Multiple Androgen Response Elements and a Myc Consensus Site in the Androgen Receptor (AR) Coding Region Are Involved in Androgen-Mediated Up-Regulation of AR Messenger RNA

Jennifer M. Grad, Jia Le Dai1, Shu Wu2 and Kerry L. Burnstein

Department of Molecular and Cellular Pharmacology University of Miami School of Medicine Miami, Florida 33101


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgen receptors (ARs) are ligand-activated transcription factors that belong to the steroid/thyroid hormone receptor gene superfamily (reviewed in Refs. 1, 2, 3). Androgen-mediated gene regulation typically occurs through AR interaction with specific sequences within androgen target genes termed androgen response elements (AREs). The location, sequence, and number of AREs associated with a given androgen target gene varies (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17), although androgen-responsive regions typically contain multiple nonconsensus AREs (7, 15, 16, 17). AREs have been identified in regions proximal to the target gene promoter, several kilobases upstream from the promoter, in introns, and, in at least two cases, exons (4, 5, 6, 7, 8, 9, 10, 11, 12). Additionally, several kilobases separate individual AREs in some androgen-regulated genes. For example, the androgen-regulated prostate-specific antigen (PSA) gene contains multiple upstream AREs that are separated by approximately 4.5 kb (11, 15). Some genes contain AREs that form part of a complex regulatory region containing binding sites for other transcription factors. Regulatory regions of the rat 20-kDa protein gene, the rat C3(1) gene of the prostatic binding protein, the mouse sex-limited protein (Slp) gene, and the human prostatic-specific kallikrein gene contain other regulatory sequences in addition to multiple AREs (7, 8, 10, 16, 17, 18, 19, 20, 21).

Androgens regulate the expression of a variety of genes including the AR gene itself (autoregulation) (22, 23, 24). Androgen treatment of castrated animals or treatment of AR-containing cell lines results in tissue- and cell-specific AR mRNA autoregulation. In many tissues and cell lines, androgen promotes down-regulation of AR mRNA; however, androgen-mediated up-regulation occurs in several cell lines and tissues (24, 25, 26, 27, 28). The mechanism of AR mRNA autoregulation was studied in detail in the human prostate cancer cell line, LNCaP (29). Nuclear run-on assays showed that down-regulation of AR mRNA is due to decreased AR mRNA transcription (29, 30). However, studies utilizing several cell lines failed to identify AREs in the AR gene promoter and up to 7 kb of upstream sequences (30, 31, 32). Analysis of androgen regulation of AR promoter activity in U2OS, an osteosarcoma cell line that exhibits androgen-mediated AR mRNA up-regulation (3- to 4-fold), showed only a very slight induction (<2-fold) of reporter gene activity (24). Thus, the promoter and upstream regions of the AR gene appear to lack AREs sufficient to account for AR mRNA autoregulation.

We have previously shown that AR mRNA autoregulation is reproduced in cells expressing a human AR cDNA and that this response is due to AREs within the cDNA (4, 33, 34). Androgen-mediated down-regulation of the AR cDNA-encoded mRNA occurs in certain transfected cell lines including COS 1 and LNCaP (34). In contrast, androgenic up-regulation of cDNA-encoded AR mRNA occurs in the human prostate cancer cell lines PC3 and DU145 (33). This androgen-mediated up-regulation of AR mRNA is transcriptional and cell specific (Refs. 4, 33 and our unpublished data). Our laboratory has focused on understanding the molecular determinants of androgen-mediated up-regulation of AR mRNA. We found that a 350-bp fragment of the human AR cDNA confers full androgen-induction of a reporter gene in PC3 and DU145 cells. Two AREs, ARE-1 and ARE-2, were identified as essential for maximal androgen inducibility of the 350-bp region (4).

In the present study, we show that ARE-1 and ARE-2 are required for androgen-mediated up-regulation of AR mRNA encoded by the cDNA. An AR genomic fragment of approximately 6.5 kb containing ARE-1 and ARE-2, like the 350-bp cDNA fragment, was androgen regulated, indicating that these AREs function in the context of the native AR gene. In vitro binding studies with PC3 cell nuclear extracts and an AR fusion protein coupled with reporter gene assays showed that two additional AREs and a Myc site are required for AR binding and regulation of the 350-bp region. The requirements for the Myc consensus site and functional Myc and Max proteins for maximal androgen regulation of the 350-bp region suggests novel cross-talk between AR and a Myc-Max heterodimer in the autoregulation of AR mRNA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Silent Mutation of ARE-1 and ARE-2 in the AR cDNA Eliminates Androgen Regulation of AR mRNA
To determine the role of ARE-1 and ARE-2 in androgenic up-regulation of AR mRNA encoded by the AR cDNA, we created silent mutations of ARE-1 and ARE-2 in the context of the entire AR cDNA. Nucleotides were chosen for mutagenesis that would destroy each ARE without altering the amino acid sequence of the resulting AR protein (35). The mutant AR cDNA bearing these silent mutations was cloned into the identical CMV promoter-driven cDNA expression vector used to express the wild-type AR and termed CMVhARmut. CMVhARmut encodes a functional receptor as assessed by reporter gene assays using mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) (Fig. 1AGo). ARs encoded by the wild-type expression vector, CMVhAR, and CMVhARmut transactivate MMTV-CAT in an R1881 dose-responsive manner (Fig. 1AGo). RNAse protection assays showed that, unlike the wild-type AR expression vector, androgen treatment of PC3 cells transiently expressing CMVhARmut did not result in up-regulation of AR mRNA (Fig. 1BGo). Basal levels of AR mRNA expression were not affected by mutation of ARE-1 and ARE-2 (compare lanes CMVhAR and CMVhARmut in the absence (-) of R1881). Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in PC3 cells expressing the AR cDNA.



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Figure 1. ARE-1 and ARE-2 Are Required for Androgen-Mediated Up-Regulation of AR mRNA

Panel A, PC3 cells were transfected with MMTV-CAT, CMVß-gal, and either the wild-type AR expression vector, CMVhAR, or an AR cDNA bearing silent mutations of ARE-1 and ARE-2, CMVhARmut. Cells were cultured in the presence or absence of R1881 for 40 h. CAT assays were performed using cell extracts containing equivalent amounts of ß-galactosidase activity. Panel B, PC3 cells were transfected with CMVhAR or CMVhARmut. Twenty four hours after transfection, cells were cultured for 20 h in the presence (+) or absence (-) of R1881. RNase protection assays were performed to measure AR mRNA and GAPDH mRNA levels. Radiolabeled antisense RNA specifically protected by AR mRNA and GAPDH mRNA were 200 nucleotides and 110 nucleotides, respectively. The experiments were performed twice and the results were the same.

 
The finding that MMTV-CAT induction is equivalent in cells expressing the wild-type or silent mutant AR cDNA suggests that sufficient levels of AR are produced by the transfected mutant cDNA for target gene transactivation. Androgen-mediated up-regulation of AR protein occurred in PC3 cells expressing the silent mutant AR (data not shown) and is likely due to posttranscriptional stabilization of AR by ligand binding (36).

