Repressors of Androgen and Progesterone Receptor Action*

Received for publication, May 16, 2003Published, JBC Papers in Press, May 27, 2003, DOI 10.1074/jbc.M305153200

Irina U. Agoulnik‡, William C. Krause‡, William E. Bingman III‡, Hassan T. Rahman‡,Mojghan Amrikachi§, Gustavo E. Ayala§, and Nancy L. Weigel‡¶

From the Departments of ‡Molecular and Cellular Biology and §Pathology, Baylor College of Medicine,Houston, Texas 77030

Androgen and progesterone receptors (AR and PR)are two determining factors in gonadal differentiationthat are highly expressed in developing and mature go-nads. Loss of AR results in XY sex reversal and muta-tions causing reduced AR activity lead to varying de-grees of defects in masculinization. Female PR knockoutmice are infertile due to ovarian defects. While muchhas been discovered about positive regulation of thesereceptors by coactivators little is known about repres-sion of the transcriptional activity of AR and PR in thepresence of agonists. In this study we assessed the effectof SMRT and DAX-1 on AR and PR activity in the pres-ence of both agonists and partial antagonists. We showthat SMRT and DAX-1 repress agonist-dependent activ-ity of both receptors, and the mechanism of repressionincludes disruption of the receptor dimer interactionsrather than recruitment of histone deacetylases. Wedemonstrate that endogenous agonist-bound PR andDAX-1 in T47D breast cancer cells and endogenous ARand DAX-1 in LNCaP prostate cancer cells can be coim-munoprecipitated suggesting that the interaction isphysiological. Surprisingly, although DAX-1 repressespartial antagonist activity of AR, it was ineffective inrepressing partial antagonist induced activity of PR. Incontrast to most reported repressors, the expression ofDAX-1 is restricted. We found that although DAX-1 isexpressed in normal human prostate, its expression isstrongly reduced in benign prostatic hyperplasia sug-gesting that DAX-1 plays a role in limiting AR activityin prostate.

Nuclear receptors are regulated both by coactivators and bycorepressors. Although steroid receptor coactivators have beenstudied extensively, less is known about corepressors of agonistactivated steroid receptors. Androgens, acting through AR,1

play a role in both benign prostatic hyperplasia (BPH) and

prostate cancer. In both cases, reduction in AR activity is animportant component of treatment, although fully effectivetreatments are not yet available. Two well characterized core-pressors of thyroid receptor (TR) activity, NCoR (nuclear recep-tor co-repressor) and SMRT (silencing mediator for retinoidacid receptor (RAR) and TR), have been identified previously(1, 2). They appear to work through binding to TR aporeceptor,recruiting complexes containing histone deacetylases(HDACs); they dissociate from the receptor upon agonist bind-ing allowing coactivator complexes to form (3). In the case ofestrogen receptor (ER), these repressors interact with the an-tagonist bound receptor through a region that largely overlapswith the coactivator binding interface, and recruit HDACs (4).An orphan nuclear receptor DAX-1 (dosage sex reversal, adre-nal hypoplasia congenita critical region on the X chromosome,gene 1) has been reported to inhibit steroidogenic factor 1(SF-1) and ER activity (5, 6). DAX-1 is an atypical nuclearreceptor containing an ssDNA/RNA binding domain in its Nterminus and a multihelical C-terminal domain, a putativeligand binding domain (LBD) (7). DAX-1 is an X chromosomalgene, amplification of which results in XY sex reversal, a phe-notype similar to AR inactivation (8). Opposing effects of thegene dose of DAX-1 and AR as well as a similar pattern ofexpression suggested that DAX-1 might be an AR repressor.AR shares many common features with other steroid receptorsbut differs in some aspects. First, whereas ER and PR formparallel homodimers through interactions within their LBD (9,10), AR forms antiparallel dimers that are stabilized by inter-action between the N and C termini (11). Second, while moststeroid receptors such as TR or ER rely strongly on theiractivation function-2 (AF-2) in the receptor hormone bindingdomain for recruitment of coactivators (12), the N-terminalAF-1 function of AR is more important (13, 14). We have stud-ied the functional and physical interactions of corepressorswith AR and PR. We found that SMRT and DAX-1 were effec-tive repressors of agonist-dependent activity of AR and PR, butDAX-1 did not repress the activity of the vitamin D receptor(VDR), a class II nuclear receptor. Moreover, endogenous ago-nist-bound PR and AR each interact with DAX-1. To betterunderstand the mechanism of repression by candidate core-pressors we compared their effects on AR and PR. In this studywe show that both AR and PR are repressed by DAX-1 in thepresence of their respective agonists, but exhibit opposite co-regulation patterns when bound to their common partial an-tagonist RU486. In contrast, SMRT represses both AR and PRin the presence of either agonist or antagonist. During thepreparation of this article, Holter et al. (15) reported thatDAX-1 represses AR activity and suggested that DAX-1 cancause mislocalization of AR to the cytoplasm in the presence ofagonist. However, our studies using a promoter interferenceassay, suggest that DAX-1 does not disrupt the interaction ofeither AR or PR with DNA. We find that the DAX-1 mechanism

* This work was supported by Specialized Program of Research Ex-cellence in Prostate Cancer Grant CA 58204, DAMD fellowship 17-01-1-0018, and Training Grant in Reproductive Biology HD 07165. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

¶ To whom correspondence should be addressed: Dept. of Molecularand Cellular Biology, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6234; Fax: 713-790-1275; E-mail: nweigel@bcm.tmc.edu.

1 The abbreviations used are: AR, androgen receptor; ER, estrogenreceptor; VDR, vitamin D receptor; TR, thyroid receptor; SMRT, silenc-ing mediator for retinoid acid receptor and TR; NCoR, nuclear receptorco-repressor; DAX-1, dosage sex reversal, adrenal hypoplasia congenitacritical region on the X chromosome, gene 1; LBD, ligand bindingdomain; DBD, DNA binding domain; GFP, green fluorescent protein;HDAC, histone deacetylases; I, input; BPH, benign prostate hyperpla-sia; CAT, chloramphenicol acetyltransferase; RAM, rabbit anti-mouseantibody.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 33, Issue of August 15, pp. 31136–31148, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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of action includes interference with N/C-terminal interactionsin AR and hormone binding domain interactions in PR.

