were cotransfected by the calcium phosphate/DNA precipitation method with chimeric constructs (15 µg), carrying different lengths of mE-RABP gene promoter ligated to the CAT reporter gene, and either AR and GR expression vector (5 µg). Cells were incubated with the appropriate hormone (10-7 M DHT or DEX) for 48 h. CAT assays were performed as described in Materials and Methods. The CAT activity was measured in triplicate determinations (dpm/min per mg protein)

These results taken together suggested that a cis-DNA regulatory element or elements that are important for the amplitude and hormonal specificity of the androgen responsiveness are located, in part, within the mE-RABP gene 5' flanking region, -543/+26.

Androgen Receptor Binds to the Androgen-Specific Response Region -543/+26

The binding of AR to the -543/+26 promoter fragment was first investigated by electrophoretic mobility shift assays (EMSA) using an AR fusion protein. This fusion protein contains the DNA-binding domain and the hinge region of the rat AR as well as the C-terminus of the glutathione-S-transferase [26]. The EMSA experiments were carried out using two overlapping DNA fragments that were subcloned from the mE-RABP gene promoter (Fig. 3A). Both DNA fragments were able to bind the GST-AR2 fusion protein (Fig. 3B).



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  		FIG. 3. A) Schematic representation of the mE-RABP gene promoter (-543/+26). The broken arrow indicates the initiation start site (+1). The overlapping restriction fragments (SmBg and NB) used in gel retardation experiments are indicated. Hatched squares point out the positions of the double-stranded oligonucleotides, ARBS-0 and ARBS-1. B) AR binds to the mE-RABP androgen response region (-543/+26). Restriction fragments SmBg and NB (20 000 dpm/lane) were incubated with 100 ng of the fusion protein GST-AR2 containing the DNA binding and the hinge domains of the rat AR linked to the glutathione-S-transferase C-terminus (see Materials and Methods). Arrows indicate retarded complexes and the asterisk indicates a nonspecific band. C) AR binds to ARBS-1. End-labeled double-stranded oligonucleotides (20 000 dpm/lane) ARBS-1 was incubated with increasing amounts of GST-AR2 (0–200 ng). Three retarded complexes (arrows) are observed, indicating that AR binds to ARBS-1. D) AR binds specifically to ARBS-1. End-labeled double-stranded oligonucleotides (20 000 dpm/lane) ARBS-1 was incubated in the absence (lane 1) or presence of 100 ng GST-AR2 (lanes 2–8). Competitions were carried out using a 1-, 10-, and 100-fold molar excess (1x, 10x, 100x, respectively) of cold synthetic double-stranded oligonucleotides carrying the ARBS-1 site or a consensus DNA-binding site for the transcription factor, NF-1. DNA-protein interactions progressively disappear in the presence of a molar excess of specific competitor ARBS-1 but not in the presence of a molar excess of nonspecific competitor NF-1. E) AR binds to ARBS-0. End-labeled double-stranded oligonucleotides (20 000 dpm/lane) ARBS-0 was incubated in the absence (lane 1) or presence of increasing amounts of GST-AR2 (6.25–200 ng). Two retarded complexes were obtained (arrows), indicating that rat AR binds to ARBS-0

Methylation protection assays were carried out to precisely localize the nucleotides involved in AR DNA binding (Fig. 4). Three guanines at position -449, -458, and -459 on the coding strand and one guanine at position -446 on the noncoding strand were protected from methylation in the presence of AR (Fig. 4A). These nucleotides are spread over 15 bp, suggesting interaction with an AR homodimer. This AR recognition site was named androgen receptor binding site 1 (ARBS-1). Analysis of ARBS-1 nucleotide sequence showed that it consisted of an imperfect palindrome (5'-GGATTAtgaTGTTCT-3') with 80% sequence similarity to the GRE/ARE consensus sequence (5'-GGTACAnnnTGTTCT-3').