ARE-1 and ARE-2 Are Functional in the Context of a 6.5-kb Genomic Fragment of the AR Gene
ARE-1 and ARE-2 are located in exons D and E, respectively, and are separated by intron 4 in the AR gene (Fig. 2Go). To determine whether these exonic regulatory elements function in their native arrangement, we cloned the genomic fragment of the AR gene consisting of exon D, intron 4, and exon E into the thymidine kinase (tk) promoter-driven reporter plasmid pBLCAT2. This 6.5-kb fragment was obtained using genomic DNA as a template and long-range PCR with primers targeting the 5'-end of exon D and the 3'-end of exon E of the human AR gene. A single band of approximately 6.5 kb was detected and subsequently cloned into pBLCAT2 upstream of the tk promoter (data not shown). To confirm that the 6.5-kb genomic fragment contains the appropriate region of the AR gene, we sequenced approximately 0.7 kb on each end. The exonic sequences, intron/exon splice sites, and the first 50 bp of each end of intron 4 were identical to those sequences reported by Lubahn et al. (37).



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Figure 2. Location of ARE-1 and ARE-2 in the Human AR Gene

The hatched rectangles and straight lines represent AR gene exons and introns, respectively. ARE-1 and ARE-2 are noted with asterisks. The 6.5-kb genomic androgen-responsive region contains exon D, intron 4, and exon E. This 6.5-kb fragment was inserted upstream of the tk promoter in the reporter plasmid pBLCAT2, resulting in I4-CAT.

 
The reporter plasmid containing this 6.5-kb AR genomic fragment (I4-CAT) is androgen regulated in PC3 and an osteosarcoma cell line U2OS (Fig. 3Go), but not in the human prostate cancer cell line LNCaP (data not shown). Regulation of I4-CAT is comparable to that of the reporter plasmid containing the 350-bp cDNA fragment, 350WT-CAT (~8 fold; P > 0.05), suggesting that ARE-1 and ARE-2 can function in their genomic arrangement. Furthermore, I4-CAT exhibits the same cell specificity as 350WT-CAT (Fig. 3Go and data not shown). This cell specificity is significant because, as we have shown, androgen treatment results in the up-regulation of AR mRNA encoded by a wild- type AR cDNA in PC3 cells (Fig. 1BGo and Ref. 33) and up-regulation of native AR mRNA in U2OS cells (Ref. 24 and J. Grad and K. L. Burnstein, unpublished data). In contrast, both the native (23, 29, 30) and cDNA-encoded AR mRNAs are down-regulated in LNCaP cells (34). Therefore, these findings are consistent with a role for the exonic elements ARE-1 and ARE-2 in cell-specific, androgen-mediated up-regulation of native AR mRNA.



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Figure 3. Androgen Regulation of the 6.5-kb AR Genomic and 350-bp AR cDNA Fragments Containing ARE-1 and ARE-2

PC3 and U2OS cells were cotransfected with CMVhAR, CMVß-gal, and 350WT-CAT or I4-CAT and cultured for 40 h in the absence (-) or presence (+) of R1881 (50 nM). 350WT-CAT contains the 350-bp AR cDNA fragment, and I4-CAT contains the 6.5-kb genomic AR fragment, both ligated upstream of the tk promoter in the parent vector pBLCAT2. CAT assays were performed using cell extracts containing equivalent amounts of ß-galactosidase activities. Representative experiments (three for PC3 and five for U2OS) are shown.

 
DNAse I Footprinting of the 350-bp Androgen-Responsive Region Reveals an Additional AR Binding Site between ARE-1 and ARE-2
We previously demonstrated by gel shift assay that an AR fusion protein consisting of the human AR DNA and steroid-binding domains fused to glutathione-S-transferase (GST-AR) expressed in E. coli binds specifically to the 350-bp fragment (4). To map potential sequences within the 350-bp region involved in contacting AR, we performed DNAse I footprinting studies using the amino terminal-deleted AR, produced by thrombin cleavage of GST-AR (Fig. 4Go). These experiments revealed the presence of three AR-binding sites encompassing ARE-1, ARE-2, and a region between these AREs termed intervening sequence (IVS) (Fig. 5AGo and 5BGo, lanes labeled "350WT"). AR binding to these regions was examined by using 350-bp probes labeled at the upstream HindIII site (to visualize ARE-1 and IVS, Fig. 5AGo) and at the downstream XbaI site (to visualize ARE-2, IVS, and ARE-1, Fig. 5BGo). The first pair of lanes in each panel shows the footprints obtained with a wild-type 350-bp probe (350WT). To assess the contribution of ARE-1 and ARE-2 to AR binding, we analyzed DNAse I footprinting of 350-bp fragments harboring mutations at either ARE-1 (mARE-1) or ARE-2 (mARE-2). Mutation of ARE-1 (lanes marked mARE-1) resulted in no footprint at the region encompassing ARE-1 and substantially decreased (50–90%) AR binding to both ARE-2 and IVS (Fig. 5Go, A and B; hatched lines mark the corresponding regions protected on 350WT). Similarly, mutation of ARE-2 (mARE-2) resulted in loss of the footprint at the region encompassing ARE-2 and decreased (50–75%) footprinting at ARE-1 and IVS (Fig. 5Go, A and B; hatched lines mark the corresponding regions protected on 350WT probe). The same results were obtained when a GST-AR fusion protein was used in footprinting analysis of wild-type and mutated 350-bp fragments (data not shown). The end-labeled IVS region alone is not bound by GST-AR (Fig. 5CGo; hatched lines show the corresponding region protected on the wild-type fragment) suggesting that AR binding to the IVS region requires the presence of ARE-1 and ARE-2. The results of the DNAse I footprinting experiments are summarized in Fig. 6Go.



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Figure 4. Isolation of an Amino Terminal-Deleted AR by Thrombin Cleavage of GST-AR

Coomassie-stained gel of GST-AR (DNA- and ligand-binding domains) fusion protein, and amino terminal-deleted AR produced by thrombin cleavage is shown. GST-AR bound to glutathione Sepharose 4B was incubated in the absence (-) or presence (+) of thrombin as described in Materials and Methods. AR protein was recovered in the flowthrough. Size markers are indicated (M). GST-AR is a protein of approximately 78 kDa and the amino terminal-deleted AR is about 52 kDa. The protein of about 30 kDa is probably GST. A contaminating Escherichia coli protein (non-spec) of approximately 64 kDa was frequently present. Preparations of GST-AR chosen for use without further purification contained a higher amount of GST-AR relative to contaminating proteins.