EXPERIMENTAL PROCEDURES

Constructs and Supplies—GRE2-E1b-Luc reporter was provided byDr. Carolyn Smith, Baylor College of Medicine (16). This reporter con-tains 2 AR response elements inserted in front of the coding region forluciferase in the pBL3 basic vector (Promega, Madison, WI). VDRE Lucwas obtained from Dr. Elizabeth Allegretto, Ligand Pharmaceuticals(17). Full-length DAX-1 cloned in pSG5 was obtained from Dr. EckardtTreuter, Karolinska Institutet, Sweden (18), and subsequently re-cloned into a mammalian expression vector pCR3.1 (Invitrogen, Carls-bad, CA). The N terminus of DAX-1 consisting of amino acids 1–260 andthe C terminus of DAX-1 consisting of amino acids 236–470 with analanine introduced before amino acid 236 were cloned into the pCR3.1expression vector. The N terminus of PR-B containing the N terminus,DNA binding domain, and hinge region (PR�LBD, amino acids 1–684)cloned into pCR3.1 was provided by Dr. Ramesh Narayanan, BaylorCollege of Medicine, Houston, TX. SRC-1e in pCR3.1 was prepared byDr. Martin Dutertre Baylor College of Medicine. SRC-1a in pCR3.1vector, SRC-1 fused to the VP16 activation domain and the GAL4 DBDwere described previously (19). ARA70 was provided by Dr. Chawns-hang Chang, University of Rochester, Rochester, NY (20). The C termi-nus of the AR, GAL-AR DH, contains AR amino acids 624–919 fused toamino acids 1–147 of the GAL4 DNA binding domain. The N terminus,VPAR ABC, contains AR amino acids 1–660 fused to amino acids411–456 of the VP16 transactivation domain. Both vectors were kindlyprovided by Dr. Elizabeth M. Wilson, University of North Carolina andwere previously described (21). Full-length PR fused to the VP16 acti-vation domain and the DBD and LBD of PR (amino acids 634–933)fused to GAL DBD were provided by Dean Edwards, Univ. of ColoradoHealth Sciences Center, Denver (22). The AR�LBD (amino acids 1–660)in pCMV vector was provided by Dr. Marco Marcelli (Baylor College ofMedicine, Houston, TX). The promoter interference CMV GRE3 CATreporter was provided by Dr. Dean Edwards (University of ColoradoHealth Sciences Center, Denver, CO) who prepared it by substitutingGRE elements for EREs in a construct originally provided by Dr. BenitaKatzenellenbogen, University of Illinois, Champaign). Full-length hu-man NCoR cloned in pSG5 was a generous gift from Dr. David Moore(Baylor College of Medicine, Houston TX). Human SMRT was obtainedfrom Dr. Ronald Evans (Salk Institute, La Jolla) and recloned intopCR3.1 by Dr. Carolyn Smith (Baylor College of Medicine, Houston TX)(4). The p53 mammalian expression vector and the MDM Luc reporterwere provided by Dr. Guillermina Lozano, UT MD Andersen CancerCenter, Houston, TX (23). Trichostatin A was obtained from SigmaAldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA),Methyltrienolone (R1881) and Promegestone (R5020) from Perkin-Elmer Life Sciences (Boston, MA). Monkey kidney COS-1 cells andHeLa (ATCC, Manassas, VA) were maintained in Dulbecco’s modifiedEagle’s medium in the presence of 5% fetal calf serum (Invitrogen).

Transient Cotransfection Experiments—Transient cotransfectionswere performed essentially as was described previously (24). Briefly,HeLa or COS-1 cells were plated the day before transfection at 50–60%confluency in DME media (Invitrogen) supplemented with 5% charcoal-stripped fetal bovine serum. The next day, the indicated amounts ofDNA were transfected using polylysine-coupled adenovirus for 2 h inserum-free Dulbecco’s modified Eagle’s medium (Invitrogen). An equalvolume of 10% charcoal-stripped fetal bovine serum was added to givea final concentration of 5% and cells were then treated with the indi-cated ligand or not for 24 h. Cells were harvested and assayed forreporter activity or used for Western blot analysis. For activity assayseach data point was done in triplicate and the average and standarddeviation calculated. Each experiment was performed a minimum ofthree times and a representative example is shown.

Immunoprecipitations—LNCaP (for AR interactions) or T47D (for PRinteractions) cells were grown to 70% confluence on 10 cm dishes. Cellswere treated for 1 h with the indicated hormone, harvested and proteinextracted by 3 freeze/thaw cycles in TESH buffer (10 mM TRIS, 1 mM

EDTA, 12 mM thioglycerol, pH 7.7) supplemented with 0.3 M NaCl andprotease inhibitors (25). Protein extracts were spun at 100,000 rpm ina TLA 100.3 rotor (540,000 � g) for 10 min at 4 °C. Fat was aspiratedand the insoluble material discarded. Clarified protein lysate was dia-lyzed against TESH buffer for 2 h at 4 °C, centrifuged at 100,000 rpmfor 10 min at 4 °C, and precleared (incubated) with 70 �l of a 1:1 proteinA-Sepharose suspension in TESH for 30 min at 4 °C. Sepharose beadswere spun and discarded. For SMRT interaction, protein A-Sepharosebeads were incubated with either 3 �g of SMRT-1542 monoclonal an-

tibody (Gene Tex, San Antonio, TX) and 5 �g of rabbit anti-mouse IgG(RAM) (Zymed Laboratories Inc., San Francisco, CA) or RAM alone, and1–4 mg of precleared protein extract overnight at 4 °C. For DAX-1interaction, protein A-Sepharose beads were incubated with 3 �g ofDAX-1 K17 rabbit polyclonal antibody or 3 �g of RAM (Santa CruzBiotechnology), and 1–4 mg of precleared protein extract overnight at4 °C. Beads were collected by brief centrifugation in an Eppendorfcentrifuge and were washed three times with TESH/0.1 M NaCl. Thesamples were transferred to fresh tubes prior to the last centrifugation,centrifuged, and the beads eluted with 2� Laemmli SDS sample buffer.Eluates were resolved by electrophoresis on 6.5% SDS-PAGE gels,transferred to nitrocellulose, and proteins detected by Western blotanalysis.

In Vitro Transcription/Translation and in Vitro Interaction—DAX-1was translated in vitro using TNT Quick Coupled Transcription/Trans-lation System (Promega, Madison, WI) and [35S]methionine (AmershamBiosciences) exactly as described in the manufacturer’s protocol. 75 �lof protein A-Sepharose slurry (1:1) and 100 �l of TESH/0.1 M NaCl wereincubated with 3 �g of either DAX-1 or rabbit antimouse IgG for 1 h onthe rocker at 4 °C. To measure binding of PR to DAX-1, beads werewashed twice with TESH/0.1 M NaCl, and 50 �l of DAX-1, 100 �l ofTESH/0.1 M NaCl, and 1 �g of purified baculovirus-expressed polyhis-tidine-tagged PR (25) were added. The mix was incubated for 2 h on arocker at 4 °C, washed twice and proteins eluted. Protein eluates werethen resolved on 6.5% SDS-PAGE and analyzed for PR by Westernblotting as described below. To measure DAX-1 binding to PR, 100 �l ofa 1:1 protein G-Sepharose (Amersham Biosciences) slurry in TESH and100 �l of TESH/0.1 M NaCl were incubated with 5 �g of PR6 antibody(a mouse monoclonal antibody that recognizes the B specific region ofPR) (26) for 1 h at 4 °C. Protein G-Sepharose beads were washed twicewith TESH/0.1 M NaCl, and 100 �l of TESH/0.1 M NaCl, 50 �l of DAX-1with or without 2 �g of purified polyhistidine-tagged PR were added.After a 2-h incubation on a rocker at 4 °C, samples were washed once inTESH/0.1 M NaCl, eluted with 2� Laemmli SDS sample buffer, andresolved on SDS-PAGE electrophoresis. DAX-1 coimmunoprecipitatedwith PR antibody was visualized following transfer to nitrocellulose byexposing the membrane to Kodak X—OMAT AR Film (Fisher, Pitts-burgh, PA).