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  		FIG. 4. Two AR binding sites are located within the mE-RABP gene promoter at positions -459/-445 and -102/-88. DMS methylation protection assays were performed as described in Materials and Methods to identify guanines involved in AR/DNA interactions. A) Three guanines at position -449, -458, and -459 on the coding strand and one guanine at position -446 on the noncoding strand were protected from DMS methylation in the presence of AR, demonstrating their involvement in AR DNA binding. These guanines spread over a 15-bp imperfect palindrome that was named ARBS-1. B) Two guanines at positions -92 and -102 on the coding strand and one guanine at position -89 on the noncoding strand were also protected from DMS methylation in the presence of AR-DBD. Once again, these nucleotides stretch out a 15-bp imperfect palindrome that was named ARBS-0. Both ARBS-0 and ARBS-1 showed homology to the GRE/ARE consensus sequence (73% and 80%, respectively). Guanines that were protected from DMS methylation are labeled with an asterisk. Inverted hexamer repeats forming the imperfect palindromes are indicated with thick vertical lines

Three other guanine residues at position -89 and -92 on the coding strand and -102 on the noncoding strand were also protected by AR (Fig. 4B). Once again, these nucleotides resided in a 15-bp imperfect palindrome that was named ARBS-0. The ARBS-0 nucleotide sequence (5'-GGCTTAcagTGTGCT-3') was similar to that seen in ARBS-1 and in the GRE/ARE consensus sequence (86% and 73% similarity, respectively).

To confirm the specificity of AR/DNA interaction, EMSA was carried out using double-stranded oligonucleotides encompassing the protected area as probes (Fig. 3, C through E). When labeled duplex oligomers encompassing ARBS-1 were used as probes, addition of the GST-AR2 fusion protein to the incubation mixture led to the formation of retarded complexes (Fig. 3C). The specificity of the DNA binding was determined by using heterologous and homologous unlabeled duplex oligomers as competitors (Fig. 3D). A 10-fold to 100-fold molar excess of the heterologous competitor (NF-1 binding site) did not decrease the amount of retarded complexes whereas a 10-fold to 100-fold molar excess of the homologous oligomer (ARBS-1 site) did. When a duplex oligomer, encompassing ARBS-0 was used as a probe, two retarded complexes were obtained after addition of the GST-AR2 fusion protein in the incubation mixture (Fig. 3E).

To determine the relative binding affinity of GST-AR2 to ARBS-0, ARBS-1, and a classical GRE from the tyrosine aminotransferase gene (GREtat: [36]), EMSA was carried out using a fixed amount of GST-AR2 and increasing molar excess of cold, double-stranded oligonucleotides (Fig. 5). The results showed that ARBS-1 had a two times higher binding affinity for GST-AR2 than ARBS-0. However, ARBS-1 had a 13 times lower binding affinity for GST-AR2 compared with that of the classical GREtat.



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  		FIG. 5. AR relative binding affinity for ARBS-0, ARBS-1, and a classical GRE identified within the tyrosine aminotransferase gene (GREtat: [36]). End-labeled double-stranded oligonucleotide (75 fmol) encompassing ARBS-1 was incubated in the presence of a fixed amount of GST-AR2 (50 ng) and increasing molar excess of cold, double-stranded oligonucleotides encompassing ARBS-1 and GREtat (A) or ARBS-1 and ARBS-0 (B). The relative density of the bands corresponding to AR/DNA complexes was determined using densitometry (BioRad model GS-670) and Molecular Analyst software. The slope of each resulting competition curves was determined using Microsoft Excel (version 8.0) software. The AR relative binding affinity between two ARBSs was determined by calculating the ratio between the corresponding slopes. Note that ARBS-1 affinity for AR is two times higher than that of ARBS-0 but is 13 times lower than that of GREtat

In summary, our results suggest that the androgen-specific responsiveness of the mE-RABP promoter may involve the binding of AR to at least two low-affinity AR binding sites.

ARBS-1 Is a Key Element for the Androgen Response

To determine whether all AR binding sites were involved in the androgen responsiveness, point mutations within the right-half site (5'-TGTTCT-3') of the imperfect palindromes ARBS-1 and ARBS-0 were carried out to change bases that are known to be critical for AR DNA binding (Table 1). Wild-type and mutant constructs were cotransfected either with AR and GR expression vector into HeLa cells and incubated with or without 10-7 M DHT or DEX. As shown in Figure 6, mutation of ARBS-0 led to a slight decrease of the androgen responsiveness compared with the control construct, -543/+26 (66.1 ± 12.7 versus 95.5 ± 24.9). This suggests that ARBS-0 may be involved in the magnitude of the androgen response, but it is not an essential regulatory element. Mutation of ARBS-1 abolished the DHT-mediated activation that was observed with the control construct, -543/+26 (1.6 ± 0.4 versus 95.5 ± 24.9). Cotransfection of the mutated constructs with human glucocorticoid receptor (hGR) expression vector did not induce CAT activity in the presence of DEX.