 


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Figure 5. Mutation of Individual AREs Decreases AR Binding at All Other AREs

DNAse I footprinting studies were performed with the wild-type 350-bp region (350WT), 350-bp fragments with mutations at ARE-1 (mARE-1) or ARE-2 (mARE-2), and the intervening region of the AR cDNA between ARE-1 and ARE-2 (IVS). The designated fragments (350WT, mARE-1, mARE-2) were end labeled, incubated with BSA or an amino terminal-deleted AR (consisting of the DNA-binding domain and ligand-binding domains) and subjected to in vitro DNAse I footprinting. The IVS fragment was incubated with GST-AR or GST. Protected regions (footprints) detected using the 350WT probe are designated by solid vertical lines. When mutant probes (mARE-1, mARE-2) were used, the regions corresponding to footprints on 350WT are designated by hatched lines. Data were quantified by phosphorimage analysis of dried gels. The amount of radioactivity in each protected region in the AR lanes was compared with the analogous region in the BSA or GST control lane and normalized to a region outside the footprint. A, The 350-bp fragments (350WT, mARE-1, and mARE-2) were end labeled at artificially generated 5'-HindIII sites to view footprints over ARE-1 and IVS. B, The 350-bp fragments (350WT, mARE-1, and mARE-2) were end labeled at the 3'-XbaI site to view footprints over ARE-2 and IVS. C, No binding of GST-AR to the IVS was observed.

 


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Figure 6. Summary of AR Footprinting of the 350-bp AR cDNA Fragment

DNA sequences protected from DNAse I digestion are shown in boldface and AREs are double underlined.Putative AREs are single underlined. The footprint located between ARE-1 and ARE-2 is designated IVS. Numbering is based on the human AR cDNA (58 ) with +1 located 42 bp 5' to the translation start site.

 
DNAse I footprinting results suggest the presence of AREs in addition to ARE-1 and ARE-2. The IVS binding site contains a sequence with similarity to the consensus ARE [(5-GAGACAgctTGTACA-3') in which the consensus nucleotides (35) are underlined]. The consensus ARE is a bipartite, partially palindromic element with a three-nucleotide spacer. However, binding of AR to the IVS region requires ARE-1 and ARE-2, as the IVS alone is not protected by GST-AR (Fig. 5CGo). Furthermore, as shown in Fig. 1BGo, mutations of ARE-1 and ARE-2 in the AR cDNA are sufficient to abrogate androgen regulation of AR mRNA encoded by the mutated cDNA, suggesting that an ARE in the IVS cannot function independently. The DNAse I footprinting experiments described above showed that the ARE-1 footprint is approximately 57 bp, which is larger than other reported AR binding sites (e.g. Refs. 12, 13). Thus, an additional ARE may be located near ARE-1 and contribute to the large protected region. A 15-bp sequence with homology to the ARE consensus is located within the ARE-1 footprint, [(5-GGTGTAgtgTGTGCT-3'); consensus nucleotides are underlined (35)]. Although neither of these putative AREs possess the well conserved "right half" sequence, TGTYCT, found in the majority of AREs, the two elements are fairly similar to the consensus bipartite ARE (8 of 12 nucleotides) (35).

Two Additional AREs Contribute to AR Binding and Androgen Regulation of the 350-bp Androgen-Responsive Region
To determine the contributions of these putative AREs to androgen regulation of the 350-bp AR cDNA fragment (350WT-CAT), we introduced mutations in each putative response element in the 350-bp region. A mutation was also made outside the footprinted areas as a mutagenesis control (mc350). These 350-bp fragments, mARE-1A (mutation in the putative ARE near ARE-1), mIVSARE (mutation in the element within the IVS), and mc350, were inserted upstream of the tk promoter in the reporter plasmid pBLCAT2. The resulting reporter plasmids, mARE1A-CAT and mIVSARE-CAT, displayed a substantial reduction of androgen inducibility (80% and 87% decrease, respectively) compared with the 350WT-CAT and mc350-CAT (Fig. 7AGo). These data suggest that both ARE-1A and IVSARE are bona fide AREs and are required for maximal androgen regulation of the 350-bp region.



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Figure 7. Two Additional AREs (ARE-1A and IVSARE) Are Required for Maximal Androgen Regulation and AR Binding of the 350-bp Androgen-Responsive Region

Panel A, PC3 cells were cotransfected with CMVhAR, the indicated CAT construct containing the 350-bp androgen-responsive region (350WT), ARE-mutant (mIVSARE, mARE-1A) or mutagenesis control (mc350) and CMVß-gal. Cells were cultured in the presence (+) or absence (-) of R1881 (50 nM) for 40 h. CAT assays were performed using cell extracts containing equivalent amounts of ß-galactosidase activities. Representative autoradiograms are shown. Fold induction is the ratio of CAT activity observed in the presence and absence of R1881 treatment. Each bar represents the mean ± SEM of three to four experiments. Panel B, Gel shift assays were performed as described in Materials and Methods using a purified GST-AR fusion protein (500 ng) and radiolabeled 350WT fragment in the presence of increasing amounts (0-, 50-, 100-, or 250-fold molar excess) of unlabeled wild-type or mutant 350-bp fragments. Purified GST is included as a control. The free probe and AR-DNA complexes are indicated in a representative autoradiogram. Radioactivity in DNA-protein complexes was quantified by phosphorimage analysis and the percent of GST-AR binding (binding in presence of competitor/binding in the absence of competitor) plotted in the lower portion of panel B. A comparably sized fragment of the AR cDNA that does not contain AREs served as a nonspecific competitor. In addition, a 350-bp fragment harboring a mutation in sequences adjacent to the IVSARE footprint (mutagenesis control, mc350) was tested. Each point represents the mean ± SEM of three independent determinations for each concentration of the indicated competitor, except for the nonspecific and mc350 competitors, which were performed once.

 
The effects of mutating these AREs (ARE-1A and IVSARE) on AR binding were evaluated by gel shift assay using radiolabeled 350WT and increasing amounts of unlabeled mutated 350-bp fragments as competitors. The 350-bp fragments containing mutations in ARE-1A or IVSARE were less effective competitors of AR-350 bp complex formation than the wild-type fragment (Fig. 7BGo). A nonspecific competitor (a comparably sized portion of the AR cDNA that lacks AREs) acts as a very poor competitor (Fig. 7BGo). Although both of the mutated 350-bp fragments were weaker competitors than 350WT, there were subtle differences between the ability of each mutant fragment to compete for AR binding (e.g. mARE-1A vs. mIVSARE), suggesting that the AREs are not bound equally by the recombinant AR. Although these studies do not provide quantitation of the affinity of AR binding to each ARE, they do illustrate that mutation of either ARE decreases DNA-AR interaction. Together, these studies and our previously published results (4) demonstrate that ARE-1, ARE-2, ARE1-A, and IVSARE each contribute to maximal AR protein binding and androgen regulation of the 350-bp region.