Western Blot Analysis—PR Western blot analysis was performedusing the 1294 antibody (generously provided by Dr. Dean Edwards,University of Colorado Health Sciences Center, Denver, CO) as de-scribed previously (27). AR Western blots were done with AR 441antibody as described in Nazareth et al. (24). Actin Western blots wereperformed as was previously described (28).

Promoter Interference Assay—Cells were transfected with 15 ng ofCMV GRE3 CAT, and 100 ng of GRE2-E1b-Luc reporters, the indicatedreceptor or not, and increasing concentrations of either DAX-1 orSMRT. Hormone (3 nM R1881 for experiments with AR and 10 nM

R5020 in experiments with PR) was added 2 h after transfection, cellswere harvested 24 h later, and cellular lysates were assayed for CATand luciferase activity.

Luciferase Assay—Cells were washed with phosphate-buffered salineonce and 200 �l of lysis buffer (provided by the manufacturer) added toeach well in a 6-well plate. After a 15-min incubation, 20 �l of the lysatewas used to measure luciferase activity as recommended by the man-ufacturer (Promega).

CAT Assay—CAT assays were carried out as was described previ-ously (29). Briefly, cells were harvested in TEN buffer (Tris, EDTA,NaCl), spun, and the buffer was removed. Cells were then lysed in 0.25M Tris (pH 7.5), supplemented with 0.4 M NaCl if the lysates were laterused for western as well as CAT assay, by freezing and thawing. 10–20�g of the protein were used to determine CAT activity.

�-Gal Assay—�-Galactosidase activity was measured to normalizeactivity of the receptors. The �-galactosidase gene cloned in the sameexpression vector (pCR 3.1) as PR and AR was used to aid in controllingboth for transfection efficiency as well as any effects of treatment on theactivity of the expression plasmid promoter. Ten percent of cellularlysates prepared for the luciferase assay was used to perform the�-galactosidase activity assay as previously described (30).

Tissue Microarray—Tissue arrays were made in the Baylor ProstateSPORE. Samples were harvested as previously described (31). Tissuearrays containing cores from normal peripheral zone and areas of BPHwere analyzed for intensity and labeling frequency. The intensity of thegrading scale ranged from no detectable signal (0) to a strong signalseen at low power (3). Two corresponds to a moderate signal seen at lowto intermediate power, while one corresponds to a weak signal seen onlyat intermediate to high power. Labeling frequency was scored as 0 (0%),1 (1–33%), 2 (34–66%), or 3 (67–100%).

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Immunohistochemistry—Briefly, the slides were deparaffinized, re-hydrated, and then heated in 10 mM citrate buffer pH 6.0 for 30 minusing a steamer. The slides were blocked with 10% normal rabbit serumfor 30 min. After washing in PBS, the slides were incubated with therabbit anti-DAX-1 polyclonal antibody (1:100) for 1 h at room temper-ature. Then the secondary biotinylated anti-rabbit IgG was applied for30 min followed by 30 min of incubation with streptavidin peroxidase(LSAB kit, DAKO). After rinsing, slides were visualized by diamino-benzidine chromogen solution (DAKO) and counterstained withroutine hematoxylin.

RESULTS

DAX-1, SMRT, and NCoR Repress Both Agonist-and PartialAntagonist-dependent AR Activity—To characterize the effectsof DAX-1, SMRT, and NCoR on AR activity, we transientlytransfected HeLa cells with a constant amount of receptor andreporter expression plasmids and increasing amounts of re-pressor plasmid balanced with the corresponding vector con-trol. As shown in Fig. 1A, DAX-1 inhibits agonist (R1881)-de-pendent activity of AR as recently reported (15) as does SMRT.Both DAX-1 and SMRT also repress the activity of AR in the

presence of the partial antagonist RU486 (Fig. 1B). Both ofthese cofactors repressed AR activity in HeLa and COS-1 cellsto a similar extent (data not shown). Surprisingly, NCoR re-pressed AR activity in the presence of RU486 and R1881 butonly in a cell-specific context. Under identical transfection con-ditions AR is repressed by NCoR in COS-1 (Fig. 1D) but not inHeLa cells (Fig. 1C). To determine if changes in receptor activ-ity are due to changes in receptor levels we performed Westernblots using samples from the transrepression experiments. Aspreviously reported (32), addition of agonist increases AR ex-pression through stabilization of the protein. However, expres-sion of repressors did not change levels of AR expression(Fig. 1E).

DAX-1 and SMRT Repress PR Activity—It has been reportedpreviously that SMRT preferentially represses partial antago-nist bound PR activity (33). However, under our transfectionconditions PR was repressed by SMRT in the presence of eitheran agonist or a partial antagonist to a similar extent in bothHeLa (Fig. 2, A and B) and COS-1 (data not shown) cell lines.

FIG. 1. SMRT, DAX-1 and NCoR repress AR activity. A, HeLa cells were transfected with 5 ng of pCR3.1 AR, 400 ng of GRE2 E1b Lucreporter, 30 ng pCR3.1 �-Gal, and either 150 ng of pCR3.1 vector or increasing concentrations of pCR 3.1 SMRT or DAX-1 supplemented with thepCR3.1 vector to a total amount of 150 ng. After transfection, cells were either left untreated (open bars) or treated with 3 nM R1881 (filled bars).24 h later cells were harvested and assayed for luciferase and �-galactosidase activity. Relative luciferase units (RLU) were normalized for�-galactosidase activity. Each data point was done in triplicate, and the average and S.D. calculated. B, HeLa cells were transfected and assayedas in A, except after transfection they were either left untreated (open bars) or treated with 100 nM RU486 (gray bars). C, HeLa cells weretransfected with 8 ng of AR and identical amounts of reporter, pCR3.1 �-galactosidase, and the indicated amounts of NCoR balanced with the pSG5vector to 200 ng. Cells were treated (no treatment, open bars; 3 nM R1881, filled bars, 100 nM RU486, gray bars) and assayed as in A. D, COS-1cells were transfected as in C. After transfection, cells were either left untreated (open bars), treated with 3 nM R1881 (filled bars) or treated with100 nM RU486 (gray bars). E, 40 �g of cellular extracts from the transrepression experiment in A and B were resolved on SDS-PAGE, transferredto a nitrocellulose membrane and AR detected as described under “Experimental Procedures.”

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The effect of DAX-1 on PR activity has not been reportedpreviously. Interestingly, DAX-1 appeared to be a much stron-ger repressor of PR than AR in transient cotransfection assays,since much less DAX-1 DNA was necessary to repress theagonist-dependent PR activity although repression by SMRTwas similar to AR (Fig. 2A). While SMRT represses PR antag-onist dependent activity as expected, most intriguing was theeffect observed with increasing concentrations of DAX-1 on thePR bound to partial antagonists (Fig. 2B). DAX-1 slightly in-creased the RU486-dependent activity of PR. Note that al-though DAX-1 potentiates RU486-dependent activity of PR, themaximal activity is still less than that of agonist-bound PRtreated with DAX-1. As seen from Fig. 2C, the expression ofRU486-bound PR is slightly higher in the presence of increas-ing concentrations of DAX-1, which may explain the increase inactivity of partial antagonist bound PR (compare Fig. 2, B andA). The opposite effect of DAX-1 on PR in the presence ofdifferent ligands suggests that its mechanism of repressionmay be different from that for AR. In contrast to AR, theexpression levels of PR are reduced in response to agonist (34).As in the case of AR, levels of agonist-bound PR are not reducedby the addition of increasing concentrations of DAX-1 andSMRT (Fig. 2C), and therefore this does not contribute todecreasing PR activity.

