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An Androgen-Dependent Upstream Enhancer Is Essential for High Levels of Probasin Gene Expression

Departments of Urologic Surgery (J.F.Z., N.G., S.K., R.J.M.), Cell and Developmental Biology (J.F.Z., N.G., S.K., R.J.M.), and Cancer Biology (S.K., R.J.M.), The Vanderbilt Prostate Cancer Center (S.K., R.J.M.), and The Vanderbilt-Ingram Cancer Center (S.K., R.J.M.), Vanderbilt University Medical Center, Nashville, Tennessee 37232-2765; and The Prostate Centre (K.R., C.N.), The Jack Bell Research Centre, Vancouver, British Columbia, Canada V6H 3Z6

Address all correspondence and requests for reprints to: Robert J. Matusik, Ph.D., Department of Urologic Surgery, A-1302 Medical Center North, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2765. E-mail: robert.matusik{at}vanderbilt.edu.

	Abstract

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Previously we reported that a small rat probasin (PB) promoter (-426 to +28 bp, -426PB) would target androgen- regulated prostate-specific expression in transgenic mice. Later we demonstrated that a large (L) fragment (-10806 to +28 bp, LPB) of the PB promoter would target high levels of gene expression to the prostate in transgenic mice. These results suggested that optimal transcription of the PB gene depended on the presence of enhancer regions upstream of the proximal promoter. To identify these enhancers, the LPB fragment was sequenced and the enhancer activities of restriction fragments were characterized in cell lines. Two nonconventional androgen receptor binding sites (ARBSs), ARBS-3 and ARBS-4, in an upstream androgen-dependent enhancer of the PB gene were identified. One site functions as a weak steroid response element in both LNCaP and MCF-7 cells; another site acts as a strong steroid response element, which preferentially responds to androgen and is preferentially activated in LNCaP cells. These two new ARBSs interact in a cooperative manner with the previously described androgen response region (ARR) (defined by -244 to -96 bp) that contains ARBS-1; ARBS-2; and two lower-affinity ARR binding sites, G-1 and G-2 sites. We conclude that the context in which the ARR binding sites are present is pivotal in determining their effect on transcriptional regulation. Thus, the -705/+28 PB promoter contains a second ARR, PB enhancer element (-705/-426 PB), in addition to the first described ARR. The PB promoter creates a model that contains six AR binding sites that function in a cooperative manner for maximum androgen-regulated prostate-specific gene transcription.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE ANDROGEN RECEPTOR (AR), glucocorticoid receptor (GR), progesterone receptor, and mineralocorticoid receptor belong to the same class I nuclear receptors. These steroid receptors share a highly conserved DNA-binding domain (DBD) and can bind to an identical steroid responsive element (SRE) (1, 2, 3). A common palindrome SRE from the murine-mammary-tumor virus (MMTV) long- terminal-repeat promoter can bind AR, GR, MR, and PR to regulate steroid hormone-induced transcription by glucocorticoids, progesterones, mineralocorticoids, and androgens, respectively (2). However, the expression of genes with SREs is often regulated by only one steroid hormone in vivo (4). These observations raised the question of how the specificity of transcriptional responses to the different steroid hormones is achieved. Several mechanisms have been proposed to explain the steroid receptor specific response on the target gene transcription: the cell type-specific steroid metabolism and/or the tissue-specific expression of receptor result in receptor-specific transcriptional regulation (5, 6); the cell type-specific and/or the receptor-specific coactivators may enhance a particular type of steroid receptor activation (7, 8); and nonreceptor transcription factors that bind next to receptor-binding sites in a complex steroid hormone response unit may govern the relative responsiveness of one receptor over another (6, 9, 10, 11). Local chromatin structure may also be important. For example, the MMTV long-terminal-repeat promoter responds to all class I receptors in transient transfections; however, activation of a chromosomal integrated template may be limited to the GR (12, 13). The cooperation of multiple receptor binding sites results in receptor-specific activation (14, 15, 16, 17, 18). A steroid receptor binding to a single nonconventional response element results in a receptor- specific response (9, 14, 19, 20, 21, 22). Only recently have specific SREs been described that can distinguish AR from other steroid receptors (14, 23, 24).

The probasin (PB) promoter has been used as a model system to help explain androgen-specific regulation of gene expression (8, 13, 18, 25, 26). Deoxyribonuclease (DNase) I footprinting and transient transfection assays have identified two androgen receptor binding sites (ARBSs), ARBS-1 between positions -236 to -223 bp and ARBS-2 between positions -140 and -117 bp, within the PB 5'-flanking region (-426 to +28 bp) (14). The AR binding affinity of ARBS-2 is greater than ARBS-1; however, DNase I footprinting assays have demonstrated that both sites are always equally protected, even at the lowest concentration of AR tested (15, 27). Transfection experiments have shown that neither ARBS function independently and that deletion of the DNA sequence between the sites results in a 60% loss of androgen inducibility (15, 27). Therefore, the region required for androgen-induced regulation of PB gene expression was named as the PB androgen responsive region (ARR, -244 to -96 bp) in which the binding of AR to ARBS-1 and ARBS-2 occurred in a cooperative, mutually dependent manner (15, 27). Recently two additional low-affinity AR binding sites (G-1 located at -209 to -196 bp, G-2 located at -107 to -93 bp) with atypical half-site sequences have been identified near the ARBS-1 and ARBS-2 sites, respectively (28). Reid et al. (28) postulated that these low-affinity AR binding sites stabilize AR binding to adjacent ARBS-1 and ARBS-2 and result in synergistic transcriptional activity and increased hormone sensitivity. Cooperation between multiple AR binding sites also has been described within the distal promoter of the secretory component gene (SC) (29); the distal and proximal promoter of the prostate-specific antigen gene (PSA) (17, 28, 30); and the 120-bp DNA enhancer region of the sex-limited protein (SLP) (9, 31, 32, 33). These observations demonstrate that cooperative binding of ARs to multiple, adjacent low-affinity sites and the resulting synergistic effects on gene expression are a common mechanism for ensuring androgen specificity in the transcriptional response.