A Consensus Site for Myc Is Required for Maximal AR Binding and Androgen Regulation of the 350-bp Region
Since auxiliary transcription factors contribute to the regulation of many steroid target genes, we performed DNAse I footprinting experiments with nuclear extracts from PC3 cells to determine whether non-AR transcription factors interact with the 350-bp region. These studies revealed subtle protection of a region that contains a perfect consensus site for a variety of transcription factors (CACGTG; E box), including the protooncogene Myc and the related proteins Max, Mad, and Mxi (reviewed in Refs. 38, 39) (Fig. 8AGo). The E box overlaps with the IVSARE by 2 bp. To determine whether PC3 nuclear protein complexes bind specifically to this Myc consensus sequence, we performed additional DNAse I footprinting experiments with a 350-bp fragment in which the Myc consensus site was mutated. No footprint was observed over the mutated Myc site (Fig. 8BGo). To confirm the specificity of the DNA-nuclear protein interaction, gel shift analyses were performed using 350WT as a probe and unlabeled oligonucleotides containing a consensus Myc site or mutated Myc site (CAGCTG; bolded nucleotides were mutated) as competitors. These studies identified two DNA-protein complexes that were specifically competed by the Myc consensus site oligonucleotide, but not by the mutated oligonucleotide (Fig. 8CGo). Thus, proteins present in PC3 cell nuclear extracts specifically recognized the Myc consensus sequences in the 350-bp region. To determine the contributions of the Myc consensus site to androgen regulation of the 350-bp region and AR binding to this fragment, the site was mutated in the context of the 350-bp fragment without disrupting the IVSARE. The resulting plasmid, mMyc-CAT, displayed decreased androgen inducibility (89% decrease) compared with 350WT-CAT in PC3 cells (Fig. 9AGo). Similar to fragments containing a mutated ARE, the mMyc 350-bp fragment was also a less effective competitor than 350WT for binding to GST-AR in gel shift assays (Fig. 9BGo). It is important to note that all four core ARE sequences are intact in the mMyc-CAT construct. Similarly, the minimal Myc sequences are intact in the mIVSARE-CAT construct.



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Figure 8. DNAse I Footprinting and Gel Shift Analysis Reveal the Presence of a Consensus Binding Site for the Myc Family of Protooncogenes within the 350-bp Androgen-Responsive Region

A, DNAse I footprinting of the 350-bp fragment (end-labeled at the artificially generated 5'-HindIII site) was performed using nuclear extracts from PC3 cells (20 µg). Nuclear extract buffer control (equal amount of heat-inactivated nuclear extract), GST, and GST-AR lanes are shown for comparison. M13 primers were included as size markers. The thin vertical line in the GST-AR lane indicates the IVSARE footprint. The thick bar in the PC3 nuclear extract lane (PC3 NE) marks the region protected from DNAse digestion, which contains a consensus site for the Myc family of protooncogenes. The Myc consensus sequences are underlined, and regions that overlap with the GST-AR-protected IVSARE are boldface. B, DNAse I footprinting of the 350WT fragment and a 350-bp region with a mutated Myc site (mMyc) (end-labeled at the artificially generated 3'-XbaI site) was performed using nuclear extracts from PC3 cells (10 µg and 20 µg). M13 primers were used as size markers but are not shown. Nuclear extract buffer control (20 µg of heat-inactivated nuclear extract) is shown for comparison. The region indicated by a bracket corresponds to the same protected region observed in panel A. C, Gel shift assays were performed as described in Materials and Methods using nuclear extracts from PC3 cells (5 µg) and radiolabeled 350WT fragment in the presence or absence of 25- and 50-fold molar excess of the indicated unlabeled oligonucleotide competitors [Myc binding site oligo (WT) or a mutated Myc binding site oligo (mutant)]. Protein-DNA complexes that were specifically competed away are indicated by arrows.

 


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Figure 9. A Myc Consensus Site Is Required for Maximal Androgen Regulation and Androgen Binding of the 350-bp Androgen-Responsive Region

A, PC3 cells were cotransfected with CMVhAR, the indicated CAT construct [350WT (wild type) or mMyc (mutated Myc)] and CMVß-gal and treated for 40 h with 50 nM R1881. CAT assays were performed using samples containing equivalent amounts of ß-galactosidase activities. Fold induction is the ratio of CAT activity observed in the presence and absence of R1881 treatment (50 nM). Each bar represents the mean ± SEM of three to four experiments. B, Gel shift assays using purified GST-AR fusion proteins in the presence of increasing amounts of unlabeled wild-type or mutant Myc fragments were performed as described in Materials and Methods. The gels were quantitated by phosphorimage analysis, and the percent of total binding (GST-AR binding in presence of competitor/binding in absence of competitor) was determined. Nonspecific DNA competitor and the mc350-bp fragment (mutagenesis control) are included. Each point represents the mean ± SEM of three experiments except for experiments testing the nonspecific and mutagenesis control (mc) fragments, which were conducted once and shown in Fig. 7BGo.

 
To determine whether a functional Myc/Max heterodimer contributes to androgen regulation of the 350-bp androgen-responsive region, we used expression vectors encoding dominant negative Myc and dominant negative Max (Ref. 40 ; generously provided by Dr. B. Amati). A transcriptionally active Myc complex is a heterodimer comprised of Myc and its binding partner Max. Myc and Max proteins interact via a basic helix-loop-helix leucine zipper (HLH-LZ) domain. Dominant negative mutants of Myc (MycRx) and Max (MaxRx) were created by reciprocal exchange of the HLH-LZ domains of Myc and Max (40). Thus, the MycRx mutant contains the Max HLH-LZ domain and binds tightly to wild-type Myc. The resulting MycRx-Myc complex does not bind DNA and is inactive (40). MycRx thereby prevents wild-type Myc from forming functional complexes with wild-type Max. The MaxRx mutant contains a Myc HLH-LZ domain and forms nonfunctional complexes with wild-type Max. The presence of MaxRx results in the titration of wild-type Max via the formation of stable but transcriptionally inactive MaxRx-Max heterodimers (40). When MycRx and MaxRx mutants are introduced together, MycRx-MaxRx heterodimers can be formed and these heterodimers are functional (40).

We found that introduction of either MycRx or MaxRx (1.0 µg per 60-mm dish) abrogates androgen regulation of reporter plasmids containing either the 350-bp cDNA or 6.5-kb genomic fragment-containing reporter plasmids (Fig. 10Go, A and B). The same results were obtained when 0.5 µg of dominant negative plasmids was used (data not shown). Androgen regulation was restored when MycRx and MaxRx were transfected together. These data suggest that functional Myc and Max are required for androgen regulation of these responsive regions. To test the possibility that dominant negative Myc or Max influences the regulation of other androgen-responsive regions, we examined androgen regulation of MMTV-CAT in cells cotransfected with wild-type Myc and Max, as well as the dominant negative expression vectors, MycRx and MaxRx. None of these vectors had any effect on androgen-mediated transactivation of MMTV-CAT (Fig. 10CGo). Androgen regulation of a 3xHRE-CAT construct was also not affected by introduction of these expression vectors (data not shown). These studies suggest that Myc and Max (possibly as a heterodimer) are involved in androgen regulation of the 350-bp and 6.5-kb regions.