DAX-1 Does Not Repress VDR and p53 Activity—To deter-mine if DAX-1 shows specificity in repression of transcriptionfactors we asked whether DAX-1 would repress the ubiqui-tously expressed transcription factor p53, or the activity of thevitamin D receptor (VDR) another member of the nuclear re-ceptor family. As shown in Fig. 3A DAX-1 does not repress p53activity (Fig. 3A). Interestingly, VDR which lacks an N-termi-nal domain and thus AF-1 is practically unaffected by increas-

ing concentrations of DAX-1 with or without 1,25 D (Fig. 3B).We used protein levels to normalize the activity of VDR andp53, because VDR was endogenously expressed and p53 was ina different vector from that of �-Gal. However, as we observedfrom our previous experiments, neither DAX-1 nor SMRT af-fected �-galactosidase expression at any DNA level tested andtherefore either protein concentration or �-galactosidase activ-ity can be used to normalize the experiments.

AR and PR Interact with DAX-1 and SMRT—Although otherinvestigators have also shown functional interactions betweenAR and SMRT or DAX-1 and we have shown functional inter-actions between agonist-bound PR and both SMRT and DAX-1,the physical interactions are not as well characterized. Holteret al. (15) has shown in vitro interaction between AR andDAX-1, Dotzlaw et al. (35) has shown in vitro interaction be-tween AR and SMRT and Liao et al. (36) has shown an in vivointeraction between AR and SMRT. In contrast, only the invitro interaction between PR and SMRT has been shown (33).To confirm that a physical interaction occurs between endoge-nous AR and DAX-1 in vivo we performed coimmunoprecipita-tion experiments using the LNCaP cell line. As shown in Fig.4A DAX-1 but not rabbit anti-mouse IgG (RAM) antibody im-munoprecipitated AR. To determine if DAX-1 and PR are in-volved in direct protein-protein interactions we first incubatedin vitro translated DAX-1 and purified polyhisidine-taggedPRB and performed coimmunoprecipitation with either DAX-1or PR antibody. As shown in Fig. 4B, PRB was co-precipitatedmuch better by DAX-1 antibody than by RAM antibody. Con-versely, DAX-1 was coimmunoprecipitated by PR antibodypreferentially in the presence of PR (Fig. 4C). To confirm thephysical interaction of endogenous PR with DAX-1 and SMRT

FIG. 2. DAX-1 and SMRT repression of pure agonist- and partial antagonist-bound PR activity. HeLa cells were transiently transfectedwith 5 ng of pCR3.1 PR, 400 ng of GRE2 E1b Luc reporter, 30 ng pCR3.1 �-galactosidase, and either 150 ng of pSG5 vector or increasingconcentrations of pSG5 DAX-1 supplemented with vector to a total amount of 150 ng. A, repression of PR by SMRT and DAX-1 was determinedwithout ligand (open bars) or with 10 nM of the agonist R5020 (filled bars). B, in the same experiment as A, regulation of PR by SMRT and DAX-1was determined without the ligand (open bars) or with 100 nM of the partial antagonist RU486 (gray bars). C, 40 �g of protein from cellular extractsof samples shown in panels A and B were resolved on SDS-PAGE, transferred to a nitrocellulose membrane, and PR detected as described under“Experimental Procedures.”

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in vivo we performed coimmunoprecipitation experiments us-ing T47D cells. As shown in Fig. 4D DAX-1 but not RAMantibody precipitates PR. We were able to detect modest SMRTinteraction with PR in extracts from both R5020 and RU486-treated cells relative to the control RAM (Fig. 4E). The fact thatonly the A isoform of PR is detectable is probably in part aresult of a much lower level of isoform B expression in ourT47D strain, as seen from the input lanes.

TSA Does Not Relieve Repression of AR by SMRT—SinceSMRT has been reported to physically interact with multipleHDACs and repression has been attributed to the activity ofHDACs (3, 37) we asked whether the HDAC inhibitor, tricho-

statin A (TSA) would block SMRT-mediated repression. Asshown in Fig. 5A, the fold repression of AR by SMRT remainsunchanged in the presence of R1881 alone or in combinationwith TSA, although TSA increases overall activity. Thus, TSA-sensitive HDACs do not appear to be involved. Even thoughDAX-1 has neither HDAC catalytic activity nor a putativeHDAC binding site, there is a report that DAX-1 can interactweakly with NCoR, a protein that does interact with HDACs(18, 38). Thus, we asked whether an HDAC inhibitor, TSA,would relieve repression by DAX-1. Similar to SMRT, althoughthe overall activity increased, no difference in repression levelwas observed for DAX-1 (data not shown).

FIG. 3. DAX-1 does not repress p53or VDR activity. A, each well in a 6-wellplate of HeLa cells was transfected with 5ng of p53 expression vector, 400 ng ofMDM Luc reporter, and either vectoralone or increasing concentrations ofpSG5 DAX-1 supplemented with vector toa total concentration of 100 ng. Cells wereassayed for luciferase activity the nextday and normalized for protein levels. B,HeLa cells were transfected with 400 ngof VDRE Luc reporter, and either vectoralone or increasing concentrations ofDAX-1. Cells were either left untreated ortreated with 10 nM 1,25-dihydroxyvita-min D3. The activity of endogenous VDRwas determined the next day and normal-ized for protein.

FIG. 4. Analysis of physical interaction of AR and DAX-1 and PR with SMRT and DAX-1. A, protein extract (4 mg) prepared from 3 nM

R1881-treated LNCaP cells was immunoprecipitated with either DAX-1 rabbit polyclonal antibody or nonspecific rabbit anti-mouse IgG (RAM)antibody. Immunoprecipitated proteins were resolved on SDS-PAGE and analyzed by Western blotting with AR 441 antibody. I, 10 �g of LNCaPprotein extract. B, 1 �g of polyhistidine-tagged PRB and 50 �l of [35S]methionine labeled in vitro translated DAX-1, were coimmunoprecipitatedwith either DAX-1 or RAM antibody. Precipitated proteins were eluted and run on an SDS-PAGE gel along with the input. PR was detected byWestern blotting with 1294 PR antibody. I, 100 ng of PR; C, 50 �l [35S]methionine labeled DAX-1 was coimmunoprecipitated with PR6 antibodyin the presence or absence of 2 �g of polyhistidine-tagged PRB. The eluted proteins were run on an SDS-PAGE along with 2 �l of starting35S-labeled DAX-1 in vitro translation mix (I), transferred to nitrocellulose, and the membrane was exposed to X-OMAT AR film for 30 min to detect35S-labeled DAX-1. D, 3 mg of cleared protein extract from T47D cells treated with 100 nM R5020 were used in an immunoprecipitation with eitherDAX-1 or RAM antibody. Proteins were extracted as described in methods, run on a 6.5% SDS-PAGE gel and PR detected by Western blot usingthe 1294 antibody. I, 5 �g T47D extract. E, 2 mg of precleared T47D cell extract prepared from cells treated with either 100 nM R5020 (left panel)or 10 nM RU486 (right panel) were used for immunoprecipitation with either SMRT and RAM antibody or RAM antibody only. On the left of eachgel are 5 and 1 �g of protein extracts from T47D cells treated with the indicated ligand. On the right are the immunoprecipitated proteins.Precipitated PR was detected by Western blot analysis using 1294 PR antibody.