The conventional high-affinity palindromic steroid receptor binding sites do not discriminate between binding of the DNA-binding domains of the AR and the GR in vitro binding assays (21). The ARBS-2 of PB, androgen responsive element (ARE) 1.2 of SC, scARE (ARE for secretory component gene) of SC, and SRE2 of the SLP gene are slightly different from the consensus high-affinity binding sites and have been reported to bind ARs preferentially over GRs (15, 22, 27, 34, 35). Claessens et al. (36) have postulated that the AR recognizes these AR-specific binding sites as a direct repeat of two 5'-TGTTCT-3'-like core sequence instead of the classical inverted repeat.

A number of prostate-specific genes, such as prostate-specific antigen (PSA), human kallikrein, prostate-specific membrane antigen (PSMA), and prostatic binding protein C3 (1) gene, carry enhancer(s) that are distantly located from the proximal promoter (37, 38, 39, 40). For example, an androgen- responsive enhancer with a prostate-specific function has been described for the PSA gene that is located approximately 4 kb upstream of the PSA transcriptional start site (17, 38, 41, 42), in which other important transcription factors modulates androgen action (11, 43). The androgen-regulated, prostate-specific enhancer of PSMA is located within intron 3 approximately 12 kb downstream from the transcriptional start site (40). These results led us to hypothesize that optimal transcription of the PB gene, like that of many genes, might depend on the presence of more enhancer regions at some distance from the proximal promoter. Therefore, to gain a greater understanding of the PB gene regulation, we searched for additional PB gene regulatory elements. We focused on the large fragment of the PB promoter (LPB) fragment of the rat encompassing -10806 to +28 bp of the 5'-flanking sequence of the PB promoter, which acts as a strong enhancer in transgenic mice (44, 45). Here we report the identification and characterization of two nonconventional AR binding sites in an upstream androgen-dependent enhancer of the PB gene. One site functions as a weak SRE in both LNCaP and MCF-7 cells; another site acts as a strong SRE, which preferentially responds to androgen and is preferentially activated in LNCaP cells. These two new AR binding sites (ARBS-3 and ARBS-4) interact in a cooperative manner with the proximal ARR to activate transcription of a reporter gene in response to androgen treatment.

	Materials and Methods

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Plasmid construction
The -10806 to +28 bp or -426 to +28 bp 5'-flanking sequence of the rat PB sequences were fused to the chloramphenicol acetyltransferase (CAT) and simian virus-40 cleavage and polyadenylation sequences to generate -426PBCAT and LPBCAT constructs, as previously described (14, 44).

MMTV-luciferase (LUC) construct has been described previously (27). Plasmid -286PBLUC, -53PBLUC, and TKLUC, consisting of -286 to +28 bp or -53 to +28 bp of the PB 5'-flanking region, or thymidine kinase (TK) promoter sequences fused upstream of the LUC gene in pGL3-basic vector (Promega, Madison, WI) were constructed. The DNA fragments derived from -10806 to +28 bp PB sequence were generated by digesting with the appropriate restriction enzymes and cloned immediately upstream of the -286PB promoter in pGL3-basic vector. The orientation of the inserted fragments was verified by restriction enzyme digestion. The PB enhancer elements (PBEs, -705 to -426 bp), either wild-type (wt) or mutant, were cloned immediately upstream of promoters in the -286PBLUC, -53PBLUC, and TKLUC constructs. Two oligonucleotide primers were used for PCR amplification of the wt and mutant PBEs: -705 primer, 5'-GAGCTCTTTCTGGATATTTCTTGGATTATA-3'; and -426 primer, 5'-GCTAGCTTTCTGATGTTGGCACAAATGACA-3'. For PCR amplification of the wt and mutant -705 to +28 PB fragment, -705 primer and +28 primer, 5'-AAGCTTGGAGGTATCTGGACC-3' were applied. The DNA sequence of the wt and mutant ARBSs are presented in Table 1. Mutations of the AR binding sites were generated by site-directed mutagenesis as described by Chen and Przybyla (46). All final PCR products were subcloned into pGEM-T^easy vector (Promega) and verified by sequence analysis. DNA sequencing was performed (by DNA Sequencing Core Laboratory, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center) as described in the Thermo sequence fluorescent-labeled primer cycle sequencing kit (Perkin-Elmer, Norwalk, CT). One, two, or three copies of an oligonucleotide for ARBS-3 (positions at -507 to -529 bp) or ARBS-4 (positions at -554 to -577 bp) were placed in front of the TK promoter in TKLUC constructs.