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Figure 10. Dominant Negative Myc or Max Blocks Androgen Regulation of the Androgen-Responsive Regions

Panel A, PC3 cells were cotransfected with CMVhAR, 350WT-CAT, CMVß-gal, and 1.0 µg of the indicated plasmid [parent vector (pBJ3), dominant negative Myc (MycRx), or dominant negative Max (MaxRx)]. Cells were treated for 40 h with 50 nM R1881. CAT assays were performed using samples containing equivalent amounts of ß-galactosidase activities. Fold induction is defined as the ratio of CAT activity observed in the presence and absence of R1881 treatment. Each bar represents the mean ± SEM of three to four experiments. Panel B, The experiment was conducted as described in panel A except I4-CAT was used in place of 350WT-CAT. A histogram of data from three independent experiments is shown. Panel C, The experiment was conducted as described in panel A except MMTV-CAT was tested in place of 350WT-CAT. A histogram of data from two independent experiments is shown.

 
To determine whether Myc and Max bind to the 350-bp region, we tested the ability of anti-Max and anti-Myc antibodies to alter the mobility of protein-DNA complexes in gel shift experiments (supershift). Although inclusion of anti-Max antibodies did result in a supershifted band, we failed to detect any up-shifted bands using several different anti-Myc antibodies (data not shown). Since it is possible that the available anti-Myc antibodies recognize denatured Myc protein but not the native form, we used gel shift assays coupled with Western blotting to determine whether Myc and Max bind to the 350-bp region. For these studies, we conducted gel shift assays using PC3 cell nuclear extracts and radiolabeled 350WT fragment. The DNA-protein complexes and the indicated regions of the gel were excised and subjected to SDS-PAGE and immunoblotting. Anti-Max antibodies specifically detected a protein with a similar molecular mass as Max (~22-kDa); and anti-Myc antibodies specifically detected a protein with a similar size to Myc (~62 kDa) (Fig. 11Go). Although the gel was UV cross-linked, only free Myc or Max protein was detected, as any Myc/Max-DNA complexes would have migrated more slowly. These experiments show that Myc and Max bind to the 350-bp region.



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Figure 11. Myc and Max Bind to the 350-bp DNA Fragment

Preparative gel shifts (100 µg of PC3 nuclear extracts) were conducted as described in Materials and Methods. Note that the gels were run longer to further separate and distinguish the nuclear protein-DNA complexes (duplicate preparative gels were run; a representative autoradiogram is shown). The 5% nondenaturing gels were exposed to x-ray film, and DNA-protein complexes were visualized by autoradiography of the wet gel (not shown). The bands corresponding to those specifically competed by Myc oligonucleotides (Fig. 8CGo) as well as the indicated additional bands were excised and subjected to SDS-PAGE, transferred to nitrocellulose, and probed with anti-Max or anti-Myc antibodies. A single 22-kDa band (Max) was detected by the anti-Max antibody; the anti-Myc antibody detected a 62-kDa band (Myc).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The overall goal of this work is to identify the relevant regulatory sequences involved in androgen-mediated up-regulation of AR mRNA. Since AREs have not been identified in the AR gene promoter and upstream flanking region (30, 31, 32), and because autoregulatory elements for both glucocorticoid and estrogen receptors are present in the cognate receptor gene-coding region (41, 42), we investigated the role of AR gene exonic sequences in the up-regulation of AR mRNA induced by androgen. We previously reported that the AR cDNA contains AREs and is sufficient for the cell-specific up-regulation of AR mRNA (4, 33). Here we focused on identifying the specific exonic sequences, both AREs and non-AR transcription factor-binding sites, that contribute to androgen-mediated up-regulation of AR mRNA. We identified four AREs and a Myc consensus site in the AR coding region that are involved in AR binding and androgen-mediated up-regulation of AR mRNA encoded by the AR cDNA. It is of interest that these AREs and Myc site appear to be well conserved (0 or 1 mismatches) among a number of species from rodents and monkeys to birds and frogs (data not shown). While it is difficult to interpret the significance of this conservation given the exonic location of these sequences, conservation of these sequences is consistent with a role for these regulatory elements in autoregulation of AR mRNA.

One caveat of the AR binding studies with the 350-bp AR cDNA fragment is that the AREs are not in their native arrangement, where three AREs are present on exon D and one ARE is present on exon E. Thus, the effects of mutating one ARE on AR binding to adjacent AREs may not extend to the native gene. However, since the AR genomic fragment encompassing these elements confers androgen inducibility to a reporter gene, the exonic elements are functional when separated by intron 4. Furthermore, the 6.5-kb genomic and 350-bp cDNA androgen-responsive regions show the same cell specificity: both regions are androgen regulated in U2OS and PC3 cells (Fig. 3Go), but not in LNCaP cells (data not shown). These findings are significant because U2OS and PC3 cells exhibit androgen-mediated up-regulation of AR mRNA (24, 33), whereas LNCaP cells exhibit androgen-mediated down-regulation of AR mRNA (23, 29, 30). Thus, these androgen-responsive regions maintain the cell specificity that is predicted for elements involved in the up-regulation of the native AR gene.

The androgen-responsive region of the AR gene contains multiple, nonconsensus AREs located in two exons separated by an intron of approximately 6.2 kb. Multiple, dispersed steroid-response elements appear to be a characteristic of many steroid-regulated genes. In several steroid hormone regulated genes, response elements are located several kilobases from each other and from the target gene promoter. For example, estrogen regulation of the rat progesterone receptor (PR) gene occurs via multiple, synergistic estrogen response elements (EREs) dispersed over more than 4 kb of the rat PR gene (43). Several of these EREs are not estrogen regulated individually, but require the presence of the other EREs (43). Maximal androgen regulation of the PSA gene requires two distinct ARE-containing regions, one proximal to the promoter and one about 4.2 kb upstream from the proximal regulatory region (11, 15, 44). Thus, our finding that multiple AREs contained in an intragenic 6.5-kb fragment confer androgen regulation is consistent with some features of other steroid-regulated genes. Androgen regulation of target gene expression typically involves cooperativity among multiple nonconsensus AREs (7, 14, 15, 16, 17, 44). The finding reported here that mutation of any ARE or of the Myc consensus site results in a drastic reduction of androgen regulation of the 350-bp region is consistent with such cooperativity.

The internal regulatory region we identified in the human AR gene is located a great distance from the AR promoter. This arrangement of regulatory sequences is not unprecedented, as a variety of transcription factors (including steroid receptors) act via regulatory sequences that are downstream from the transcription start site and separated from gene promoters by many kilobases (reviewed in Ref. 45). For example, the {alpha}-globin gene contains an internal regulatory region (encompassing exons 1 and 2 and intron 1) that cooperates with upstream 5'-flanking sequences to confer tissue-specific gene expression (46). Studies in transgenic mice revealed that androgen regulation of the ß-glucuronidase gene involves regulatory sequences present within a large genomic fragment (encompassing intron 3 through intron 9) located approximately 6.4 kb downstream from the ß-glucuronidase gene promoter (5, 6). Androgen-responsive regions have also been described within intron 1 of the rat 20-kDa protein and intron 1 of the prostatein C3 subunit gene (7, 8, 9).

