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Endocrinology Vol. 142, No. 3 1107-1116
Copyright © 2001 by The Endocrine Society

ARTICLES

The Androgen Receptor (AR) Amino-Terminus Imposes Androgen-Specific Regulation of AR Gene Expression via an Exonic Enhancer1

Jennifer M. Grad2, Leah S. Lyons, Diane M. Robins and Kerry L. Burnstein

Department of Molecular & Cellular Pharmacology (J.M.G., L.S.L., K.L.B.), University of Miami School of Medicine, Miami, Florida 33101; Geriatric Research, Education and Clinical Center (K.L.B.), Miami VA Medical Center, Miami, Florida 33125; Department of Human Genetics (D.M.R.), University of Michigan School of Medicine, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Kerry L. Burnstein, Ph.D., Department of Molecular & Cellular Pharmacology (R-189), P.O. Box 016189, Miami, Florida 33101. E-mail: kburnste{at}miami.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen and glucocorticoid receptor (AR, GR), two closely related members of the nuclear receptor superfamily, can recognize a similar cis-acting DNA sequence, or hormone response element (HRE). Despite this apparent commonality, these receptors regulate distinct target genes in vivo. The AR gene itself is regulated by AR but not GR in a variety of cell types, including osteoblast-like cells, as shown here. To understand this specificity, we first identified the DNA sequences responsible for androgen-mediated up-regulation of AR messenger RNA. A 6.5-kb region encompassing exon D, intron 4, and exon E of the AR gene contains four exonic HREs and exhibits cell type-specific, AR-mediated transcriptional enhancement when placed upstream of a heterologous promoter and reporter gene. A 350-bp fragment consisting of just exons D and E exhibits the same cell- and androgen-specificity as the 6.5-kb region, as well as the native AR gene. Consistent with a role for the exonic HREs, androgen regulation via this intragenic enhancer requires the HREs as well as a functional receptor DNA binding domain. A panel of AR/GR chimeric receptors was used to test which AR domains (amino-terminal, DNA binding or ligand binding) confer androgen-specific regulation of the 350-bp enhancer. Only chimeric receptors containing the amino-terminus of AR induced reporter gene activity from the AR gene enhancer. Further, a constitutively active AR consisting of only the AR amino-terminus and DNA binding domain (AA{phi}) retained the capacity to activate the internal responsive region, unlike a constitutively active chimera harboring the GR amino-terminus and AR DNA binding domain (GA{phi}). Thus, the AR amino terminus is the sole determinant for androgen-specific regulation of the AR gene internal enhancer. These findings support a model in which the amino termini of ARs bound to HREs within the AR gene interact with an exclusive auxiliary factor(s) to elicit androgen-specific regulation of AR messenger RNA. This is the first example of androgen-specific response in which the necessary and sufficient distinguishing capacity resides within the AR amino terminus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGEN and glucocorticoid receptors (ARs, GRs) are ligand-activated transcription factors that elicit highly selective, tissue-specific effects through regulation of divergent target genes (reviewed in Ref. 1). These receptors are members of a closely related subgroup of the nuclear receptor family including progesterone and mineralocorticoid receptors. The conservation in the DNA binding domains (DBDs) of this subgroup underlies recognition of a common hormone response element (HRE) (reviewed in Ref. 2). Despite the common consensus sequence, AR mediates physiological actions that are distinct from GR’s, even though GRs and bioactive glucocorticoids are present in virtually all androgen target tissues (3). Based on analysis of a few androgen-specific target genes, diverse mechanisms have been proposed to explain the basis for AR specificity, including: 1) preferential binding of the AR DBD to androgen response elements (AREs) distinct from the consensus HRE (4, 5, 6, 7, 8); 2) differential interaction between AR and auxiliary nonreceptor factors (coactivators and/or transcription factors) that participate in receptor-mediated gene regulation (9, 10, 11, 12, 13, 14, 15); and 3) intra and interreceptor interactions that support cooperative binding of AR to multiple HREs within androgen-specific enhancers (16).

Subtle differences in individual HREs may underlie differential receptor DNA binding resulting in specific gene regulation in some cases. The prostatic gene probasin is regulated preferentially by AR in prostate cells. This regulation is exerted through two AREs in an upstream enhancer (17, 18) that, in vitro, reveal selective binding by the AR compared with the GR DBD (4, 5). Androgen-specific regulation of the secretory component gene also appears to derive from preferential AR binding to a distinct ARE in the promoter (6). Thus, specific androgen regulation of some genes may be explained by the capacity of the AR DBD to discriminate among HREs.