Transient transfection assays by calcium phosphate-DNA coprecipitation method
Cells were plated at an initial density of 3–5 x 10⁵/100-mm dish or 2–3 x 10⁴/well in 24-well tissue culture plates and transfected by the calcium phosphate-DNA coprecipitation method as previously described (14). Briefly, the cells were exposed to the DNA precipitate for 6 h, followed by removal of the medium and the addition of steroids for 24 h. The rat androgen receptor (rAR) expression vector and the rat glucocorticoid receptor (rGR) expression vector were used in the transfection experiments. Both the rAR and rGR expression vectors use the Simian virus 40 promoter to direct expression of these transgenes. The absolute level of protein expression from each vector may vary from cell line to cell line because of the concentrations of transcription factors that may regulate the Simian virus 40 promoter, changes in stability of the mRNA, and stability of the receptor proteins. This can ultimately influence the level of induction of the reporter gene. The transfection conditions were initially optimized by cotransfecting MMTV-CAT with increasing concentrations of rAR and rGR expression vectors (27). These experiments determined that the optimal amounts of each steroid receptor expression vector were similar, and, therefore, all cotransfection experiments were performed using an equal concentration of plasmid (5 µg/ml), which was the optimal rAR and rGR concentrations for the maximal response of MMTV-CAT to dihydrotestosterone (DHT) or DEX treatment, respectively (27). The cells transfected with promoter/reporter constructs were harvested after 30 h of incubation from the beginning of the transfection for CAT or LUC activity assays. The transfection efficiency was determined by cotransfecting pRL-CMV containing the Renilla LUC reporter gene (Promega Biotech, Madison, WI). The Renilla LUC activity was determined using the dual-luciferase reporter assay system (Promega). The values plotted represent the mean of at least three individual samples ± SD.

CAT and luciferase reporter assays
The cells transfected with PBCAT plasmids were harvested after 30 h of incubation from the beginning of the transfection, and CAT activity was determined by the two-phase flour diffusion assay using [³H]acetylcoenzyme (5.9 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) as the substrate (14). The levels of PBCAT gene expression in transgenic mouse lines was compared (at least five mice per group). Tissue samples were microdissected for the determination of CAT activity. Proteins were extracted by homogenization in buffer [0.1 M Tris (pH 7.8), 0.1% Triton X-100], and protein concentrations were determined by protein assay (Bio-Rad Laboratories, Hercules, CA). For luciferase assays, cells were harvested by removing the medium, washing the cells once with PBS, and incubating with 100 µl passive lysis buffer (Promega) for 1 h at room temperature. Both firefly and Renilla LUC activity were determined in a lumicounter (LUM/star, BMG Lab Technologies, Inc., Durham, NC) by using the dual-luciferase reporter assay system (Promega).

DNase I footprinting analysis
The PBE fragment (-705 to -426 bp) was generated by digestion of LPB sequence with HindIII, cloned into the HindIII site of the plasmid Bluescript pBS-SK^+/- II (Stratagene, La Jolla, CA), and was verified by sequence analysis. DNase I footprinting analyses were performed essentially as described by Fink et al. (47). Briefly, a 10 fmol -[³²p]dCTP-labeled PBE probe was incubated with 0–3 µg of purified AR-DBD fusion protein (AR₂HisTag, which contains DNA binding domain of AR), and 0.5 µg poly (dI-dC) for 10 min on ice. The samples were subsequently removed from the ice, RQ DNase I was added, and digestion was carried out for 75 sec at room temperature. The reaction was stopped by adding 100 µl of 2 x proteinase K buffer [0.2 M Tris (pH 7.5), 25 mM EDTA, 0.3 M NaCl, 2% (wt/vol) SDS, 5 µg tRNA, and 10 U proteinase K] and incubated at 37 C for 30 min. The samples were extracted with phenol/chloroform, precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol, and redissolved in 5 µl of loading buffer [80% (vol/vol) formamide, 1 mM EDTA, 0.1 (wt/vol) xylene cyanol, and 0.1% (wt/vol) bromphenol blue]. The samples were heated at 90 C for 3 min, cooled on ice for 1 min, and electrophoresed on a urea-7% polyacrylamide gel in Tris-borate EDTA (TBE) buffer. Gels were run at room temperature in 1 x TBE and dried before being autoradiographed.

Dimethyl sulfate (DMS) methylation protection assay
Methylation protection of the PBE (-705 to -426 bp) was performed by a modified protocol (28). In brief, histidine-tagged AR-DBD (3.6 µg, 6.8 µM) was incubated at room temperature with 2.0 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and DNA binding buffer [20 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 1 mM dithiothreitol]. To each reaction 350,000 dpm (26.5 fmol) of ³²P-single-end-labeled DNA probe was added, and the binding reaction was brought to equilibrium at room temperature. To methylate the protein-bound DNA probe, DMS was added to a final concentration of 19 mM and incubated at room temperature for exactly 2 min. The methylation process was stopped by loading the reaction onto a 5% (29:1) polyacrylamide gel containing 0.5 x TBE while it was running at 16 V/cm at room temperature. DNA treated in the same manner, but without AR-DBD added, was used as a control. After separation by PAGE, bands indicating protein-bound and protein-free probes were excised and the DNA was eluted. The methylated DNA was cleaved using 1 M piperidine, and the denatured fragments were separated on a 6% (29:1) denaturing polyacrylamide gel containing 1 x TBE and 8.3 M urea. Gels were dried and autoradiographed, and the developed images were scanned using a 6300-dpi resolution scanner (Hewlett-Packard, Portland, OR). Bands were quantitated and compared using ImageQuant (5.0, Molecular Dynamics, Sunnyvale, CA).