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DAX-1 Repression Is Reversed by Coactivators That Act Pri-marily through AF-1 (SRC-1e) or AF-2 (ARA70)—Next wewished to determine whether AR repression by DAX-1 could becompeted by AF-1 or AF-2 interacting coactivators. We testedSRC-1e, a splice variant of the more commonly studied SRC-1a,that lacks the C-terminal LXXLL motif of SRC-1a and isthought to interact with AR exclusively through AF-1 (39) andARA70, which interacts through the ligand binding domain(40) for their ability to relieve repression of AR by DAX-1. Asshown in Fig. 5B, repression by DAX-1 can be reversed byincreasing concentrations of either ARA70 or SRC-1e coactiva-tors. Two explanations are possible for the coactivator reversalof repression of AR by DAX-1. First, coactivators could becompeting for the same binding interface on the AR dimer.Alternatively, the corepressors and coactivators can act inde-pendently of each other and the final activity of the receptor isa result of the balance between coactivators and corepressors.

DAX-1 Does Not Disrupt the Interaction of AR and the Coac-tivator SRC-1a in the Mammalian Two-hybrid Assay—To de-termine whether DAX-1 competes with the AF-1 coactivatorbinding site we measured the ability of DAX-1 to interruptinteraction between the AR N terminus and SRC-1a in a mam-malian two-hybrid assay. The schematic of the constructs usedin the interaction assay is shown in Fig. 6D. As shown in Fig.6A no disruption of the interaction between the N terminus andSRC-1 was observed. This suggests that DAX-1 and possiblyother AF-1-interacting coactivators have distinct binding sites.Steroid receptors interact with coactivators in part throughLXXLL motifs, which interact with AF-2. Since DAX-1 has oneLXXLL motif and 2 LXXLL-like motifs, it is possible that itcompetes with the coactivators for binding to the LBD of thereceptor. However, no interference by DAX-1 was detected in amammalian two-hybrid assay between SRC-1a and the C ter-minus of AR (Fig. 6B). Moreover, DAX-1 did not affect interac-tion between full-length SRC-1a and full-length AR in a mam-malian two-hybrid assay showing that the combined AF-1 andAF-2 interaction of AR with SRC-1a is not compromised byDAX-1 (Fig. 6C). Note that there is a significant hormone-independent interaction between full-length AR and SRC-1,presumably through AF-1 and that this is increased byhormone.

Both SMRT and DAX-1 disrupt the N-C-terminal Interactionof AR—AR dimers are formed in an antiparallel orientationunlike other steroid receptors (11). Interaction between the Nand C-terminal domains of the receptor stabilizes the dimer. Todetermine whether DAX-1 and SMRT disrupt the contact be-tween the N and C terminus of AR we performed mammaliantwo hybrid experiments with Bind ARDH and Act ARABC (SeeFig. 6D for construct design) alone or with increasing concen-trations of DAX-1 or SMRT. As shown in Fig. 7, A and B, bothSMRT and DAX-1 disrupt interaction between N- and C-ter-minal fragments of AR.

DAX-1 Affects PR Dimer Interactions but Not Interactionwith SRC-1—Recent reports suggest that the LXXLL motifs ofcoactivators such as SRC-1 bind to agonist-bound receptors ina region that overlaps with the site in antagonist bound recep-tor that is recognized by CoRNR box motifs of SMRT and NCoR(41). Although DAX-1 has no perfect consensus CoRNR motifswe asked if its mechanism of action is similar. Interestingly,DAX-1 did not compete with SRC-1 for PR interaction (Fig. 7C).PR is a parallel dimer stabilized by interaction between LBDsand possibly hinge domains that can be detected in a mamma-lian two-hybrid assay (9). DAX-1 disrupted PR intramolecularinteractions between full-length PR (Act PR) and the DBD LBDportion of PR (Bind PRLBD) (Fig. 7D).

DAX-1 Represses the Activity of the AR�LBD Mutant but Notof the PR�LBD Mutant, While SMRT Represses Both PR andAR �LBD Fragments—To identify the regions of AR and PRnecessary for functional interaction with DAX-1 and SMRT welooked at DAX-1 and SMRT repression of constitutively activeAR and PR mutants lacking the LBD (amino acids 1–660 forAR and 1–684 for PR) and therefore AF-2. As shown in Fig. 8Atruncated AR is repressed by both DAX-1 and SMRT thoughpossibly to a slightly lesser extent than wild-type AR. However,PR lacking the LBD is repressed only by SMRT (Fig. 8D) andnot by DAX-1 (Fig. 8C) although both (Fig. 8B) repress full-length PR.

The N Terminus of DAX-1 Is Sufficient to Repress PR Activitybut Not AR Activity—Nuclear receptor-interacting domains inSMRT have been mapped previously (36). To determine whichpart of DAX-1 is necessary for AR and PR repression, we clonedthe N-terminal and helical C-terminal domains into the mam-

FIG. 5. TSA does not relieve repression of AR by SMRT and coactivators relieve AR repression by DAX-1. A, HeLa cells weretransfected with 5 ng of pCR3.1 AR, 30 ng of pCR3.1 �-galactosidase, 400 ng of GRE2 E1b Luc reporter, and either pCR3.1 vector or increasingconcentrations of pCR3.1 SMRT. After transfection cells were either left untreated (open bars), treated with 3 nM R1881 (filled bars), or 3 nM R1881and 400 nM Trichostatin A (TSA) (gray bars). B, cells were transfected with 5 ng of pCR3.1 AR, GRE2 E1b Luc reporter, 30 ng pCR3.1 �-Gal, 65ng of pCR3.1 or pSG5 or DAX-1, and increasing concentrations of SRC-1 balanced with pCR3.1 to a total of 300 ng, or increasing concentrationsof ARA70 balanced with pSG5 to a total of 300 ng of DNA. Cells were either left untreated or treated with 3 nM R1881. Cellular extracts wereassayed for luciferase and �-galactosidase activity, and RLU values were normalized for �-galactosidase activity. Filled bars, R1881-treatedsamples balanced with pCR3.1 for analysis of SRC-1 activity.

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malian expression vector pCR3.1 and used them in the trans-repression assay. As shown in Fig. 8E only full-length DAX-1 iscapable of repressing AR. On the other hand both full-lengthand the N terminus of DAX-1 repress PR. It is evident however,that full-length DAX-1 is much more effective in repressing PR(Fig. 8F).