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  		TABLE 1. Nucleotide sequences of wild-type and mutant ARBS-0 and ARBS-1 sites. Bases (G and C) that were replaced after mutation are underlined in the wild-type nucleotide sequences. The thymidine bases that were generated after mutation are presented in lower case in the mutated nucleotide sequences



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  		FIG. 6. HeLa cells were cotransfected as described in Figure 1 with chimeric constructs (15 µg), carrying wild-type (pCATSm) or a mutated AR binding site (pCATmARBS-0 and pCATmARBS-1). Black squares represent the wild-type AR binding sites (ARBS-0 and ARBS-1), whereas white squares indicate the mutated sites. Cells were incubated with the appropriate hormone (10-7 M DHT or DEX) for 48 h. The CAT activity was measured in triplicate determinations and is presented as the mean ± SEM (dpm/min per mg protein). The induction factor was determined by calculating the ratio between the CAT activity measured in DHT- or DEX-treated and untreated cells. Note that mutation at contact sites on ARBS-0 led to a slight decrease of the androgen responsiveness, whereas mutation of the contact sites on ARBS-1 abolished DHT-mediated induction

Altogether, these results indicate that ARBS-1 is likely the most important motif in the androgen response region (-543/+26) of the mE-RABP gene and that although ARBS-0 is not essential for the androgen-specific response, it is required to further increase the magnitude of response to androgen treatment.

ARBS-1 Can Act as a Weak Androgen Response Element When Isolated from Its Promoter Context

To address whether ARBS-1 may act as a functional ARE when it is isolated from the endogenous mE-RABP promoter context, one to three copies of a duplex oligomer encompassing ARBS-1 were ligated in front of the thymidine kinase promoter driving the CAT reporter gene. These constructs were cotransfected into HeLa cells with either hAR or hGR expression vector as described earlier (Fig. 7). No DHT- or DEX-mediated induction of CAT activity was observed despite the presence of one or two copies of ARBS-1. However, an increase of the CAT activity was detected when the construct carrying three copies of ARBS-1 was incubated in the presence of either 10-6 M DHT or DEX (10.6 ± 4.3- and 16.2 ± 5.2-fold induction, respectively). This suggests that ARBS-1 alone can act as a weak ARE and GRE. We also conclude that ARBS-1 alone may not account for the androgen-specific responsiveness of the mE-RABP gene promoter.



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  		FIG. 7. One to three copies of the double-stranded oligonucleotide ARBS-1 were ligated into the pBLCAT2 vector in front of the tk promoter driving the CAT reporter gene. Chimeric constructs (2 µg) were cotransfected in HeLa cells as described in Figure 1 except that cells were incubated in the presence of 10-6 M DHT or DEX for 48 h. CAT activity was determined from three independent assays and is presented as the mean ± SEM (dpm/min per mg protein). No significant increase of the CAT activity was observed in the DHT- or DEX-treated cells that were cotransfected with chimeric constructs containing none, one, or two copies of ARBS-1 (pBLCAT2, 1xARBS-1 and 2xARBS-1 constructs, respectively). However, the CAT activity was highly increased in DHT- or DEX-treated cells that were cotransfected with the construct 3xARBS-1 carrying three copies of ARBS-1. These results indicate that ARBS-1 behaves as a weak ARE and GRE outside the mE-RABP gene promoter context. Horizontal arrows indicate the sense orientation (5' to 3') and the black squares represent the location of the double-stranded oligonucleotide, ARBS-1. The broken arrow points out the transcription initiation site of the tk promoter

Analysis of 3' Deletions of the mE-RABP Gene Promoter

In an attempt to delineate the minimal DNA fragment required to confer an efficient androgen response, 3' deletions of the -543/+26 promoter fragment were carried out using appropriate restriction enzymes. The truncated DNA fragments were ligated in front of the heterologous thymidine kinase (tk) promoter of the herpes virus driving the CAT reporter gene. The chimeric constructs were transfected in HeLa cells as described earlier (Fig. 8). No DHT- or DEX-mediated induction of the CAT activity was observed with the constructs containing the DNA fragments -543/-317 or -543/-166. More surprising, the DNA fragment -543/-88 containing both ARBS-0 and ARBS-1 confers a limited twofold induction in response to androgen treatment. These results suggest that the proximal mE-RABP promoter fragment -87/+26 that was removed from the last constructs contains important cis-DNA regulatory elements for the androgen responsiveness.