EMSA
Oligonucleotides for EMSA were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The sense strands of ARBS-3 and ARBS-4 are given in Table 1. The probes were end-labeled with T4 polynucleotide kinase (New England Biolabs) [-³²P]ATP and purified by 15% PAGE. Binding reaction involved a 10-min incubation of 200,000 cpm of radiolabeled probe, 0.05 µg glutathione-S-transferase (GST)-AR fusion proteins (11), and buffer D (20 mM HEPES-NaOH, pH 7.9; 100 mM KCl; 0.2 mM EDTA; 1.5 mM MgCl₂; 1 mM dithiothreitol; 20% glycerol; and 1 mM phenylmethylsulfonyl fluoride), in a total volume of 20 µl. Complexes were resolved by electrophoresis for 2.5 h at 160 V on a 5% native polyacrylamide gel, which was later dried and processed for autoradiography.

	Results

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Identification of enhancer region(s) in the LPB promoter
The endogenous rat PB gene is expressed at very high levels in prostatic epithelial cells (8). The -426PB promoter directs hormonally and developmentally regulated transgene expression specifically to the prostate in transgenic mice. However, reporter gene activity is low, suggesting that -426PB promoter is missing regulatory regions necessary for maintaining high levels of PB gene expression (48). The LPB promoter linked to the CAT reporter gene conferred high levels of androgen-regulated transgene expression to the transgenic mouse prostate, indicating the existence of positive regulatory sequences upstream of -426 bp (44). At 9 wk of age, CAT activity in the anterior, dorsolateral, and ventral prostate prostate lobes of LPBCAT line I were 24-fold, 33-fold, and 41-fold higher, respectively, compared with the -426PBCAT line-4248 (Fig. 1B), which was the highest transgene-expressing line achieved with the -426PB fragment (48). In LPBCAT line VI, we also detected higher levels of CAT activity than in -426PB lines (44). These data indicate that important regulatory DNA sequences are located in the LPB fragment for maximal transgene expression. Although the endogenous PB gene is expressed at high levels in the dorsal-lateral prostate, PB DNA promoter fragments tend to target the ventral prostate, suggesting lobe-specific elements are missing.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Structure and activity of the -426PBCAT and LPBCAT constructs introduced in transgenic mice and transfected cells. A, Schematic representation of constructs -426PBCAT and LPBCAT. B, CAT activities were measured in 25 µg protein obtained from the anterior (AP), dorsolateral (DLP), and ventral prostate (VP) of 9-wk-old -426PBCAT line-4248 and LPBCAT line I transgenic mice. CAT activities are presented as the mean ± SD of five mice per group. The transcriptional activities of -426PBCAT or LPBCAT in LNCaP cells (C) and MCF-7 cells (D) were determined as described in Materials and Methods. P values were calculated for the comparisons of CAT activities of -426PBCAT and LPBCAT. (P < 0.0001 with DHT treatment and P < 0.0018 with DEX treatment in LNCaP cells; P < 0.0013 with DHT treatment and P < 0.0015 with DEX treatment in MCF-7 cells.)

To localize the enhancer-type element(s), the LPB fragment was sequenced (GenBank accession no. AY370611), and nine overlapping restriction enzyme DNA fragments (Fig. 2A) were generated and linked to a LUC reporter gene driven by the androgen-dependent PB proximal promoter (-286 to +28 bp, -286PBLUC) (Fig. 2B). These constructs would allow for the analysis of SREs that functioned cooperatively with the previously identified ARR of the probasin promoter. The -286PBLUC construct alone served as the control in these experiments for androgen induction by the proximal promoter. Thus, the -286PBLUC construct and constructs containing the LPB consecutively overlapping fragments were assayed for LUC activity in transiently transfected human prostatic carcinoma cell lines LNCaP (Fig. 2C) and PC-3 (Fig. 2D) with or without DHT treatment.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Identification of enhancer regions in the LPB sequence. A, The LPB DNA fragment examined for enhancer activity in this study is shown as a hatched box. Individual fragments, subcloned into reporter constructs, are shown as single lines labeled with the fragment position from 5' to 3' end. B, Schematic drawing of the PB proximal promoter-luciferase reporter gene constructs used to test the enhancer activity of fragments from the LPB. Two AR binding sites of -286PB are shown as an open oval circle. Transcriptional activity of reporter constructs was determined in LNCaP cells (C) and PC-3 cells (D) as described in Materials and Methods.

Because -1557/-286 PB induced the highest levels of enhancer activity in both LNCaP and PC-3 cells, it was subjected to further deletion analysis to map the positions of any putative functional enhancer elements within this fragment (Fig. 3). A series of deletion mutants of the -1557/-286 PB fragment were constructed as shown in Fig. 3A. In the presence of DHT, the -426/-286PB fragment inhibited 67% of reporter gene level in contrast to the -286PBLUC control. This observation is consistent with previous studies that this region functions as a negative motif (14, 49). In addition, the -1557/-1088 PB and -1088/-705 PB fragments only marginally increased reporter gene expression with 10^-8 M DHT treatment. However, the 280-bp fragment -705/-426 PB showed strong enhancer activity in both LNCaP and PC-3 cells with DHT treatment. The levels of LUC activity induced by -705/-426 PB were similar to those induced by -1557/-286 PB fragment (Fig. 3C), indicating that -705/-426 PB contained the core enhancer region within the larger fragment. Therefore, -705/-426 PB was designated as the PBE.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Deletion analysis of the -1557- to -286-bp enhancer region. The PB proximal promoter-luciferase reporter gene constructs used to test the enhancer activity of the -1557- to -286-bp sequences are shown as top panel (A). The hatched portions represent the tested DNA fragment; and the single line portions represent DNA sequences that were deleted from the -1557- to -286-bp fragment. Transcriptional activity of reporter constructs was determined in LNCaP cells (B) and PC-3 cells (C) as described in Materials and Methods.