Steroid receptor function is strongly influenced by coactivator proteins, which interact directly with receptor and with the transcriptional machinery (reviewed in Refs. 19, 20, 21). A coactivator that interacts with one receptor in preference to others could provide a basis for specificity, but most coactivators identified thus far interact broadly with members of the nuclear receptor family. One coactivator, ARA 70, preferentially interacts with and increases the transcriptional activity of AR (22). However, this specificity of ARA 70 is not observed in all cell systems or assay conditions (23, 24, 25).

Heterotypic interactions with nonreceptor transcription factors may contribute to androgen specificity (9, 10, 11, 12, 13, 14, 15, 26, 27, 28). Such interactions represent an important component of tissue-specific gene regulation and differential protein-protein interactions may form the basis for steroid specificity of some target genes (9, 10, 11, 12, 13, 14, 15, 26, 27, 28, 29). For example, the transcription factor AML3/CBF{alpha}1 binds to a site adjacent to an HRE in the androgen-specific sex-limited protein (Slp) gene enhancer and interacts better with AR than GR in vitro (9). The amino termini of the receptors may be involved in these interactions as this domain is highly divergent even among closely related nuclear receptors. When the AR amino terminus is fused to the GAL-4 DBD, transcriptional activity is higher in a cell line in which HREs are preferentially regulated by AR (29). However, an absolute requirement for the AR amino terminus in androgen specificity has yet to be shown.

In addition to functions intrinsic to the three major receptor domains, intramolecular interactions are requisite for AR function. Interaction between the AR amino- and carboxyl-termini participates in ligand binding, gene transactivation, and regulation of AR stability (30, 31, 32, 33, 34). Furthermore, androgen-specific regulation of Slp is also dependent on AR domain interactions (16). Mapping of the AR domain(s) required for Slp enhancer specificity revealed a complex requirement for amino-carboxyl terminal interactions that promote cooperative binding of AR, but not GR, dimers to multiple HREs (16).

Although studies of androgen specificity have been limited to a small number of genes, several mechanisms appear to be used. In this report, we identify a new mechanism that operates in the androgen-specific regulation of AR messenger RNA (mRNA). An internal enhancer of the AR gene directs androgen-specific transcription, even when upstream of a heterologous reporter, in a cell-specific fashion (35, 36). Receptor chimeras comprised of amino-terminal, DBDs and ligand binding domains (LBDs) of AR and GR were used to define the domain requirements for androgen-specific regulation. We found that the information required for androgen-specific regulation of this unique enhancer resides solely in the AR amino terminus. Thus, the AR gene itself provides the long sought example for which steroid-specific regulation is determined by the highly divergent receptor amino terminus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, transfection, and CAT assays
U2OS cells (American Type Culture Collection, Manassas, VA) were routinely cultured in McCoy’s media, 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 as previously described (35). Supercoiled plasmid DNA used for transfection was purified with QIAGEN columns (Santa Clarita, CA). For reporter gene assays, all cells were cultured in 60-mm plates and received 500 ng CMVhAR, RSVhGR, or the indicated chimera, 10.0 µg reporter plasmid, and 1.0 µg {beta}-galactosidase expression plasmid (CMV{beta}-gal) (to normalize for transfection efficiency). Cells were incubated with the calcium-phosphate precipitates for 5–7 h, glycerol shocked for 45 sec and then cultured in McCoy’s media supplemented with 2% charcoal-stripped FBS (css FBS). The indicated hormone [50 nM methyltrienolone (R1881), 200 nM dexamethasone] or vehicle control was added immediately after. For the experiments using constitutively active receptors no hormone was added. Forty hours after hormone treatment, transfected cells were harvested in 1x reporter lysis buffer (Promega Corp., Madison, WI). {beta}-Galactosidase and CAT activities from cell extracts were measured as previously described (35, 36). 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 a Student’s t test and SigmaPlot software (Jandel Scientific).

Reporter plasmids and expression vectors
Chimeric receptors containing combinations of mouse AR (mAR) or rat GR (rGR) receptor domains were constructed as previously described (16). The {Delta}F581, {Delta}R614, and R614H constructs were obtained from Dr. L. Pinsky (37); the {Delta}538–614 construct was obtained from Dr. E. Wilson (38). The 350 wild-type (WT)-CAT reporter plasmid consists of exons D and E of the human AR gene linked upstream from the tk promoter and CAT gene in the vector pBLCAT2 and was described previously (35). 2XHRE-CAT (obtained from Dr. J. Cidlowski (NIEHS, Research Triangle Park, NC) contains two tandem copies of an HRE (GRE) consensus sequence linked to the minimal E1b promoter and CAT (39). The I4-CAT reporter plasmid consists of exon D, intron 4 and exon E of the human AR gene linked upstream from the tk promoter and CAT gene in the vector pBLCAT2 (36) and was generated using an exonuclease III digestion strategy as previously described (36).