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  		FIG. 8. HeLa cells were cotransfected as described in Figure 1 with chimeric constructs (2 µg), carrying different restriction fragments of mE-RABP gene promoter (-543/+26) ligated in front of the tk promoter in the sense orientation. Hatched squares represent the AR binding sites (ARBS-1 and ARBS-0). The CAT activity was measured in triplicate determinations and is expressed as the mean ± SEM (dpm/min per mg protein). The induction factor was determined by calculating the ratio between the CAT activity measured in DHT- or DEX-treated and untreated cells. No significant increase (P < 0.001) of the CAT activity was observed in hormone-treated cells with any of the chimeric constructs, pCATtkSmBg and pCATtkARR. However, construct pCATtkARR2 showed a limited twofold increase of CAT activity after androgen treatment. This suggests that cis-DNA regulatory elements, which are important for full androgen responsiveness of the mE-RABP gene reside within the -87/+26 proximal promoter fragment

DISCUSSION

In this study, the characterization of an androgen-specific response region located within the first 600 bp of the mE-RABP gene promoter is described. We have recently shown that a 5 kb DNA fragment of the 5' flanking region of the mE-RABP gene, linked to the CAT reporter gene, mimics the tissue-specific, cell-specific, and androgen-regulated expression of the endogenous mE-RABP gene in transgenic mice [27]. Therefore, the mE-RABP ARR (-543/+26), we identified here, in vitro, may account for the androgen-specific responsiveness of this gene in vivo.

At least two AR binding sites were localized within the DNA region, -543/+26, of the mE-RABP gene promoter. The first binding site for AR was found at position -459/-445 and named ARBS-1. Point mutation analysis showed that ARBS-1 was required for the androgen responsiveness of the mE-RABP gene promoter. The structure of ARBS-1 is an imperfect palindrome 5'-GGAtagTGTTCT-3'. Although this sequence is highly homologous to the GRE/ARE consensus sequence (5'-GGTACAnnnTGTTCT-3'), three deviations at positions 3, 4, and 5 were observed in the left half site (underlined bases). Two of them, at positions 4 (T instead of A) and, more importantly, at position 5 (T instead of C) were located at the most critical bases for AR binding and functional activity [37]. These observations may account for 1) the low binding of AR to ARBS-1 as observed in the EMSA, and 2) the absence of androgen and glucocorticoids responsiveness of the chimeric constructs carrying one or two synthetic ARBS-1 sites ligated in front of the tk promoter.

Within the probasin gene promoter, two AR binding sites, named ARBS-1 and ARBS-2, cooperate to give rise to an efficient androgen responsiveness [26, 38]. Synergism between multiple AR binding sites was also described within the first intron of the cysteine-rich protein 2 (CRP2) gene [25], the distal promoter of the secretory component (sc) gene [39], and the distal and proximal promoter of the prostate specific antigen (PSA) gene [24, 40]. A second AR binding site was localized within the proximal mE-RABP promoter region at position -101/-88 and was named ARBS-0. The sequence of ARBS-0 (5'-GGCAcagTGTGCT-3') is highly similar to ARBS-1 (86% homology) and to the GRE/ARE consensus sequence (75% homology). Once again, two bases important for AR binding and functional activity are not conserved at position 4 (T instead A) and 5 (T instead of C). Only three contact points with AR were observed instead of four for ARBS-1. Point mutations of ARBS-0 only slightly affect the androgen responsiveness of the mE-RABP gene ARR (-543/+26), suggesting that ARBS-0 was poorly functional within the mE-RABP promoter context. Nonfunctional and poorly functional AR binding sites have also been described within the promoter region (site A) and intron 1 (site B) of the C3(1) gene encoding the prostate-specific steroid binding protein (PSBP) [41]. Nevertheless, De Vos et al. [42,43], showed that nuclear extracts from a variety of tissues enhance binding of AR to intron fragments of the C3(1) PSBP gene. Therefore, we cannot exclude that other cofactors may be required for ARBS-0 function in vivo. The fact that mouse ARBS-0 and ARBS-1 are conserved in the promoter of the gene encoding the rat orthologue of the mE-RABP protein (Table 2) is also suggestive of the functional importance of these two AR binding sites in the androgen-regulated gene transcription.