PBE was subcloned 5' of the TK, -53PB, and -286PB promoters and linked to the LUC reporter gene to determine whether PBE is prostate cell specific and/or required for the androgen-specific response. These constructs were cotransfected with the rAR or rGR expression vectors into LNCaP prostate cells (Fig. 4, A, C, and E) or into the nonprostatic MCF-7 cell line (Fig. 4, B, D, and F). The induction of LUC activity in the presence of 10^-8 M DHT or 10^-8 M DEX was compared with LUC activity in the absence of hormone treatment. The addition of PBE resulted in a 68-fold increase in androgen-induced PBE/TKLUC activity and a 31-fold increase in glucocorticoid-induced activity in LNCaP cells, whereas the corresponding rise in reporter gene activity was 43-fold with androgen treatment and 27-fold with glucocorticoid treatment in MCF-7 cells (Fig. 4, A and B). These results suggest that the induction of LUC gene expression by PBE is neither prostate cell nor androgen specific. However, higher levels of both total activity and fold induction induced by PBE/TKLUC in LNCaP cells in response to steroid hormone treatment was seen, compared with the nonprostatic MCF-7 cells.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Activities of the PBE with the TK promoter, -53PB promoter, and -286PB promoter in prostatic LNCaP cells and nonprostatic MCF-7 cells. The LUC activities of the PBE/TKLUC, PBE/-53PBLUC, and PBE/-286PBLUC were determined in LNCaP cells (A, C, E) and MCF-7 cells (B, D, F) in the presence or absence of 10-8 M DHT or 10-8 M DEX as described in Materials and Methods. A nonspecific GRE/ARE reporter gene construct, MMTV-LUC, was used as a transfection assay control. P values were calculated for the comparisons of LUC activities of PBE with different promoters with DHT treatment and DEX treatment (in LNCaP cells, P < 0.0329 with TK promoter, P < 0.0003 with -53PB promoter, and P < 0.0007 with -286PB promoter; in MCF-7 cells, P < 0.0104 with TK promoter, P < 0.0238 with -53PB promoter, and P < 0.0003 with -286PB promoter). P values were calculated for the comparisons of LUC activities of PBE with different promoters in the LNCaP and MCF-7 cells (with DHT treatment, P < 0.0133 with TK promoter, P < 0.0003 with -53PB promoter, and P < 0.0002 with -286PB promoter; with DEX treatment, P < 0.0404 with TK promoter, P < 0.0031 with -53PB promoter, and P < 0.0001 with -286PB promoter).

The selectivity of PB ARR (-244 to -96 bp) as an AR-dependent, androgen-regulated enhancer has been demonstrated both in cell culture (19, 49) and the transgenic mouse model (28, 51). When PBE was placed 5' of -286PB, reporter gene expression was further enhanced by 3.8-fold (from 4514 RLU/min·µg protein to 17,000 RLU/min·µg protein) in LNCaP cells with androgen treatment (Fig. 4E). In contrast, a nonspecific glucocorticoid responsive element (GRE)/ARE reporter MMTV-LUC showed higher levels of induction to glucocorticoid than to androgen treatment (Fig. 4, E and F). These results indicate that PBE preferentially regulates reporter gene expression in response to androgen treatment in LNCaP cells. Taken together, these observations suggest that cooperation between the proximal promoter and upstream enhancer might not only enhance PB promoter activity but may also augment both androgen and cell specificity.

Identification of two ARBSs in the PBE
To identify candidate AR binding sites, DNase I footprinting was performed with the PBE fragment, using the purified AR-DBD fusion protein (AR₂HisTag). A clear protected region was observed at -529 to -507 bp, over the sequence 5'-AAGGCTAGAACCTCCAGTTCCAC-3' (Fig. 5A). The protected area contained the sequence AGAACCtccAGTTCC, which shows some homology (overall 8 of 12 bp), with the consensus sequence GGT/AACAnnnTGTTCT that is reported to give high-affinity AR binding (52). This new AR recognition site was named ARBS-3.

View larger version (47K): [in this window] [in a new window]   FIG. 5. Two AR binding sites are located within the PBE at position -507 to -529 and -577 to -554. A, DNase I footprinting analysis of the PBE region was performed using the AR-DBD fusion protein (line 1, 0 µg; line 2, 1.5 µg; line 3, 3 µg). Protected regions are marked, and their nucleotide sequence and positions are indicated. B, DMS methylation protection assays were performed as described in Materials and Methods to identify guanines involved in AR/DNA interactions. Arrows indicate half-site location and orientation. Open circles represent protected guanines residues, whereas solid circles represent guanines hypersensitive to DMS. C, EMSA shows that both ARBS-3 and ARBS-4 interact with in vitro purified GST-AR fusion proteins (AR-DBD and AR-NT/DBD).