The mI4-CAT reporter plasmid contains the 6.5-kb genomic fragment harboring mutations at ARE-1 and ARE-2. ARE-1 is changed from 5'-TGTCCT-3' to 5'-CGTCTT-3' and ARE-2 is changed from 5'-AGTACT-3' to 5'-AATATT-3' (altered base pairs are underlined) (36). The reporter plasmid was created using an exonuclease III digestion strategy similar to that described for I4-CAT (36) First, the AR genomic fragment encompassing intron 4 was amplified by PCR using human genomic DNA (Roche Molecular Biochemicals, Indianapolis, IN) as template and primers targeted to the 5' and 3' ends of intron 4. The primers also contained NotI sites as well as 10–12 bp of sequence complementary to the intron 4/exon D and exon E boundary within the AR gene. The forward primer was 5'-GTGGGCCAAGGCCTTGCCGCGTAAGGAAAAGGGAAGT-3'; the reverse primer was 5'-CCACGTGTAAGTTGCCGCGGCCCTGGAGAAGAAGAGG-3'. The 6.2-kb PCR product was gel purified and ethanol precipitated. Second, a NotI site was introduced into the intron/exon splice site of the WT 350-bp region or into a 350-bp region harboring mutations in both ARE-1 and ARE-2 (35), using the Clone Amp system (Life Technologies, Inc.). A first round of PCR was performed to generate the 5' and 3' halves of each mutated 350-bp region, respectively, (underlined nucleotides are mutated) using the mARE-1/mARE-2 mutant 350 bp construct as template (35). The primers for the first round of PCR were the forward (F) primer F-H3CAU (5'-CAUCAUCAUCAUGCCAGTGCCAAGCTTAAC-3') paired with the reverse (R) primer: R-350-NOT (5'-GTTGCGCGGCCGCGGCAAGGCCTTG-3') to produce the 5' fragment. The 3' fragments were generated using the reverse primer R-XbaCUA (5'-CUACUACUACUAGGATCCTCTAGACGGTAC-3') paired with the forward primer (underlined nucleotides are mutated): F-350NOT (5'-CCTTGCCGCGGCCGCGCAACTTACAC-3') to produce the 3' fragment. 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 fragments were cloned into the pAMP vector using uracil DNA glycosylase. The NotI-containing 350 bp regions were liberated by digestion with HindIII and XbaI and subcloned into a HindIII/XbaI digested pBLCAT2 vector for reporter gene assays. 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 (40). The resulting constructs were called 350NotI-CAT and m350NotI-CAT. Each construct was linearized by digestion with NotI and gel purified. The intron fragment generated by PCR (described above) (50ng) and the NotI-digested 350NotI-CAT and m350NotI-CAT vectors were incubated with Exonuclease III (Stratagene, La Jolla, CA) for 45 sec. The total reaction volume was raised to 50 µl with TE buffer (pH 8.0), the ligated DNA phenol/chloroform extracted and ethanol precipitated and used for transformation of Epicurian Coli XL-10 Gold Ultracompetent cells (Stratagene, La Jolla, CA).

RNA isolation and analyses
Northern blot analyses and poly-A isolation were performed as previously described (41, 42). RNase protection assays were performed as previously described (35). Probe fragments specifically protected by AR and GAPDH mRNA are 200 and 110 nucleotides, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen but not glucocorticoid receptor up-regulates AR mRNA in osteoblastic cells.
Androgen promotes increased expression of AR mRNA in a tissue- or cell-specific manner. One cell type that displays this positive autoregulation is osteoblastic (osteosarcoma) cells and this induction of receptor levels correlates with enhanced cellular responsiveness to androgen (43–45, Burnstein, K. L., J. M. Grad, and C. A. Maiorino, unpublished). We chose to assess the steroid specificity of AR mRNA up-regulation in osteosarcoma cells because these cells have endogenous AR and GR. Further, androgens and glucocorticoids elicit distinct effects on bone, suggesting that these cells possess relevant mechanisms to ensure steroid specificity (reviewed in Refs. 46, 47).