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  		TABLE 2. The nucleotide sequence and location of the androgen receptor binding sites ARBS-0 and ARBS-1 are well conserved within the promoter of the ESPI gene encoding the rat orthologue of the mE-RABP protein

The replacement of the proximal mE-RABP promoter with the heterologous tk promoter (construct -543/-88) nearly eliminated the androgen-induced response even in the presence of both ARBS-1 and ARBS-0. Within the intron 1 of the C3(1) PSBP gene, the functional activity of a GRE/ARE-like sequence (named core II or site C) is enhanced by surrounding sequences [44]. Similar observations were also reported for the complex androgen response region of the sex limited protein (Slp) gene [45]. This androgen response region resides within the 5' long terminal repeat of an ancient endogenous provirus that is inserted 2 kb upstream from the Slp promoter. The functional activity of a GRE/ARE-like sequence is enhanced or decreased depending on deletions of DNA fragments present in its vicinity [45]. Functional cooperativity was demonstrated between steroid receptors and recognition sequences for transcription-modulating factors, including NF-1, SP1, CCAAT box, OTF, and CACCC box-binding proteins [46]. Several of these putative cis-DNA regulatory elements (CACCC boxes, NF-1, and SP-1 binding sites) are present within the DNA fragment -87/+26 of the mE-RABP promoter [18]. Therefore, it is possible that the functional activity of ARBS-1 requires interactions between the AR and ubiquitous transcription factors binding to the -87/+26 DNA region.

One the most interesting features of the mE-RABP ARR -543/+26 is that it is activated by AR and not by GR in HeLa and PC-3 cells. Hormonal specificity may depend, in part, on the affinity of the steroid receptor for its cognate response element. The AR binding site 2 of the probasin ARR binds differentially with AR and GR, such that it behaves as an androgen-specific response element [38, 47, 48]. However, when ARBS-2 is placed inside the physiologic probasin promoter context, it cooperates with another AR binding site (ARBS-1) and binds AR and GR [38]. Similarly, another androgen-specific unit has been described recently within the human secretory component (sc) gene [39]. This ARR is also constituted by several weak ARBSs that cooperate with a core element, named ARE1.2. This element, consisting of an imperfect direct repeat separated by a three-nucleotide spacer, displays selectivity for the AR that accounts for the androgen-specific responsiveness of the sc gene [39, 49]. In contrast, we have shown that both AR and GR were able to enhance the activity of ARBS-1 outside the mE-RABP promoter context in transient transfection assays. It is therefore unlikely that the androgen-specific behavior of mE-RABP ARR (-543/+26) is due only to the specific binding of AR to ARBS-1.

Several androgen response regions bind both AR and GR. Some of them, like the three ARRs found in the 5' flanking region of the PSA gene, can be activated by AR and GR in transfection assays [24]. Others, like the Slp [23], Crp2 [25], and probasin [26] ARR have a highly androgen-specific behavior in vitro. Although AR remains a more efficient activator than GR, the androgen-specific behavior of Crp2, Slp, and probasin ARR performs best in specific cell lines. This suggests that the cellular content of accessory factors (coactivators and corepressors), transcription factors, or both may modulate the hormonal specificity and magnitude of gene induction. Several molecular mechanisms have been proposed for steroid receptor induction of gene transcription. One of them postulates that steroid receptors bound to DNA temporally modify DNA structure, allowing a rapid exchange with other transcription factors that in turn enhance gene transcription. Androgen-dependent DNA-binding proteins have been demonstrated in the intron 9 of the mouse ß-glucuronidase gene [50], in the promoter region of the mouse RP2 gene [51], and in the androgen-dependent enhancer of the mouse Slp gene [52].