ARBS-3 is a stronger androgen response element than ARBS-4
Androgen-regulated gene expression was abolished when either ARBS-1 or ARBS-2 were mutated in -286PB (14, 15). Although one copy of each ARBS-1 and ARBS-2 linked to TKCAT resulted in strong reporter gene activity, three copies of either ARBS-1 or ARBS-2 were ineffective in response to steroid hormone treatment (27). Clearly, neither ARBS-1 nor ARBS-2 could function as an independent androgen-responsive element. To discover whether ARBS-3 and ARBS-4 could function as an independent SRE, one, two, and three copies of a double-stranded oligomer encompassing either ARBS-3 or ARBS-4 were ligated upstream of the TKLUC reporter gene. These constructs were cotransfected into LNCaP cells with either the rAR or rGR expression vectors as described earlier. LUC gene expression increased in a dose-dependent manner, resulting in a 8.9-fold, 43.7-fold, and 91.8-fold rise in LUC activity with one, two, and three copies of ARBS-3, respectively, (Fig. 6B). The corresponding increases in transcription after DEX treatment were 7.5-fold, 25.5-fold, and 57.3-fold, respectively (Fig. 6B). These results demonstrate that ARBS-3 alone is a functional ARE and GRE, with DHT-stimulated activity consistently higher than that induced by DEX with every construct tested. This selectivity for androgen over glucocorticoid is not due to different expression levels of cotransfected AR vs. GR because a parallel MMTV-LUC reporter showed higher levels of induction to DEX treatment (Fig. 6, B and C). The same constructs were tested in MCF-7 cells (Fig. 6C); LUC gene expression increased 3.9-fold, 20.7-fold, and 44.6-fold with DHT treatment and 3.8-fold, 9.5-fold, and 19-fold with DEX treatment. Once again, ARBS-3 showed a preference for androgen, compared with glucocorticoid-induced gene expression.

View larger version (21K): [in this window] [in a new window]   FIG. 6. ARBS-3 functions as a strong SRE. One, two, and three copies of the double-stranded oligonucleotide ARBS-3 were ligated into the pGL3-basic vector in front of the TK promoter driving the LUC reporter gene (A). The LUC activities of the resulting constructs were measured in the LNCaP cells (B) and MCF-7 cells (C) as described in Materials and Methods. P values were calculated for the comparisons of LUC activities of one, two, and three copies ARBS-3 with TK promoter with DHT treatment and DEX treatment. (In LNCaP cells, P < 0.0013 one copy, P < 0.0409 two copies, and P < 0.0473 three copies; in MCF-7 cells, P < 0.0188 one copy, P < 0.0172 two copies, and P < 0.0003 three copies). P values were calculated for the comparisons of LUC activities of one, two, and three copies ARBS-3 with TK promoter in the LNCaP cells within the MCF-7 cells (with DHT treatment, P < 0.0001 one copy, P < 0.0089 two copies, and P < 0.0094 three copies; with DEX treatment, P < 0.0033 one copy, P < 0.0017 two copies, and P < 0.0043 three copies). MMTV-LUC was used as a SRE-positive control.

View larger version (23K): [in this window] [in a new window]   FIG. 7. ARBS-4 functions as a weak SRE. One, two, and three copies of the double-stranded oligonucleotide ARBS-4 were ligated into the pGL3-basic vector in front of the TK promoter driving the LUC reporter gene (A). The LUC activities of the resulting constructs were measured in the LNCaP cells (B) and MCF-7 cells (C) as described in Materials and Methods. P values were calculated for the comparisons of LUC activities of three copies ARBS-4 with TK promoter with DHT treatment and DEX treatment. (In the LNCaP cells, P < 0.0002; in the MCF-7 cells, P < 0.0025.) P values were calculated for the comparisons of LUC activities of three copies ARBS-4 with TK promoter in the LNCaP cells within the MCF-7 cells (with DHT treatment, P < 0.7218; with DEX treatment, P < 0.8137).

View larger version (28K): [in this window] [in a new window]   FIG. 8. Mutation analysis of PBE on TK promoter. The sequence of the wt and seven mutations of ARBS-3 or ARBS-4 were given in Materials and Methods (Table 1). The PBEs, which contain mutations of ARBS-3 (A), ARBS-4 (B), or double mutants of ARBS-3 and ARBS-4 (C), were ligated into the pGL3-basic vector in front of the TK promoter driving the LUC reporter gene. The transient transfection and LUC assays were performed as described in Materials and Methods. P values were calculated for the comparisons of LUC activities of mutants of ARBS-3 and mutants of ARBS-4 with their wt controls with DHT treatment (ARBS-3, P < 0.0143 mt1, P < 0.0013 mt2-mt7; ARBS-4, P < 0.3532 mt1, P < 0.0754 mt2, P < 0.0697 mt3, P < 0.0125 mt4, P < 0.0165 mt5, P < 0.0182 mt6, P < 0.014 mt7).

View larger version (25K): [in this window] [in a new window]   FIG. 9. Mutation analysis of PBE on PB promoter. The sequence of the wt and seven mutations of ARBS-3 or ARBS-4 were given in Materials and Methods (Table 1). The PBEs, which contain mutations of ARBS-3 (A), ARBS-4 (B), or double mutants of ARBS-3 and ARBS-4 (C), were ligated into the pGL3-basic vector in front of the -286PB promoter driving the LUC reporter gene. The transient transfection and LUC assays were performed as described in Materials and Methods. P values were calculated for the comparisons of LUC activities of mutants of ARBS-3 and mutants of ARBS-4 with their wt controls with DHT treatment (ARBS-3, P < 0.0147 mt1, P < 0.0003 mt2, P < 0.0002 mt3, P < 0.0002 mt4, P < 0.0003 mt5, P < 0.0003 mt6, P < 0.0034 mt7; ARBS-4, P < 0.0971 mt1, P < 0.0019 mt2, P < 0.0017 mt3, P < 0.0001 mt4, P < 0.0003 mt5, P < 0.0004 mt6, P < 0.0002 mt7).