To determine whether the AR gene is specifically regulated by androgen in human osteosarcoma cells, we analyzed AR mRNA levels in U2OS cells following androgen (R1881) or glucocorticoid (dexamethasone) treatment. Northern blot analysis demonstrated that androgen treatment promoted a 2- to 2.5-fold increase in AR mRNA in U2OS cells (Fig. 1AGo), as has been shown previously in osteosarcoma cell lines (43, 44, 45). In contrast, RNase protection assay showed that dexamethasone had no significant effect on AR mRNA levels (Fig. 1BGo). RNase protection assay was used as this method is more sensitive than Northern blotting. The failure of glucocorticoid to regulate AR mRNA was not due to absence of functional GR in this cell line because dexamethasone treatment led to induction of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR), which responds to androgens and glucocorticoids in most cell lines (48, 49) (Fig. 1CGo). Androgen did not induce CAT activity from the MMTV-LTR (Fig. 1CGo), consistent with lower levels of AR than GR in osteosarcoma cells (50, 51, 52). The same results were obtained using a reporter plasmid consisting of two copies of an HRE linked to a minimal promoter and CAT (2XHRE-CAT) (data not shown).



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  		Figure 1. Androgen-specific regulation of AR mRNA in the human osteosarcoma cell line, U2OS. U2OS cells were treated with vehicle or 50 nM R1881 for 72 h (A); or with vehicle or; 200 nM dexamethasone (Dex) for 48 h (middle lane) or 72 h (right lane) (B). A, Poly-A enriched RNA (~5 µg) was analyzed by Northern blotting using AR and GAPDH cDNA probes. B, Total RNA (20 µg) was analyzed by ribonuclease protection assay. GAPDH mRNA was assessed in each experiment to control for loading. C, U2OS cells were cotransfected with MMTV-CAT and a CMV{beta}-gal expression vector and treated with dexamethasone (Dex, 200 nM) or R1881 (50 nM). (Neither a GR nor AR cDNA expression vector was transfected.) CAT assays were performed as described in Materials and Methods. Representative data from two to three experiments are shown.

 
Exonic HREs of the internal enhancer of the AR gene are essential for androgen regulation
An enhancer internal to the AR gene is involved in androgen-mediated up-regulation of AR mRNA and can confer androgen inducibility when placed upstream of a heterologous promoter and CAT reporter gene (35, 36). This enhancer resides within an approximately 6.5-kb fragment consisting of exon D, intron 4, and exon E (36; Fig. 2Go). This region was linked to the heterologous tk promoter and CAT gene to assess androgen activation in U2OS cells cotransfected with an AR complementary DNA (cDNA) expression vector, CMVhAR. As shown previously (36), the resulting reporter construct, I4-CAT, was induced by androgen (Fig. 2Go). Cotransfection with an AR cDNA was required as endogenous levels of AR are low and not sufficient for activation of reporter genes (Fig. 1CGo, 50). These data suggest that despite the location of this enhancer downstream from the site of transcription initiation, this region functions in androgen-mediated transcriptional regulation of AR mRNA.



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  		Figure 2. Androgen regulation of the internal enhancer of the AR gene is dependent on HREs. U20S cells were cotransfected with a human AR cDNA expression vector (CMVhAR), CMV{beta}-gal and the indicated reporter plasmid containing the WT (I4-CAT) or mutated (mI4-CAT) 6.5-kb internal enhancer region of the AR gene or the 350bp fragment representing exons D and E (350WT-CAT). Cells were treated with vehicle or R1881 (50 nM) and CAT assays performed as described in Materials and Methods. A representative experiment is shown [I4-CAT (n = 12), mI4-CAT (n = 3), 350WT-CAT (n = 15)].

 
To determine whether previously identified HREs (35, 36) are required for androgen regulation of the 6.5-kb enhancer, two of the HREs, which are both half-sites, were mutated to generate the reporter plasmid mI4-CAT. One HRE in exon D (ARE-1, TGTCCT) was mutated (CGTCTT) and one HRE in exon E (ARE-2, AGTACT) was mutated (AATATT). The resulting reporter plasmid, mI4-CAT, with mutations of ARE-1 and ARE-2 was cotransfected with CMVhAR into U2OS cells. Because no androgen regulation of the mutated enhancer was observed, androgen regulation of the 6.5-kb internal enhancer of the AR gene is dependent on the exonic HREs (Fig. 2Go). Consistent with this conclusion, a 350-bp fragment with only exons D and E linked to tk-CAT (350WT-CAT) was regulated by androgen to the same extent as I4-CAT (36 and Fig. 2Go, quantitation shown in Fig. 3BGo).



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  		Figure 3. The internal enhancer of the AR gene exhibits androgen-specific regulation. A, U2OS cells were cotransfected with a human GR cDNA expression vector (RSVhGR), CMV{beta}-gal and the indicated reporter plasmid: 350WT-CAT (containing the 350-bp exonic enhancer region; n = 9), I4-CAT (containing the 6.5-kb genomic enhancer region; n = 6), or MMTV-CAT (n = 8). Cells were treated with dexamethasone (Dex, 200 nM) and CAT assays performed as described in Materials and Methods. B, Data ( ± SD) from Figs. 2Go and 3AGo were quantitated using a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA) and analyzed by Student’s t test using Sigma Plot software. Fold induction is the ratio of CAT activity observed in the presence and absence of R1881 treatment. GR did not transactivate 350WT-CAT or I4-I4-CAT (*, P < 0.05).