Despite the demonstration of internal cis-acting sequences in a variety of steroid target genes, no clear mechanism for how these sequences function has emerged. Downstream regulatory elements might modulate transcription via a variety of mechanisms including influencing the rate of transcription initiation or elongation. DNA looping and characteristics of chromatin structure may facilitate interactions between transactivators binding downstream and in the promoter region (45). Alternatively, transactivators binding to downstream regulatory elements may interact with an upstream enhancer element to influence the rate of initiation. For instance, AR binding intragenically might interact with an upstream enhancer to increase transcription initiation from the AR gene promoter. Another possible mechanism involves augmentation of transcriptional elongation. In this model an enhancer protein binds transiently to a downstream element and opens up the DNA strand, allowing more efficient elongation. Since the androgen-responsive region of the AR gene can function upstream of a heterologous promoter (350WT-CAT, I4-CAT), enhanced elongation is less likely. Bridging or adaptor proteins (coregulators) are also likely to play a role in mediating the activities of distal and proximal regulatory elements, be they intragenic or in the 5'-flanking region (47, 48).

Nonreceptor transcription factors can play a critical role in steroid-mediated gene regulation; however, the mechanism by which nonreceptor factors influence steroid receptor action is not fully understood (16, 17, 18, 19, 49, 50). These auxiliary factors may confer cell-specific effects through distinct combinations of factors acting on complex regulatory regions. The tissue-specific effects of androgens might be achieved via interactions between AR and nonreceptor DNA-binding proteins (16, 17, 18, 19, 49, 50). We describe here a novel link between the Myc family of transcription factors and AR in AR mRNA autoregulation. The members of this family of proteins (Myc, Max, Mad 1–4, Mxi, etc.) regulate gene expression from CACGTG elements through a complex array of homo- or heterodimers (reviewed in Refs. 38, 39). Members of the Myc family are deregulated in a variety of human malignancies (38, 39); and a role for this family of proteins is well established in proliferation, differentiation, oncogene transformation, and programmed cell death (38, 40, 51). Differential AR mRNA autoregulation occurring during development (52), for example, may be related to changes in the relative levels of Myc and related family members. AR/Myc interactions may underlie androgen-mediated regulation of AR mRNA in the developing prostate (52) where changes in functional Myc/Max heterodimers might augment or attenuate sensitivity to androgen. Overexpression of c-myc in prostate cancer cells (53) may influence AR levels and androgen-stimulated proliferation. Finally, a connection between androgen responsiveness (via up-regulation of AR mRNA) and Myc function is intriguing as it offers another link between AR and the cell cycle (54, 55, 56, 57).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture, Transfection, and CAT Assays
PC3 and U2OS cells (American Type Culture Collection, Manassas, VA) were routinely cultured in RPMI-1640 media or McCoy’s media, respectively, supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Life Technologies, Inc., Gaithersburg, MD), and, unless otherwise noted, 10% heat-inactivated FBS (HyClone Laboratories, Inc. Logan, UT).

Cells were transfected using the calcium phosphate coprecipitation method (4). Supercoiled plasmid DNA used for transfection was purified by CsCl2-ethidium bromide gradient centrifugation or with Qiagen columns (Valencia, CA). All cells were cultured in 60-mm plates and received 500 ng CMVhAR, 10 µg CAT reporter plasmid, and 1.0 µg ß-galactosidase expression plasmid (CMVßgal) (to normalize for transfection efficiency). For the studies using dominant negative Myc and Max, each 60-mm dish of cells also received 0.5 or 1.0 µg of the parent vector pBJ3, MaxRx, or MycRx expression vectors. Transfected cells were cultured in RPMI-1640 or McCoy’s media supplemented with 2% charcoal-stripped FBS. Charcoal-stripped FBS was prepared by incubating serum with dextran-coated charcoal to deplete endogenous steroids. Methyltrienolone (R1881, from DuPont NEN, Boston, MA) or ethanol vehicle was added to the cultures either immediately after transfection (for reporter gene assays) or 24 h after transfection (for AR mRNA studies). Forty hours after R1881 treatment, transfected cells were harvested in 1x reporter lysis buffer (Promega Corp., Madison, WI). ß-Galactosidase and CAT activities from cell extracts were measured as previously described (4). After the ß-galactosidase assays, cell extracts were heated at 65 C for 10 min. Aliquots of cell extracts containing equivalent ß-galactosidase activities were incubated with 1.0 mM acetyl-coenzyme A (Roche Molecular Biochemicals, Indianapolis, IN) and 5.87 µM [14C]-chloramphenicol (DuPont NEN, specific activity, 50.0 mCi/mmol) in a total volume of 150 µl for 2–6 h at 37 C. Acetyl-coenzyme A (5 µl; 8 mM) was added to each reaction after every 2 h of incubation. The percent conversion of chloramphenicol to acetylated forms was quantified by phosphorimage analysis using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Statistical analysis was performed using Student’s t test.

Reporter Plasmid Construction
The reporter plasmid 350WT-CAT was previously described (4). All 350-bp fragments were inserted in the 5'- to-3' orientation upstream of the tk promoter of pBLCAT2. The numbering system used for the reporter plasmids is based on the human AR cDNA (58) with +1 located 42 bp 5' to the translation start site.

The I4-CAT reporter plasmid was created using an exonuclease III digestion strategy (59). The AR genomic fragment was amplified by PCR using human genomic DNA (Boehringer Mannheim, Indianapolis, IN) as template and primers targeted to the 5'- and 3'-ends of exon D and E of the AR gene, respectively. The forward primer was 5'-TGCCTGCAGGTCGACAACCCAGAAGCTGACAGTGTC-3'; the reverse primer was 5'-TCCTCTAGAGTCGACCGGTACTCATTGAAAACCAGATC-3'. After gel purification, the 6.5-kb PCR product (50 ng) and SalI-digested pBLCAT2 were incubated with Exonuclease III (Stratagene, La Jolla, CA) for 45 sec. The total reaction volume was raised to 50 µl with Tris-EDTA (TE) buffer (pH 8.0), and the ligated DNA was phenol/chloroform extracted and ethanol precipitated. The pellet was dissolved in TE buffer (pH 8.0) and incubated at 37 C for 10 min. A 5-µl aliquot was used for transformation of Epicurian Coli XL-10 Gold Ultracompetent cells (Stratagene).

The expression vectors, Myc, Max, MycRx, and MaxRx, and the parent vector pBJ3 were generously provided by Dr. B. Amati (40).