View larger version (32K): [in this window] [in a new window]   FIG. 10. The effect of the inactivation of ARBS-1, ARBS-2, ARBS-3, and ARBS-4 on androgen regulation of the PB promoter. The sequence of the mutations of ARBS-1, ARBS-2, ARBS-3, and ARBS-4 were given in Materials and Methods (Table 1), and a diagram of -705/+28 bp PB promoter, which contains four ARBSs, is presented (A). The activity induction in response to hormone treatment was determined in LNCaP cells by comparing receptor gene activity induced by DHT or DEX to the corresponding baseline value in the absence of hormone. P values were calculated for the comparisons of LUC activities of mutants of ARBS-1, ARBS-2, ARBS-3, and ARBS-4 with wt controls with DHT treatment (ARBS-1, P < 0.0367 mt1, P < 0.0088 mt2; ARBS-2, P < 0.0011 mt1, P < 0.0006 mt2; ARBS-3, P < 0.0054 mt1, P < 0.0036 mt2; ARBS-4, P < 0.0803 mt4, P < 0.0238 mt7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The in vitro transient transfection assays have shown that in cells cotransfected with the rAR or rGR expression vectors, -426PBCAT and LPBCAT activities are induced by both DHT and DEX treatment, respectively (Fig. 1C). The ability of glucocorticoids to regulate LPBCAT and -426PBCAT transgene expression in vitro but not in vivo suggests that the activation of the integrated gene has additional specific controls that are beyond those seen in transient transfection experiments (54). Nevertheless, these results indicate AR-dependent enhancer elements are located in the LPB sequence.

Often a proximal promoter results in only low levels of gene expression, whereas optimal expression is dependent on the presence of additional regulatory regions located upstream of the proximal promoter. In the present study, we characterized a strong upstream enhancer that is required for high levels of PB gene expression and that may also contribute to both androgen-specific and prostate-specific PB gene expression. Two positive regulatory DNA fragments located at positions -9548 to -7919 bp and -1557 to -286 bp were identified by transient transfection analysis (Fig. 2). The -1557/-286 PB fragment contained a strong enhancer activity which, when further characterized, identified the PBE as residing within the smaller -705/-426 PB core region.

Although promoters can often act to promote reporter gene expression in multiple cell types, enhancer elements can modulate promoter activity by either enhancing or suppressing that activity, depending on cell type. Prostate-specific enhancers have been described for several prostate-specific expression genes, including the PSA enhancer (PSE) and the prostate-specific membrane antigen (PSMA) enhancer. The PSE has been extensively characterized (17, 37, 41, 42, 55) and is located about 4 kb upstream of the PSA transcription start site. The PSE contains multiple low-affinity, nonconsensus AR binding sites, which bind AR cooperatively and act synergistically to stimulate transcription (17). The PSE has been shown to be both necessary and sufficient for maximal and prostatic cell PSA expression (38, 42). Recently an androgen-regulated prostate-specific PSMA enhancer has been identified in intron 3 of the PSMA gene, approximately 12 kb downstream from the transcription start site. This enhancer is characterized by a 72-bp direct repeat within a 331-bp core region (40) and seems to be the key regulatory element for prostatic cell-specific expression of the PSMA gene (40). A similar organization of AR and heptocyte nuclear factor-3 binding sites in PB, PSA, and prostatic acid phosphatase regulatory regions suggest this complex is involved in maximum prostate-specific gene expression (11). Thus, these prostate-specific genes contain prostate-specific enhancer elements that direct prostate-specific gene expression.

The -705/+28 PB promoter can be divided into three regions: 1) -53/+ 28 PB contains the minimal promoter, which is weak, with no or little effect on prostate-specific expression; 2) -244/-96 PB encompasses the PB ARR, which contains the essential enhancer elements for androgen-regulated, prostate-specific PB gene expression; and 3) -705/-426 PB is the core PBE, which might confer a further level of control to induce high levels of androgen-regulated, prostate-specific PB gene expression. In each of these three fragments, binding sites for both ubiquitous and prostate-specific transcription factors may be located. However, other DNA elements are also important for PB gene transcription. Previously we reported that the -426PB promoter had weak androgen-regulated activity in prostatic cell lines (19). When the sequences from positions -426 to -287 bp were removed, a significant increase in androgen-induced activity was seen (14), suggesting that -426/-287 PB contained a negative regulatory element. However, we now demonstrate that within the context of the -705/+28 PB promoter region, very strong androgen induction can be achieved (Fig. 3B) and that the sequences between -426/-287 PB must be important for the overall activity of the PB promoter. Therefore, an enhancer/promoter complex may form that includes AR- and prostate-specific factors binding in cooperation with the ARBS sites and additional cis-acting sequences within -705/+28 PB promoter region. Further experiments are required to elucidate all the transcription factors that bind to this DNA fragment.

To investigate the role of DNA sequence to target cell specificity by the PB upstream enhancer, we compared its activity in the prostatic cell line LNCaP to the non-prostatic cell line MCF-7. As shown in Fig. 4, strong enhancer activities were detected in both LNCaP cells and MCF-7 cells when PBE was linked to the TK, -53PB and -286PB promoters. However, the absolute activities and the highest levels of induced reporter gene expression were always greater in prostatic LNCaP cells than in non-prostatic MCF-7 cells at all points tested. Although the -286PB promoter sequence is sufficient to control prostate specificity in vivo as demonstrated in transgenic mice (51), the PBE preferentially androgen-regulated reporter gene expression in LNCaP cells suggests that PBE may both enhance PB promoter activity and also augment the prostate-specific expression of the PB gene.