 
Androgen but not glucocorticoid receptor regulates the internal enhancer of the AR gene.
To determine whether regulation of the AR gene internal enhancer, like the native AR gene, retained specificity for androgen, we examined the capacity of expressed, ligand-activated AR or GR to transactivate reporter plasmids containing either the exonic enhancer of the AR gene (350WT-CAT) or the entire 6.5-kb genomic enhancer (I4-CAT). U2OS cells were cotransfected with 350WT-CAT or I4-CAT and a GR cDNA expression vector (RSVhGR) and treated with dexamethasone. Dexamethasone treatment did not induce CAT activity in cells expressing GR (Fig. 3AGo). As a control for GR function, glucocorticoid induction of the MMTV-CAT reporter plasmid is shown (Fig. 3AGo). Quantitation of androgen vs. glucocorticoid regulation of the 350WT-CAT and I4-CAT constructs is shown in Fig. 3BGo. The failure of GR to regulate either 350WT-CAT or I4-CAT is unlikely to be due to insufficient GR levels as a robust induction of MMTV-CAT was observed in dexamethasone treated U2OS cells expressing the GR cDNA (Fig. 3AGo). This induction is comparable with that observed in androgen-treated cells expressing CMVhAR (Fig. 4Go, WT). These results indicate that the HRE-containing AR gene regions retain the androgen specificity exhibited by the native AR gene.



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  		Figure 4. A functional receptor DNA binding domain is required for androgen regulation of the AR gene internal enhancer. U2OS cells were cotransfected with CMVhAR (WT) or the indicated AR DNA binding domain mutant, MMTV-CAT or 350WT-CAT, and CMV{beta}-gal. {Delta}F581 lacks Phe at position 581 (37 ); {Delta}R614 lacks Arg at position 614 (Arg) (37 ); R614H contains an Arg to His substitution at position 614 (37 ); {Delta}538–614 contains a deletion from position 538 to 614 (38 ). Cells were treated with R1881 (50 nM) and CAT assays performed as described in Materials and Methods. Data are from a minimum of three independent experiments. Fold induction is the ratio of CAT activity observed in the presence and absence of R1881 treatment.

 
Androgen regulation of the AR gene internal enhancer requires a functional receptor DNA binding domain
Although steroid regulation typically requires receptor binding to cis-acting response elements, hormonal control of some genes involves protein-protein interactions that are independent of receptor binding to DNA (for example see Ref. 53 and references within). Such protein-protein interactions may involve the receptor DBD but not require DNA binding per se (for example see Ref. 54). To test the requirement for a functional AR DBD in the regulation of the AR intragenic enhancer, we measured reporter gene activation by several AR mutants deficient in DNA binding. Two of the AR mutants, {Delta}F581 and {Delta}R614 (37), lack Phe at position 581 and Arg at position 614, respectively. These amino acids, located just C-terminal to the first zinc finger ({Delta}F581) and second zinc finger ({Delta}R614) of AR, are highly conserved among steroid receptors and are required for AR interaction with AREs (37). A third DNA binding mutant, R614H, harbors an arginine to histidine substitution at position 614 (37). {Delta}538–614 lacks the region from position 538 to 614, which includes both zinc fingers (38). Each mutant receptor has been shown to be expressed in heterologous cells (37, 38, 54) and to bind androgen (37, 38, 54). Based on the ability of the {Delta}F581, {Delta}R614, and R614H mutant receptors to bind ligand (37), these receptors are expected to be predominantly nuclear in the presence of androgen. However, localization studies of these mutants have not been reported. The {Delta}538–614 receptor has been shown to translocate to the nucleus in the presence of androgen, albeit to a lesser extent than WT AR (38). Consistent with reports from other investigators (37, 54), we found that these mutant receptors did not transactivate reporter gene activity from the MMTV-LTR (Fig. 4Go). Similarly, the mutant receptors did not transactivate 350WT-CAT (Fig. 4Go). These data, in conjunction with our identification of HREs within the 350-bp and 6.5-kb fragments whose mutation abrogates induction (35 and Fig. 2Go), indicate that AR binding to HREs in the internal enhancer is essential for androgen regulation of this region.