Another model proposes that steroid receptors, bound to their cognate DNA element, lead to a disruption of phased nucleosomes by interacting with accessory factors such as CREB binding protein (CBP), steroid receptor coactivator 1 (SRC-1), p300/CBP associated factor (PCAF), and androgen receptor associated protein (ARA70) [53]. In a first step, the recruitment of coactivators that have an intrinsic histone acetylase activity may result in the loss of the nucleosomal structure present in the vicinity of the hormone response element. In a second step, accessory coactivators may enhance gene expression by stabilizing the assembly of the transcription preinitiation complex. Corepressors having a histone deacetylase activity may have a reversed function [54]. Some of the coactivators and corepressors enhance or decrease the transactivation mediated by a given steroid receptor. For example, whereas the coactivator ARA70 can induce the transcriptional activity of AR up to 10-fold, it can only slightly increase (twofold) the transcription of other steroid receptors, including GR, progesterone receptor (PR), and estrogen receptor (ER) [55]. In addition, a cofactor can behave as a coactivator or as a corepressor depending on the steroid receptor. Cotransfection assays have shown that SRC-1 functions as a coactivator of GR, ER, thyroid receptor (TR), and retinoids-X-receptor (RXR) [56] by increasing their transcriptional activity, while it acts as a repressor of AR-mediated transactivation [57].

The presence of a particular content of cofactors in HeLa cells can not alone account for the androgen-specific responsiveness of the mE-RABP ARR because the androgen-specific behavior of the probasin [26] and Crp-2 [25] ARR is poorly expressed in this cell line. The magnitude and specificity of the androgen responsiveness of the Slp gene is likely a combinatorial function of receptor and nonreceptor binding sites [23]. Therefore, taking into consideration the information from other studies, we speculate that the androgen-specific responsiveness of the mE-RABP promoter may be the result of a combination of molecular mechanisms involving 1) protein-DNA interactions that are allowed to select the efficient combination of transcription-modulating factors, appropriate modification of the DNA structure, or both; and 2) protein-protein interactions between the AR, cofactors, transcription factors (ubiquitous, cell-specific, or both), and the transcription preinitiation complex to enhance gene expression.

In this study, we provide evidence that the mE-RABP ARR is a valuable model to further understand the molecular mechanisms leading to an androgen-specific responsiveness.

ACKNOWLEDGMENTS

We thank the Cancer Center DNA Sequencing Core, directed by Dr. K. Bhat for valuable technical assistance. We also gratefully acknowledge D.E. Ong for helpful comments throughout the course of the study, and for the critical review of the manuscript. J.J.L. thanks Dr. A.O. Brinkmann for providing the human androgen receptor, cDNA.

FOOTNOTES

First decision: 20 March 2000.

1 This work was supported by NIH grants HD03820, HD05797, HD36900, and HD25206. Back

2 Correspondence: Marie-Claire Orgebin-Crist, Center for Reproductive Biology Research, Vanderbilt University, School of Medicine, Medical Center North, Room C-3306, Nashville, TN 37232-2633. FAX: 615 343 7797; m-c.orgebin-crist{at}mcmail.vanderbilt.edu Back

3 Current address: Institut National de la Recherche Agronomique, Station Commune de recherches en Ichtyophysiologie, Biodiversite et Environnement, Campus de Beaulieu, 35042 Rennes Cedex, France. Back

Accepted: August 11, 2000.

Received: February 23, 2000.

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J. Zhang, N. Gao, S. Kasper, K. Reid, C. Nelson, and R. J. Matusik
An Androgen-Dependent Upstream Enhancer Is Essential for High Levels of Probasin Gene Expression
Endocrinology, January 1, 2004; 145(1): 134 - 148.
[Abstract] [Full Text] [PDF]

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 J AndrolHome page
Y. Araki, K. Suzuki, R. J. Matusik, M. Obinata, and M.-C. Orgebin-Crist
Immortalized Epididymal Cell Lines From Transgenic Mice Overexpressing Temperature-Sensitive Simian Virus 40 Large T-Antigen Gene
J Androl, November 1, 2002; 23(6): 854 - 869.
[Abstract] [Full Text] [PDF]

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 Hum Mol GenetHome page