RNAse Protection Assays
RNAse protection assays were performed essentially as described previously (4) using a human AR antisense RNA probe synthesized from a 0.7-kb HindIII-EcoRI fragment of human AR cDNA subcloned into BluescriptIIKS (Stratagene). The probe template was linearized with ScaI. [{alpha}-32P]UTP (DuPont NEN, specific activity 800 Ci/mmol)-labeled antisense RNA probes were generated by in vitro transcription with T7 RNA polymerase using the Maxiscript kit (Ambion, Inc. Austin, TX). One microgram of total RNA was hybridized with both AR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antisense RNA probes simultaneously. The RNA samples were treated with 0.5 U RNAse A/20 U RNAse T1 for 30 min at 37 C. RNA was precipitated and fractionated by electrophoresis through 5% polyacrylamide gels containing 4 M urea. Probe fragments specifically protected by AR and GAPDH mRNA are 200 and 110 nucleotides, respectively.

Mutagenesis and PCR
The expression vector CMVhARmut containing silent mutations of ARE-1 and ARE-2 was generated by sequential PCR using CMVhAR as the template. To create the ARE-1 silent mutation, the forward primer F9 (5'-TAGCCCCCTACGG-CTACACT-3') and backward primer B11 (5'-CGATCGAGTTCCTTGATGTAGT-3') were paired, respectively, with a backward primer B9 (5'-GGCTCAATGGCTTCCAAGACGTTCAGAAAGATGG-3') and a forward primer F7 (5'-CCA-TCTTTCTGAACGTCTTGGAAGCCATTGAGCC-3') that each contain two mutated nucleotides (in boldface) to replace the wild-type ARE-1. The reactions resulted in two PCR products that contained overlapping sequences. These two resultant PCR products were then amplified as templates using F9 and B11 to generate a 1.1-kb fragment (+1367 to 2557) harboring an ARE-1 mutant. Similarly, to generate silent mutation of ARE-2, F9 and a backward primer B10 (5'-GAGCCCCATCCAGGAATATTGAATGACAGCC-3'), and a forward primer F8 (5'-GGCTGTCATTCAATATTCCTGGATGGGGCTC-3') and B11 were paired, respectively, using the ARE-1 mutant fragment as a template. The resulting 1.1-kb PCR product was digested with AocI and SfuI, and this fragment was used to replace the corresponding wild-type fragment of the AR cDNA in the Bluescript II vector (BSAR3.1). The AR cDNA bearing mutations in ARE-1 and ARE-2 was subcloned into the CMV2 expression vector.

The mutant reporter constructs, mARE-1A-CAT, mIVSARE-CAT, mMyc-CAT, and mc350-CAT, were generated using the CloneAmp System (Life Technologies, Inc.). The 5'-half of each ARE was mutated (boldface nucleotides) as follows: ARE-1A was mutated from TGTGCT to GAATTC (mARE-1A); IVSARE was mutated from TGTACA to GAACCA (mIVSARE). The mMyc plasmid contains a 350-bp region mutated at the Myc site CACGTG to CAGCTG. In the mutagenesis control plasmid (mc350-CAT), sequences were mutated from GGGCCA to CAGCTG (14 bp 3' to the IVSARE). The mMyc fragment used for footprinting studies (primers are noted as MYCFOOT below) is mutated from TGTACACGTG to GAATTCCGTG. To create the mutant constructs, a first round of PCR reactions was performed to generate the 5'- and 3'-halves of each mutated 350-bp region, respectively (underlined nucleotides were mutated). The primers for the first round of PCR were the forward (F) primer F-H3CAU (5'-CAUCAUCAUCAUGCCAGTGCCAAG-CTTAAC-3') paired with the indicated reverse (R) primer: R-ARE1-A (5'-GTGTCCGAATTCCACTACACCTGG CTCA-ATG-3'), R-IVSARE (5'-CCACGTGGTTCAGCTGTCTCTCTCCCAG-3'), R-MYC (CAGTACCAGCTGT ACAAGCTGT-CTCTCTCCCAG-3'), R-MC350 (5'-CCTTGGCAGCTGTGA-CCACGTGTAC-3'), R-MYCFOOT (5'-CAGTACGAATTCA-GCTGTCTCTGTCCCAGTTCATT-3') to produce the 5'-fragments of each mutant. The 3'-fragments were generated using the reverse primer R-XbaCUA 5'-CUACUACUACUAGGATCCTCTAGACGGTAC-3') paired with the indicated forward primer (underlined nucleotides are mutated): F-ARE1-A (5'-GGTGTAGTGGAATTCGGACACGACAACA ACC-3'), F-IVSARE (GAGACAGCTGAACCACGTGGTCAAGTGG-3'), F-MYC (5'CAGTACCA GCTGGTCAAGTGGGCCAAGGCCTTG-3'), F-MC350 (5'-ACGTGGTCACAGC TGCCAAGGCCTTG-3'), and F-MYCFOOT (5'-CAGTACGAATTCCGTGGTCAAGTGGGCCAAGGCC-3') to produce the 3'-fragments of each mutant. The resulting 5'- and 3'-fragments overlap approximately 10–15 bp surrounding the mutant sequences where they serve as template in the second round of PCR. The second round of PCR connects the 5'- and 3'-fragments using the F-H3CAU and R-XbaCUA primers. The resulting 350-bp fragment was cloned into the pAMP vector using uracil DNA glycosylase. The 350-bp mutant constructs were liberated by digestion with HindIII and XbaI and subcloned into a HindIII/XbaI-digested pBLCAT2 vector for reporter gene assays or used for footprinting studies. The resulting reporter gene constructs are called mARE1A-CAT, mIVSARE-CAT, and mMyc-CAT. Each inserted 350-bp fragment was sequenced to confirm that each construct was in the 5'-3' orientation and upstream of the tk promoter of pBLCAT2.

Expression and Purification of AR Proteins
GST-hAR (472–917) plasmid encodes a fusion protein containing GST linked to the DNA- and hormone-binding domains of human AR protein (GST-AR) and was provided by Dr. M. J. McPhaul (60). The expression and purification of GST-AR were done as previously described (4). The amino terminal-deleted AR lacking the GST moiety was obtained essentially as described by Roehrborn et al. (60). Briefly, purified GST-AR fusion protein was bound to glutathione Sepharose 4B (Pharmacia Biotech, Piscataway, NJ). Column-bound GST-AR was digested with thrombin (final concentration of 2 U/ml) in 100 mM Tris (pH 8), containing 150 mM NaCl and 2.5 mM CaCl2. Digestion proceeded for 8 h at 20 C, followed by an additional 2 h at room temperature. The reaction was stopped by adding EDTA to a final concentration of 5 mM; AR protein was eluted in PBS flowthrough. The purity of the AR protein was checked by SDS-PAGE (Coomassie stain), and the amount of AR protein was quantitated by the Bradford method (Bio-Rad Laboratories, Inc., Richmond, CA).