Neither DAX-1 nor SMRT Reduce the Ability of AR and PR toBind DNA in a Promoter Interference Assay—Holter et al. (15)have suggested that DAX-1 may prevent AR from localizing tothe nucleus and thus binding to DNA. To test whether DAX-1or SMRT reduce DNA binding of AR or PR, we performedsimultaneous promoter interference assays using a CAT re-porter and a transrepression assay using a luciferase reporter.The two different reporters allowed us to measure in the samecell whether amounts of DAX-1 and SMRT sufficient for repres-sion (inhibition of luciferase activity) would remove receptorsfrom the DNA in the promoter interference assay (thus bring-ing the CAT activity to the level measured in the absence of

activated AR). The model for this experiment is shown in Fig.9. The promoter interference assay is based on the fact that theCMV promoter is much stronger than the hormone responsivepromoter formed by the three ARE/PRE elements that arelocated between the CMV promoter and the CAT transcriptionstart site. The CMV ARE (PRE) CAT promoter is constitutivelyactive in the absence of AR or PR (Fig. 9A). Binding of ligandedPR or AR to the ARE/PRE response elements partially blockstranscription from the stronger upstream CMV promoter (Fig.9B). If DAX-1 or SMRT remove the receptor from the DNA, theCAT activity will return to the no receptor level (Fig. 9C).However, if SMRT or DAX-1 interacts with receptors on theDNA, the activity will remain reduced (Fig. 9D). In contrast, inthe absence of hormone the positively regulated ARE Luc pro-moter will be inactive (Fig. 9A) and addition of hormone willstimulate luciferase activity (Fig. 9B). Whether the repressorcauses release from DNA (Fig. 9C) or simply inhibits the ac-tivity of the DNA-bound receptor (Fig. 9D), luciferase activity

FIG. 6. The SRC-1 coactivator interacting site differs from the DAX-1 interacting site in AR. A, 400 ng of 17-mer Luc and the indicatedcombinations of either 500 ng of Act or Act ARABC, 100 ng of Bind or Bind SRC-1, and increasing concentrations of DAX-1 balanced with pCR3.1to a total of 100 ng were transfected; 24 h post-transfection cells were assayed for luciferase activity. RLU values were normalized to total proteinconcentration. B, cells were transfected as in A except Bind ARDH and Act SRC-1 were used and cells were either treated with 10 nM R1881 (filledbars) or left untreated (empty bars). C, cells were transfected as in A except Act AR and Bind SRC-1 were used. After transfection cells were eitherleft untreated (empty bars) or treated with 10 nM R1881. 24 h later cells were harvested and assayed for luciferase activity that was normalizedfor protein concentration. D, structure of the constructs used in the interaction assays as previously reported (11). Full-length AR was fused to Act(an activation domain of VP16). Act ARABC contains the N terminus, DNA binding domain and hinge regions (dotted area) fused to the VP16activation domain (Act). Bind ARDH contains the DNA binding domain, hinge, and LBD of AR fused to Bind (DNA4 DBD). Act PR contains theVP16 activation domain fused to full-length PRB. Bind PRLBD chimera has the GAL4 DBD fused to the hinge and LBD of PR. Bind SRC-1a is afull-length SRC-1a with the GAL4 DBD fused to the N terminus.

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will be reduced in the presence of repressor. As seen in Fig. 10under conditions where DAX-1 repressed AR (Fig. 10A, lowerpanel) and PR (Fig. 10B, lower panel) activity, both receptorscontinued to interfere with the stronger CMV promoter simi-larly to the receptor without repressors. This shows that inhi-bition by DAX-1 is not due to elimination of DNA binding. Thestatistically insignificant difference in the activity of the CMVCAT reporter in the presence of pSG5 and pSG5 DAX-1 plas-mids in the absence of ligand (Fig. 10A, upper panel) may bedue to a slight difference in squelching of these constructs. Notethat in the case of PR (Fig. 10B, upper panel) no difference isobserved since much less pSG5 DAX-1 DNA is needed to re-press PR activity. Alternatively, DAX-1 may facilitate the bind-ing of unliganded AR to the DNA. Colocalization of SMRT andAR on a target gene has been reported previously (42). Inagreement with this we see that addition of SMRT sufficient for

repression in the same assay (data not shown) does not relieveAR (Fig. 10C) and PR (Fig. 10D) interference with the CMVpromoter. The identical behavior of AR and PR in this assayfurther supports the idea that DAX-1 represses their activityby transforming an active receptor complex on the promoterinto an inactive one.

DAX-1 Is Expressed in Prostate and Its Expression Is Dimin-ished in Benign Prostatic Hyperplasia—Although the studiesshown here as well as previously published studies, suggestthat corepressors have the potential to modulate the activity ofreceptors in vivo, little is known about their roles in receptorfunction. If corepressors are important in the negative regula-tion of AR activity, then we predict that we would find reducedexpressionof corepressorunderconditionsofaberrantandrogen-dependent growth such as in BPH. To test this, we used pros-tate tissue microarrays to measure the expression of DAX-1

FIG. 7. Effect of corepressors on protein-protein interactions of AR and PR. A, HeLa cells were transfected with 400 ng of 17-mer Lucreporter and the indicated combinations of either 100 ng of Bind or 100 ng of Bind ARDH, 500 ng of Act, or 500 ng of Act ARABC, and increasingamounts of DAX-1 balanced with pSG5 vector to a total of 500 ng. Cells were either left untreated (open bars) or treated with 10 nM R1881 (filledbars). B, cells were transfected as in A and increasing amounts of SMRT balanced with pCR3.1 to a total of 500 ng were added instead of DAX-1.Cells were left untreated (open bars) or treated with 10 nM R1881 (filled bars). C, HeLa cells were transfected with 17-mer Luc reporter, and theindicated combinations of 20 ng of Bind or Bind SRC-1, 100 ng of Act, or Act PR, and increasing concentrations of DAX-1 balanced with the vector.After transfection, cells were either treated with 100 nM R5020 (filled bars) or left untreated (open bars). Cells were lysed and assayed for luciferaseactivity. RLU values were normalized to protein concentration. D, cells were transfected as in C except 20 ng of Bind PRLBD was used. Reporteractivity was normalized for protein levels.

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FIG. 8. Domains of AR, PR, and DAX-1 necessary for functional interaction. A, HeLa cells were transfected with 400 ng of GRE2 E1b Lucreporter, 40 ng of pCR3.1 �-Gal, 5 ng of AR �LBD (amino acids 1–660), and either increasing concentrations of DAX-1 balanced with its expressionplasmid pSG5 or increasing concentrations of SMRT balanced with the corresponding expression vector pCR3.1. Luciferase activity was normalizedfor �-galactosidase expression. B, cells were transfected with 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 �-Gal, and the indicated amountsof PR and repressors balanced with the corresponding vector to a total of 40 ng. Cells were either left untreated (open bars) or treated with theappropriate agonist (10 nM R5020 for PR and 3 nM R1881 for AR) (filled bars). Ratios of 1 to 2.5 and 1 to 8 were found to be optimal for PR repressionby DAX-1 and SMRT, respectively, and the repression by these levels is shown here. C, 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 �-Gal,and increasing concentrations of PR�LBD were transfected into HeLa cells in the presence of pSG5 vector to demonstrate the receptor dependenceof the luciferase activity. The highest level of receptor mutant (60 ng) was also transfected with increasing concentrations of pSG5 DAX-1 balancedwith vector to test the effects of DAX-1 on activity. Luciferase activity was normalized for �-galactosidase activity. D, cells were transfected as inC except pCR3.1 SMRT balanced with pCR3.1 vector to 500 ng was used instead of DAX-1. E, cells were transfected with 400 ng of GRE2 E1b Lucreporter, 40 ng of pCR3.1 �-Gal, 5 ng of AR, and increasing concentrations of either pCR3.1 DAX-1, or pCR3.1 DAX-11–226, or pCR3.1 DAX-1220–413,all balanced with the pCR3.1 vector to the same amount of DNA. Cells were either treated with 3 nM R1881 (filled bars) or not (open bars) for 20 h,harvested, and assayed for luciferase and �-galactosidase activity. F, cells were transfected as in E, except 5 ng of PR was used. Cells were leftuntreated (open bars) or treated with 10 nM R5020 (filled bars) for 20 h, and assayed for luciferase and �-galactosidase activity.