The amino terminus of AR is required for transactivation of the internal enhancer of the AR gene
To define AR domains responsible for the androgenspecific regulation of the AR gene internal enhancer, we used a panel of AR/GR chimeric receptors (Fig. 5AGo). In these chimeras, the amino terminal region, DBD and LBD originate from either the mouse AR or the rat GR cDNAs (16). Chimeric receptors are named by the origin of each domain with A designating AR and G, indicating GR. For example, a chimeric receptor containing an AR amino terminus, a GR DBD and a GR LBD is designated AGG. Expression of the mouse AR, rat GR and all chimeric receptors is under the control of the CMV promoter (16).



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  		Figure 5. The AR amino terminus confers androgen-specific regulation to the AR gene enhancer (350-bp exonic fragment). A, Schematic of chimeric receptors. B–D, U2OS cells were transfected with the indicated parent receptor [mouse AR (AAA), rat GR (GGG)] or chimera, the indicated reporter plasmid and CMV{beta}-gal. All receptor and chimera cDNAs were under the control of the CMV promoter. Cells were treated with either 50 nM R1881 or 200 nM dexamethasone, appropriate to the LBD of the receptor. CAT assays were performed as described in Materials and Methods. Fold induction is the ratio of CAT activity observed in the presence and absence of hormone treatment. The data (four to six experiments) were analyzed as described above [B, * differs from AAA (P < 0.05), ** differs from AGA and AAA (P < 0.05); D, ** differs from AAA P < 0.01)].

 
Examination of the regulation of 350WT-CAT by the panel of chimeric receptors, activated by ligand appropriate to the LBD, revealed that only those chimeras containing the amino terminus of AR transactivated the AR gene internal enhancer (Fig. 5BGo). In contrast, none of the chimeric receptors containing a GR amino-terminus induced 350WT-CAT (Fig. 5BGo). Consistent with earlier findings (16), all the chimeric receptors transactivated a simple, nonspecific enhancer comprised of two HREs linked to a minimal promoter and CAT (2xHRE-CAT) (Fig. 5CGo). The AGA chimera evoked a more modest induction of 350WT-CAT compared with the control (AAA) and chimeric receptors, AGG and AAG. However, the AGA chimera was also a poor transactivator of MMTV-CAT relative to the other receptors (Fig. 5DGo). Thus, this chimeric receptor may be inherently weaker at more complex regulatory regions such as the MMTV-LTR, which consists of multiple HREs as well as other transcription factor binding sites (48, 49).

To determine whether androgen-specific regulation of the genomic 6.5-kb internal enhancer also required the AR amino terminus, AGG and GAA were tested in parallel with both I4-CAT and 350WT-CAT. These chimeric receptors displayed the same specificity for I4-CAT as for 350WT-CAT; AGG but not GAA transactivated the AR gene internal enhancer (Fig. 6Go). Thus, androgen specificity is retained even though the spatial organization of HREs differs dramatically between the 6.5-kb and 350-bp enhancer regions.



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  		Figure 6. The AR amino terminus is required for regulation of the AR gene enhancer (6.5-kb genomic fragment). U2OS cells were transfected with 350WT-CAT, mARE-1,2-CAT (the 350-bp region containing mutations of the HREs, ARE-1, and ARE-2) or I4-CAT, the indicated receptor cDNA, and CMV{beta}-gal. Cells were treated with dexamethasone (AGG expressing cells) or R1881 (GAA, AAA, or AGA expressing cells) and CAT assays performed as described in Materials and Methods. A, Summary from three independent experiments. No androgen induction of 350WT-CAT or I4-CAT by GAA was observed in any experiment. Fold induction is the ratio of CAT activity in the presence and absence of hormone. The data were analyzed as described above [* (P < 0.05)].

 
The finding that AGG and AGA regulate 350WT-CAT suggests that the GR DBD binds HREs within the AR internal enhancer. To test the requirement for the HREs, we examined the ability of the AGA chimera to transactivate a 350-bp reporter plasmid harboring mutations in two HREs of the enhancer (mARE1,2-CAT, Ref. 35). Although AGA induced CAT activity from 350WT-CAT (Figs. 5AGo and 6BGo), AGA had little effect on mARE1,2-CAT (Fig. 6BGo). Because only the DBD portion of AGA is from GR, these data suggest that the GR DBD can bind to HREs within the AR gene enhancer.