Isolation of Nuclear Proteins
PC3 cells were grown to 85–90% confluency, washed with ice-cold 1xPBS, and scraped into 10 ml of PBS. Cells were centrifuged and pellets were resuspended in 1xPBS, rapidly frozen in an ETOH/dry ice bath, and then thawed at 37 C for 5 min. Cell extracts were incubated on ice in Hypotonic Solution A [10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 1 mM dithiothreitol (DTT)] for 10 min. Lysis was monitored by trypan blue exclusion. The extracts were centrifuged and the supernatant discarded. The resulting pellet was resuspended in Hypertonic Solution C [10 mM HEPES, pH 7.9; 0.4 M KCl; 1.5 mM MgCl2; 25% Glycerol; 0.2 mM EDTA; 1 mM DTT; 0.5 mM phenylmethyl sulfonylfluoride (PMSF)] and shaken on ice for 30 min. After centrifugation, the supernatant was transferred to a new tube and diluted with Buffer D (20 mM HEPES, pH 7.9; 50 mM KCl; 20% Glycerol; 0.2 mM EDTA; 1 mM DTT; 0.5 mM PMSF). Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA) and aliquots stored at -80.

DNAse I Footprinting
The AR cDNA fragments (350WT, IVS, mARE-1, or mARE-2) were labeled at either the 5'- or 3'-end by end-filling of HindIII- or XbaI-linearized plasmid using Klenow fragment and [{alpha}-32P]dCTP. The labeled fragments were subsequently released by digestion with either XbaI or HindIII and isolated after electrophoresis. BSA or amino terminal-deleted AR (20 µg), GST or GST-AR fusion proteins (20 µg), or PC3 nuclear extract or buffer control (10 or 20 µg) were incubated with 20,000 cpm of labeled DNA fragments (~5 fmol) in 40 µl DNA binding buffer containing 2.5 µg polyd(I-C) and 5 µg BSA. After 30 min incubation at room temperature, MgCl2 and CaCl2 were adjusted to 4 mM and 2.5 mM, respectively. DNAse I (0.2 ng; Boehringer Mannheim) was immediately added to the reaction mixture and the mixture was incubated for 2 min at room temperature. The digestion reactions were stopped by adding 40 µl inactivation buffer [20 mM Tris-HCl (pH 7.8), 20 mM EDTA (pH 8.0), 300 mM NaCl, 1% SDS, 100 µg/ml tRNA, and 0.5 mg/ml proteinase K] and incubated at 37 C for 1 h. The sample was phenol extracted and the DNA was precipitated with ethanol. DNA pellets were resuspended in loading buffer [95% formamide, 20 mM EDTA (pH 8.0), 0.05% bromophenol blue, 0.05% xylene cyanol FF] and resolved on an 8% sequencing gel. DNA size ladder or 350-bp sequencing product was run in parallel. The gel was then dried and subjected to autoradiography. DNAse I footprint data were quantified by phosphorimage analysis of the dried gels. The amount of radioactivity in each protected region (standardized for each gel) was determined relative to a region outside the footprint.

Gel Mobility Shift Assays
Wild-type and mutant 350-bp fragments were obtained by digestion of their respective CAT vectors (350WT-CAT, mARE1A-CAT, mIVSARE-CAT, mMyc-CAT) with HindIII and XbaI, followed by purification from agarose gels. GST, affinity-purified GST-AR fusion proteins (500 ng), or PC3 nuclear extract proteins (5 µg) were mixed with 5 µg polyd(I-C) (from Roche Molecular Biochemicals), 1 µg BSA, and 20,000 cpm 32P-labeled 350-bp fragment (~2.5 fmol) in DNA binding buffer [10 mM Tris-HCl (pH 7.9), 50 mM KCl, 10% glycerol, 0.2 mM EDTA (pH 8.0), 1 mM DTT, and 50 nM R1881] to a final volume of 20 µl, and incubated at room temperature for 20 min. For the competition studies, the proteins were preincubated for 20 min at room temperature with the indicated unlabeled 350-bp DNA fragment competitor (50-, 100-, 250-fold molar excess) or Myc oligonucleotide competitor (25-, 50-fold molar excess), followed by addition of the labeled 350WT probe. The sequences for the Myc competitor oligonucleotides were 5'-GGAAGCAGACCAC-GTGGTCTGCT-TCC-3' (consensus site is boldface) and 5'-GGAAGCAGACCAGCTGGTCTGCTTCC-3' (mutated consensus site is boldface). The samples were loaded directly onto a prerun, nondenaturing 5% polyacrylamide gel (acrylamide-bis, 29:1) in 45 mM Tris-borate (pH 8.0), 1 mM EDTA (pH 8.0), run at 200 V for 1 h and 250 V for 2–3 h at room temperature, and then dried onto a filter paper and visualized by autoradiography. The DNA-protein complexes were quantitated by PhosphorImager Analysis using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

For the gel shift-based Western blot, a preparative gel shift using 100 µg of PC3 nuclear extracts was incubated under the above conditions and separated on a 5% nondenaturing gel. The gel was run longer (1.5 h at 250 V and 4 h at 200 V) to further separate and distinguish the nuclear protein-DNA complexes. The gel was UV cross-linked (1200 joules) and exposed to film to visualize the complexes. The indicated bands and regions were cut out and incubated at room temperature in 5x Laemmli loading buffer for 20 min. The gel slices and buffer were loaded into wells of an SDS-PAGE gel (13% resolving/4% stacking) and separated. Complexes were transferred to nitrocellulose membrane and processed for immunoblotting using standard procedures. Briefly, filters were incubated in blocking solution (5% dry milk, 1% Tween) for 1 h followed by incubation with primary antibody (anti-Max sc-765, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-Myc panMyc (a generous gift from Dr. T. Littlewood) for 1 h. After washing, the blot was incubated with horseradish peroxidase-conjugated secondary antibody (antirabbit, Santa Cruz Biotechnology, Inc.), and proteins were visualized using the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK) following the supplier’s instructions.


    ACKNOWLEDGMENTS
 
We thank Dr. Marco Marcelli for providing AR cDNA clones, Dr. Michael McPhaul for the GST-AR (472–917) construct, Dr. Bruno Amati for advice and providing the pBJ3, Myc, Max, MycRx, and MaxRx vectors, and Dr. Trevor Littlewood for the pan-Myc antibody. We are grateful to Dr. Bonnie Blomberg and Ms. Carol Maiorino for helpful comments on the manuscript, Dr. Olga Hernandez for suggestions on gel shift/immunoblots, and to Dr. Melanie Palmer for advice on cloning strategies.


    FOOTNOTES
 
Address requests for reprints to: Kerry L. Burnstein, Ph.D., Department of Molecular and Cellular Pharmacology (R-189), P.O. Box 016189, Miami, Florida 33101. e-mail: kburnste@chroma.med.miami.edu.

This work was supported by NIH Grant DK-45478 to K.L.B. J.M.G. received support from the American Heart Association-Florida Affiliate (Fellowship 9604012) and NIH Grant T32 HL-07188.

1 Current Address: Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21205. Back

2 Current Address: Department of Pediatrics, University of Miami School of Medicine, Miami, Florida 33101. Back

Received for publication October 28, 1998. Revision received July 20, 1999. Accepted for publication July 23, 1999.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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