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protein in prostate. DAX-1 was expressed in the nuclei of nor-mal prostate and BPH with varying levels of expression. Innormal prostate, nuclei of basal and luminal epithelial cellsstain strongly as do the nuclei of many of the stromal cells. Themean values for both intensity and amount of cells expressingDAX-1 in normal prostatic tissues were 2.5 and 2.3, respec-tively. Meanwhile, a much lower level of expression was iden-tified in BPH tissues (1.4 and 1.4). While the vast majority ofcells in normal tissues expressed DAX-1 with very high inten-sity (68.5%), the majority of the cells in BPH were negative forDAX-1 or had low to medium levels of expression (Fig. 11).71.5% of the normal prostate samples have medium to highDAX-1 expression, while 51.5% of BPH samples have negativeto low DAX-1 expression.

DISCUSSION

In this study, we have shown that corepressors differentiallyrepress the activities of nuclear receptors and that the samecorepressor may utilize different means and domains to repressthe activities of nuclear receptors. As reported recently, DAX-1represses the activity of agonist bound AR (15) as well as thepreviously described SF-1 orphan receptor (7) and ER (18). Weshow here that DAX-1 also represses the activity of PR, butdoes not repress the activity of endogenous VDR in HeLa cells.Thus, DAX-1 is not a universal repressor for nuclear receptors.Remarkably, DAX-1 represses RU486-dependent activity ofAR, but does not repress RU486-dependent activity of PR. Wefind that SMRT, which has previously been considered to be arepressor of antagonist-bound receptors (4, 33), also repressesthe agonist activity of both AR and PR (Figs. 1 and 2). AlthoughDotzlaw et al. (35) detected both agonist and partial antagonist-dependent interaction between the C-terminal of SMRT andAR using a modified mammalian two-hybrid assay, SMRT wasnot very effective in repressing agonist bound AR activity inCV1 cells. However, Liao et al. (36) have just reported SMRTrepression of agonist bound AR in 293 cells. Thus, the ability ofSMRT to repress agonist bound AR may be cell type-dependent.Our finding that SMRT inhibits constitutively active AR lack-ing the LBD clearly demonstrates that an antagonist is notrequired for functional interaction between AR and SMRT. Wefind that both agonist- and partial antagonist-dependent activ-

ities of PR are inhibited by SMRT with SMRT being slightlymore effective in inhibiting antagonist-dependent activity.NCoR and SMRT have been reported to function in a similarmanner and both inhibit the activity of unliganded TR (1, 2).Surprisingly, NCoR does not appear to inhibit AR activity inHeLa cells, although it does inhibit AR activity in COS-1 cells(Fig. 1). Cheng et al. (43) recently reported that NCoR caninhibit the activity of AR in transfected CV-1 cells. The findingthat the interaction between NCoR and the AR requires theLBD of AR (43) whereas the SMRT interaction site has beenlocalized to the N terminus of AR (35) supports our finding ofdifferential functional interactions in HeLa cells.

Although in vitro interactions between DAX-1 and AR havebeen reported (15), there have been no reports of interactions ofendogenous DAX-1 and AR. Neither functional nor physicalinteractions between PR and DAX-1 have been reported. Usingan antibody to DAX-1, we precipitated endogenous PR fromT47D cells and endogenous AR from LNCaP cells demonstrat-ing an in vivo interaction. Although the coimmunoprecipitationwas antibody-specific (Fig. 4), the percent of receptor precipi-tated was rather low. This is, perhaps, not surprising as bothcell lines express relatively high levels of receptor and based onour Western blotting of cell extracts (data not shown), eitherthe levels of DAX-1 protein are low or the DAX-1 antibody isnot a high affinity antibody. Unfortunately, the molecular massof DAX-1 (51.7 kDa) precludes the reverse experiment as itcomigrates with the heavy chain of IgG. In vitro (35) and in vivo(36, 42) interactions of AR with SMRT have been reportedpreviously. The in vivo physical interaction between AR andSMRT is stronger in the presence of the antagonist, flutamidethan with DHT (36). We observe an interaction between eitherRU486- or R5020-bound PR and SMRT in the T47D cell linealthough the interaction relative to nonspecific was not asclean as in the case of DAX-1 (Fig. 4). Therefore, we scannedthe films from multiple experiments to better assess the bind-ing. In the course of seven experiments the intensity of theR5020 bound PR band pulled down by the SMRT antibody wason average 2.2-fold greater than that obtained with RAM an-tibody. In contrast, cells treated with RU486 produced an av-erage difference of 5.4-fold between specific and nonspecific

FIG. 9. Schematic of the promoter interference assay. A, in the absence of liganded receptor the constitutively active CMV promoter inducesCAT transcription and the (ARE)2 Luciferase promoter is silent. B, when hormone is added, activity of the stronger CMV promoter is compromisedby 3 interfering AR or PR dimers, while the GRE2 Luciferase promoter is now active. C, if the corepressor takes receptor off DNA, then activityof the CAT reporter returns to the initial level when no receptor is interfering with the CMV promoter, while luciferase reporter activity is reduced.D, if corepressor-receptor complexes remain on DNA, the CMV promoter activity remains reduced because receptor dimers remain between theCMV promoter and the CAT coding sequence and the activity of the GRE2 Luciferase promoter is reduced due to repression of the ARtranscriptional activity by corepressors. HSP-heat shock protein complex.

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binding (five experiments) as determined by densitometryscans. Thus, we believe these results reflect an in vivointeraction.

Although in some cases SMRT represses transcription byrecruiting HDACs to the promoter, the histone deacetylaseinhibitor, TSA, does not block SMRT-dependent repression ofAR demonstrating that the inhibitory mechanism for AR dif-fers. Our finding that SMRT inhibits N/C-terminal interactions

implies that SMRT bound to the N terminus prevents theN/C-terminal interaction presumably through steric interfer-ence. That SMRT inhibits the activity of partial antagonist-bound AR, which does not induce N/C-terminal interactions(44), and AR lacking its hormone binding domain suggests thatbinding of SMRT also interferes with the formation of produc-tive AR coactivator complexes. Our observation that SMRTblocks the activity of PR lacking its hormone binding domain

FIG. 10. SMRT and DAX-1 bind PR and AR without removing them from the promoter. Hela cells were transfected with GRE2 E1b Lucreporter, CMV GRE3 CAT reporter, and 10 ng of AR and increasing concentrations of DAX-1 (A); 10 ng of PR and increasing concentrations ofDAX-1 (B); 10 ng of AR and increasing concentrations of SMRT (C); 10 ng of PR and increasing concentrations of SMRT (D). Cells were either leftuntreated (open bars) or treated with the appropriate agonist for each receptor (filled bars, 3 nM R1881 for AR and 10 nM R5020 for PR). Cells wereharvested, lysed, and assayed for CAT and luciferase activity, which was normalized for protein levels. Simultaneous measurements of CAT andluciferase activity show that amounts of DAX-1 that are optimal for AR and PR repression (A and B) do not take off receptors from the identicalbinding sites between the CMV promoter and the CAT coding sequence.