The two novel AR binding sites ARBS-3 (position -529 to -507 bp) and ARBS-4 (position -577 to -554 bp) within the PBE have been identified and characterized by DNase I footprinting, EMSA, and MeP (Fig. 5). The guanine nucleotides involved in AR/DNA interactions were localized by MeP. Transient transfection analysis was used to determine whether either ARBS-3 or ARBS-4 could function as an independent SRE, and of the two sites, only ARBS-3 could strongly induce hormonally regulated transcription from the heterologous TK promoter (Fig. 6). These results suggest that ARBS-3 is a strong enhancer for the hormone specificity of the PBE. A minimum of three copies of ARBS-4 were required to increase LUC activity, suggesting that this was more of a pharmacological response rather than one seen in the endogenous PB promoter, which contains only a single copy of ARBS-4 (Fig. 7). The importance of both sites was demonstrated by mutational analysis, whereas mutations in both ARBS-3 and ARBS-4 eliminated the hormonally induced response, suggesting that there is cooperation between the sites and that both sites need to be intact for full hormonal responsiveness. Furthermore, this responsiveness did not depend on promoter context because loss of enhancer activity was observed with ARBS-3 and ARBS-4 mutations, irrespective of whether they were linked to the TK or the endogenous PB promoters. Therefore, PBE containing ARBS-3 and ARBS-4 is essential for androgen regulation of the PB gene, although whether other cofactors are required for overall activity of the PBE cannot be eliminated at this time.

A hallmark of androgen-dependent gene expression appears to be the requirement of multiple AR binding sites that result in the cooperative interaction of AR to induce gene expression. For example, within the proximal PB promoter, four AR binding sites, named ARBS-1, ARBS-2, G-1, and G-2, cooperate to give rise to efficient androgen responsiveness (14, 15, 27, 28). In the PSA upstream enhancer, multiple low-affinity, nonconsensus AR binding sites bind AR cooperatively and act synergistically to stimulate PSA gene transcription (17). We have further characterized the androgen-specific response of the PB promoter and have found that in addition to the proximal ARR, there two other ARBS sites located within the PBE region. Class I nuclear receptors are known to recognize response elements that consist of a partial palindrome of two core sequences separated by a three-nucleotide spacer and to bind such sequence as homodimers in a head-to-head way (56). However, the AR can have a different mode of binding because it can recognize direct repeats of the same'5-TGTTCT-3'-like core sequence. Indeed, the ARBS-2 in the PB proximal promoter, scARE1.2 of the human secretory component gene, and the SRE2 in mouse sex-limited protein enhancer contain such AR-selective direct repeats (14, 16, 29). The structure of ARBS-3 and ARBS-4 are imperfect palindrome 5'-AGAACCtccAGTTCC-3 and 5'-AAGACTgtaTGCTCC-3', respectively. The ARBS-3 is a direct repeat supporting the theory of an AR-selective binding to direct repeat.

In context of the -705/+28PB promoter, the combination of six AR binding sites are essential for inducing the highest levels of androgen-regulated transcription and is regulated preferentially in cells of prostatic origin. These data reflect that observed in vivo, in which in transgenic mice, when all of the characterized AR binding sites were present, LPBCAT gene expression was the highest, compared with -426PBCAT, which contained only the proximal ARR (44). Furthermore, transgene expression occurred only in the luminal epithelial cells, and androgen-specificity was clearly established because no functional GRE was detected within the LPB promoter (44).

From these findings we conclude that the context in which the AR binding sites are present is pivotal in determining their effect on transcriptional regulation. Thus, the -705/+28 PB promoter contains a second ARR, PBE (-705/-426 PB) in addition to the first described ARR -244/-96 PB), with G-1 and G-2 sites. This creates a model for PB that contains six AR binding sites that cooperate for maximum androgen-regulated gene transcription. The -705/+28PB promoter provide a powerful tool for promoting gene expression in prostate epithelial cells for both biological investigation and potential therapeutic intervention.

	Acknowledgments

 
The authors thank Mr. Tom Case and Mr. Manik Paul for their technical assistance.

	Footnotes

 
This work was supported by R01-DK-55748 from the NIH and the Frances Williams Preston Laboratories of the T. J. Martell Foundation.

Abbreviations: AR, Androgen receptor; ARBS, AR binding site; ARE, androgen responsive element; ARR, androgen response region; CAT, chloramphenicol acetyltransferase; DBD, DNA-binding domain; DEX, dexamethasone; DHT, dihydrotestosterone; DMS, dimethyl sulfate; DNase, deoxyribonuclease; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; GST, glutathione-S-transferase; LPB, large fragment of the PB promoter; LUC, luciferase; MeP, methylation protection assay; MMTV, murine-mammary-tumor virus; PB, probasin; PBE, PB enhancer element; PSA, prostate-specific antigen; PSE, PSA enhancer; PSMA, prostate-specific membrane antigen; rAR, rat AR; rGR, rat GR; SC, secretory component gene; scARE, ARE for secretory component gene; SLP, sex-limited protein; SRE, steroid responsive element; TBE, Tris-borate EDTA; TK, thymidine kinase; wt, wild-type.

Received May 7, 2003.

Accepted for publication September 10, 2003.

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