A ligand binding domain is not required for regulation of the AR gene internal enhancer
The experiments discussed above show that in the context of a full-length receptor the AR amino terminus is the primary determinant of androgen-specific regulation of the AR gene internal enhancer. To determine whether a receptor LBD is required for regulation of 350WT-CAT, constitutively active receptors lacking the LBD were employed in reporter gene assays. These constructs contain either the AR amino-terminus and an AR DBD (AA{phi}) or the GR amino-terminus and an AR DBD (GA{phi}). Both AA{phi} and GA{phi} induced reporter gene activity from the nonspecific 2XHRE-CAT and MMTV-CAT reporter constructs normalized to the parent vector CMV5 (Fig. 7AGo). In contrast AA{phi}, but not GA{phi}, activated the 350-bp region (Fig. 7BGo). Thus, the AR amino terminal domain is necessary for specificity and sufficient when coupled to a receptor DBD, even in the absence of an LBD.



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  		Figure 7. A receptor ligand binding domain is not required for regulation of the AR gene enhancer. A, U2OS cells were transfected with 2XHRE-CAT or MMTV-CAT (A) or 350WT-CAT (B), the indicated constitutively active chimera (AA{phi} or GA{phi}), and CMV{beta}-gal. CAT assays were performed as described in Materials and Methods. Fold induction is the percent conversion of chloramphenicol in cells (expressing equivalent amounts of {beta}-gal activity) transfected with AA{phi} or GA{phi} divided by percent conversion observed with CMV5, the parent vector (typically 1–2%). Data from 2 (A) and 4 (B) independent experiments are shown. The data (B) were analyzed as described above [** (P < 0.05)].

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study documents the presence of an HRE-containing enhancer within the AR coding region that confers stringent androgen-specific regulation similar to that exhibited by the native AR gene. The AR amino terminus plays a critical role in conferring steroid specific regulation of this internal enhancer. Thus, this work reveals a unique functional requirement of the amino terminal domain in an androgen-specific response.

We examined regulation of a reporter plasmid containing the 350-bp AR gene enhancer fragment, which contains exon D and E sequences linked upstream of the tk promoter and CAT gene. A panel of chimeric receptors composed of AR and GR domains was used to identify the region of the AR required for the androgen specificity exhibited by this AR gene enhancer. Only receptors containing an AR amino terminal segment (parental AAA and chimeras AGA, AGG, AAG) transactivated this enhancer (Fig. 5BGo). Similar results were obtained with the 6.5-kb genomic enhancer, which consists of exons D and E separated by intron 4, suggesting that no critical additional regulatory sequences reside in the intron and that specificity of enhancer regulation occurs when the HREs are in the context of the native enhancer. The importance of the AR amino terminus in regulating the AR gene enhancer was verified further by experiments showing that of the two truncated, constitutively active receptors tested (AA{phi} and GA{phi}), only AA{phi} activated the internal AR gene enhancer (Fig. 7BGo). This result not only confirms the requirement for the AR amino terminus but shows that the AR LBD is not specifically involved in conferring androgen selective regulation of the AR gene enhancer.

Similar to other nuclear receptors, AR contains an AF-1 (activation function 1) region in the amino terminal domain, which participates in gene transactivation. The AF-1 regions of nuclear receptors are highly variable in both sequence and length and their mode of action is still incompletely understood. One general mechanism by which the AF-1 region has been proposed to function is through recruitment of basal transcription factors and RNA polymerase to target gene promoters (55). More recent data supports indirect interaction between nuclear receptors and the transcriptional machinery via coactivators (19, 20, 21). The amino terminal AF-1 of AR contacts the p160 family of coregulators, which includes SRC-1 (25, 56, 57), and this interaction is mechanistically more important for transactivation by AR than the ligand-dependent interaction between SRC-1 and the AF-2 region of the AR LBD (25). By contrast, ligand-dependent AF-2 interaction with coactivators appears to be more critical for transactivation by other nuclear receptors including ER (19, 20, 21). A new mode of coactivator recruitment by AR has been proposed (33, 58, 59) that depends on the well established AR amino-carboxyl terminal interaction (30, 31, 32, 33, 34). The coactivators CBP and F-SRC-1 enhance the amino-carboxyl terminal interaction of the rat AR suggesting that coactivators may function in this manner (33). The human AR amino terminus contains WXXLF and FXXLF sequences that strongly interact with AF-2 (59). Unlike other nuclear receptors in which coactivators interact with receptor AF-2 regions via LXXLL sequences, SRC1 and TIF2 bind AR amino terminal and DBD regions in an LXXLL-independent manner (58, 59). Thus, the AR amino terminal WXXLF and FXXLF sequences may compete with coactivators for binding to the AR AF-2 region (58, 59). Despite these differences in the mechanisms of coactivator function for AR compared with other nuclear receptors, p160 coactivators are unlikely to confer androgen specificity as these factors interact widely with and enhance activity of numerous members of the nuclear receptor superfamily (19, 20, 21).