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also suggests that there is a region distinct from the hormonebinding domain that interacts with SMRT. The previous studyby Wagner et al. (33) showing PR interacting with the C-terminal portion of SMRT only in the presence of antagonistsutilized the PR LBD linked to GAL4 DBD. Thus, there is likelyan additional region of PR that also interacts with SMRT andinduces the agonist-dependent repression.

DAX-1 is the least well characterized of the repressors wehave examined. Although DAX-1 inhibits agonist-dependentactivity of both PR and AR, several other features suggest thatDAX-1 interactions with the two receptors differ substantially.DAX-1 inhibits the activity of the N terminus of AR but has noeffect on the activity of the N terminus of PR. This is consistentwith its failure to block antagonist-induced activity of PR,which is mediated through its N terminus. Both the N and Ctermini of DAX-1 appear to be necessary for AR repression,since neither the putative helical ligand binding domain northe N-terminal RNA/ssDNA binding motif alone are sufficientto repress AR function. Since the N terminus has 3 LXXLL-likemotifs, it is possible that this is the interacting portion ofDAX-1, although the C terminus is required for repression.Holter et al. (15) found that the N terminus of DAX-1 wassufficient to cause aberrant localization of AR in the cytoplasmimplicating this region in a physical interaction. In contrast,the N terminus of DAX-1 is sufficient to partially repress theactivity of PR albeit to a lesser extent than the full-length (Fig.7F). Thus, although the activities of both PR and AR are re-

pressed by DAX-1, the functional interactions differ substan-tially. Whereas DAX-1 inhibits both the agonist- and antagonist-dependent activity of AR as well as inhibiting an N-terminalfragment of AR, only the agonist-dependent activity of PR isinhibited and the N-terminal portion of DAX-1 is sufficient toinduce partial inhibition.

Holter et al. (15) had found that expressing high levels ofDAX-1 caused mislocalization of hormone bound GFP-AR tothe cytoplasm, leading them to propose that DAX-1 acts in partby blocking nuclear localization and, thus, DNA binding of AR.However, our promoter interference studies show that DAX-1does not reduce the DNA binding-dependent AR- or PR-medi-ated repression of a constitutive promoter while, in the samecell, preventing transactivation of a receptor responsive lucif-erase reporter (Fig. 10). Thus AR remains associated with theDNA and the mislocalization may be an artifact of overexpres-sion. Certainly, DAX-1 is predominantly nuclear in prostate(Fig. 11) suggesting that this is an unlikely mechanism.

The finding that addition of high levels of coactivators stim-ulates the activity of AR in the presence of DAX-1 suggestedthat DAX-1 might act in part by competing with coactivatorsfor binding to AR. Because DAX-1 inhibits the activity of ARlacking its hormone binding domain, we used a mammaliantwo hybrid assay to determine whether DAX-1 can block theinteraction between SRC-1 and the N terminus of AR, butfound no inhibition of binding. Moreover, DAX-1 was unable toinhibit the binding of SRC-1 to either the AR LBD or the

FIG. 11. Comparison of DAX-1 ex-pression in normal prostate andBPH. A, DAX-1 expression was detectedusing diaminobenzidine (brown/black),and the tissue was counterstained withhematoxylin (purple) as described in “Ex-perimental Procedures.” DAX-1 expres-sion in normal prostatic tissue is shown inthe left portion of the panel, while expres-sion in BPH is shown on the right. Notethe much higher intensity and percentageof cells expressing nuclear DAX-1 in boththe epithelial cells lining the glands aswell as the stromal cells in the normaltissue. (Immunohistochemistry upperpanels, �40; lower panels, �100). B, 552cores of normal prostate were graded forfrequency of high intensity of DAX-1 ex-pression and plotted against frequency. 0,no cells with high intensity staining forDAX-1; 1, 1–33% of cells with high inten-sity DAX-1 staining for DAX-1; 2, 34–66%, and 3, 67–100%. The results weremean, 2.6; S.D., 0.72. C, 554 BPH coreswere analyzed as in B, and the number ofcores with high intensity DAX-1 stainingwas plotted against the frequency. Mean,1.4; S.D., 1.18.

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full-length receptor. Similarly, DAX-1 did not block interac-tions between PR and SRC-1. Although physical interactionswith SRC-1 were not blocked, SRC-1 acts by recruiting a host ofother factors including CBP (45) and P/CAF (46) and it is likelythat the presence of DAX-1 prevents the assembly of the fullyfunctional coactivator complex. In contrast, as reported previ-ously (15) and shown here (Fig. 7), DAX-1 blocks N/C-terminalinteractions in AR. Moreover, DAX-1 inhibits the interaction ofPRLBD with PR (Fig. 7). Steroid receptors dimerize throughmultiple interaction surfaces including those measured in thisassay as well as through interactions between DNA bindingdomains (9). Our finding that DAX-1 also inhibits the activityof AR lacking the hormone binding domain as well as antago-nist-bound AR, which lack the N/C-terminal interaction, dem-onstrates that DAX-1 has multiple actions on AR. Taken to-gether, these studies suggest that DAX-1 interacts at sitesdistinct from the SRC-1 binding site and inhibits activity byreducing dimer interactions and, in addition, likely interfereswith optimal formation of complete coactivator complexes.

Although our studies and those of others have shown thatDAX-1 has the potential to negatively regulate the activity ofAR, the contribution to AR action in vivo is unknown. Interest-ingly, Gregory et al. (48) have shown that two coactivatorsSRC-1 and TIF2 are overexpressed in recurrent prostate can-cer, another androgen-dependent disease, but found no evi-dence for overexpression in BPH. Our studies of the expressionof DAX-1 in prostate and in BPH samples suggest that DAX-1,indeed, regulates AR activity. The BPH samples contain mark-edly lower levels of DAX-1 implying elevated AR activity. ARaction contributes to BPH and treatment with a 5�-reductaseinhibitor, finasteride, which reduces levels of the potent andro-gen, dihydrotestosterone is a common treatment (49). Thus, theloss of DAX-1 expression may be a significant contributor to thedevelopment of BPH while overexpression of coactivators maybe more important in prostate cancer.

Acknowledgments—We thank Anna Frolov, the Baylor SPORE inProstate Cancer biostatistician, for statistical analysis of the data onDAX-1 expression in BPH. We would also like to acknowledgeJoyoti Dey for expert technical assistance.

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Repressors of AR and PR Action31148

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Mojghan Amrikachi, Gustavo E. Ayala and Nancy L. WeigelIrina U. Agoulnik, William C. Krause, William E. Bingman III, Hassan T. Rahman,

Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.M305153200 originally published online May 27, 20032003, 278:31136-31148.J. Biol. Chem. 

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