The AR amino terminus is unique in that two different polymorphic trinucleotide repeat regions encoding polyglutamine and polyglycine tracts are present. ARs with shorter or longer than average polyglutamine repeats have been associated with altered transcriptional activity (60, 61) and receptor levels (62). These repeats may contribute to androgen-specific gene responses by directing exclusive interaction with other factors. In addition, the AR amino terminus was shown to contain a small region that is involved in interaction with the retinoblastoma protein (Rb) (63, 64). Although Rb enhances both AR and GR transactivation, AR but not GR function appears to depend on Rb (63). Because AR function is diminished in the absence of Rb, differences in Rb status are unlikely to underlie androgen specificity.

Androgen regulation of the AR gene internal enhancer regions was demonstrated with both human and mouse AR. The amino acid sequences of the mouse and human AR N termini are about 90% sequence identical (65), with largely conservative changes. Perhaps the most striking differences are in placement and length of the two polyamino acid tracts (65). The polyglutamine tract is about 100 amino acids closer to the carboxyl terminus in mouse and is disrupted by histidines (65). The polyglycine tract, located near the DBD, contains about twenty glycines in human but only four or five in mouse (65). A physiological correlate to these species differences is not known, and distinct differences (for example in transcriptional activity) between mAR and hAR in vitro have not been noted.

We have found that the carboxyl terminus of GR can substitute for that of AR because the full-length receptor chimeras, AGG and AAG, regulate the AR gene internal enhancer. Furthermore, the receptor, AA{phi}, lacking a carboxyl terminal region also activates the AR gene enhancer, suggesting that the amino-carboxyl interaction of AR is not essential for androgen-specific regulation of this region in reporter gene assays. However, the AR amino-carboxyl interaction may be important in androgen-specific regulation of native genes. The activity of AA{phi} also suggests that neither the AR nor GR carboxyl terminal regions are needed to maintain the amino terminal domain of AR in an appropriate conformation for protein-protein contacts.

Androgen-specific regulation of the AR gene enhancer occurs through a mechanism that contrasts with the other modes of androgen specificity described for androgen target genes like probasin, secretory component and Slp. Further, unlike many other androgen-specific enhancers, which GR can activate in certain cell lines, the AR gene enhancer is not regulated by GR in any cellular context investigated thus far (data not shown). One mechanism, used by the probasin gene, involves differential binding affinities of the AR and GR DBDs (5). Although not measured directly, the GR DBD appears to bind effectively to the AR gene AREs as chimeras containing the GR DBD (AGA, AGG) transactivate both the 350-bp and 6.5-kb enhancers (Fig. 6Go). Thus, androgen specificity of the AR gene is not exerted through differential DNA binding. The Slp gene is also regulated in an androgen-specific fashion. However unlike probasin, specificity derives from a complex interplay of interactions that occur between the AR domains as well as between receptor and other transcription factors, notably AML3/CBF{alpha}1 (9, 10, 16). The AR amino terminus is not sufficient for androgen- specific regulation of Slp as specificity depends in part on the ability of the AR DBD, but not the GR DBD, to evade suppressive effects exerted by neighboring factors (16). Further, interaction between AR’s amino and carboxyl termini underlies cooperative DNA binding by AR dimers (16). In contrast to Slp, androgen regulation of the AR gene enhancer occurs through an apparently simpler mechanism requiring only the AR amino terminus for specificity.

In summary, our results indicate that the AR amino terminus plays a dominant role in androgen-specific regulation of an androgen target gene. This finding supports longstanding theories that steroid-specific regulation of target gene enhancers occurs via distinct and specific associations between nonreceptor auxiliary factors and the highly divergent amino terminal regions of receptors. For the androgenspecific regulation described in this study, a putative intermediary factor appears to be selective for the AR amino terminus, as the GR amino terminus cannot substitute. This factor may be a previously described DNA binding protein or a coactivator that interacts in this case to confer stringent specificity not previously demonstrated. Although the contribution from nonreceptor transcription factors in steroid regulated gene expression is clear, no model systems have been elucidated in which the specific and perhaps broad role of receptor amino termini in maintaining steroid specific responses could be assessed. Our finding that the AR amino terminus is the sole contributor to androgen-specific regulation of the AR gene enhancer now provides the means to identify which factors impart this specificity.


   Acknowledgments
 
We are grateful to Ms. Carol Maiorino for expert technical assistance.


   Footnotes
 
1 This work was supported by the NIH [DK-45478 (to K.L.B.), DK-56356 to D.M.R.)]. Back

2 Received support from the American Heart Association-Florida Affiliate (Fellowship No. 9604012) and the NIH (T-32-HL-07188). Back

Received August 10, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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