Oct-1 Preferentially Interacts with Androgen Receptor in a DNA-dependent Manner That Facilitates Recruitment of SRC-1*

M. Ivelisse GonzalezDagger and Diane M. Robins§

From the Department of Human Genetics and Cell and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618

Received for publication, September 22, 2000, and in revised form, November 2, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gene regulation by steroid hormone receptors depends on the particular character of the DNA response element, the array of neighboring transcription factors, and recruitment of coactivators that interface with the transcriptional machinery. We are studying these complex interactions for the androgen-dependent enhancer of the mouse sex-limited protein (Slp) gene. This enhancer has, in addition to multiple androgen receptor (AR)-binding sites, a central region (FPIV) with a binding site for the ubiquitous transcription factor Oct-1 that appears crucial for hormonal regulation in vivo. To examine the role of Oct-1 in androgen-specific gene activation, we tested the interaction of Oct-1 with AR versus glucocorticoid receptor (GR) in vivo and in vitro. Oct-1 coimmunoprecipitated from cell lysates with both AR and GR, but significant association with AR required both proteins to be DNA-bound. This was confirmed by sensitivity of the protein association to treatment with ethidium bromide or micrococcal nuclease. Addition of DNA to micrococcal nuclease-treated samples restored interaction, even when binding sites were on separate DNA molecules, suggesting association was due to direct protein-protein interaction and not indirect tethering via the DNA. AR/GR chimeras revealed that interaction of the N and C termini of AR was required to communicate the DNA-bound state that enhances interaction with Oct-1. Protease digestion assays of hormone-bound receptors revealed further conformational changes in the ligand binding domain of AR, but not GR, upon DNA binding. Furthermore, these conformational changes led to increased interaction with the coactivator SRC-1, via the NID 4 domain, suggesting DNA binding facilitates recruitment of SRC-1 by the AR-Oct-1 complex. Altogether, these results suggest that the precise arrangement of binding sites in the Slp enhancer ensures proper hormonal response by imposing differential interactions between receptors and Oct-1, which in turn contributes to SRC-1 recruitment to the promoter.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Androgen receptors (AR)1 and glucocorticoid receptors (GR) are members of the nuclear receptor transcription factor superfamily (1). The steroid receptors share a common structural arrangement and have been shown to recognize a similar DNA-binding site, known as a hormone response element (HRE) (2). Nevertheless, the hormones for each receptor elicit distinct physiological functions. Multiple mechanisms have been demonstrated in several cases to enforce steroid-specific gene activation, including subtle preferences in DNA-binding site (3-5), domain interactions within and between receptors that enhance cooperative function (6, 7), and differential protein-protein interactions of the receptors with other regulatory factors (8, 9). The latter may include interactions with other DNA-binding factors (e.g. AP-1, NFI, and Oct-1) (10) or with transcriptional coactivators that do not themselves bind DNA (e.g. SRC-1, GRIP1, and TIF2) (11). These interactions sum to regulate the steroid response in a promoter- and cell context-dependent manner.

To understand hormone-specific gene regulation, we study the mouse sex-limited protein (Slp) gene, which expresses in adult male liver and kidney (12). Androgen dependence is mediated by a 120-base pair enhancer located 2 kb upstream of the gene (13). Although both GR and AR are able to bind to HREs within the enhancer in vitro, only androgens elicit a response in vivo. Previous studies have demonstrated that this androgen specificity is determined by the interaction of AR with nonreceptor factors bound adjacent to the HREs (14). One critical enhancer region, revealed functionally in transfection assays and biochemically by in vivo and in vitro protein binding studies, is called FPIV (15, 16). This complex element includes an Oct-1-like recognition sequence and can bind several other proteins as well as Oct-1 in vitro (17). Oct-1 is an intriguing candidate for interaction with AR, in part because of its well established and diverse interactions with GR (18, 19). That is, Oct-1 can enhance or prevent glucocorticoid induction dependent on the precise sequence and arrangement of the binding sites for these factors.

Oct-1 is a ubiquitous member of the POU-homeodomain family and is involved in the regulation of a wide variety of genes (20). Previous studies have demonstrated that Oct-1 can interact positively or negatively with several nuclear receptors, in a promoter-specific manner. For example, with the mouse mammary tumor virus (MMTV) (21) and gonadotropin-releasing hormone promoters (22), binding of both GR and Oct-1 is required for transactivation. In contrast, GR repression of the histone H2b promoter seems to involve sequestration of Oct-1 by GR prior to DNA binding (23). These studies reveal alternative actions of Oct-1 in nuclear receptor regulation, but the disparate mechanisms responsible are not completely understood. Analysis has been complicated in mammalian cells by pleiotropic effects of altering Oct-1 expression, making functional significance for steroid activation difficult to determine. An alternative approach utilizing expression in yeast cells has not yet overcome the transcriptional inactivity of Oct-1 in that system (24).

To examine whether Oct-1 is involved in androgen specificity, we compared physical interaction between Oct-1 and AR versus GR. Our data show that both AR and GR interact with Oct-1, but in qualitatively different manners. First, in contrast to GR, AR interacts well with Oct-1 only when both factors are bound to DNA. Second, binding to the Slp enhancer induces selective changes in the conformation of the ligand-binding domain of AR, but not GR, and leads to increased interaction with Oct-1. Finally, the coactivator SRC-1 interacts more efficiently with the AR·Oct-1 complex when both transcription factors are bound to DNA. These results suggest that DNA-dependent protein-protein interactions, functionally amplified by enhanced coactivator recruitment, may promote receptor-selective activation. Thus differential interactions among factors, rather than their stringent specificity, can confer precise hormonal response.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids-- cDNAs for the mouse androgen receptor (mAR; D. Tindall) and the rat glucocorticoid receptor (rGR; K. Yamamoto) contained in the eukaryotic expression vector, pCMV5, have been described (16). Chimeras of AR and GR (AGA, GAG, GAA, and aGAA) have also been described (7). For in vitro translation, the mouse AR cDNA was excised from the expression vector and cloned into the HincII site of pGEM4; rat GR cDNA was translated from its original pC7 vector (pC7G). The pGEX3X vectors expressing full-length Oct-1 and Brn-1 as GST-fused proteins were kindly provided by N. Segil (25) and S. Young (26), respectively. To translate the AR DBD, the sequence encoding the first 37 amino acids of AR N terminus was fused via a SmaI site to the AR DBD and inserted into pGem4. The chimeric receptors AGA, GAG, GAA, and aGAA were excised from pCMV5 at the KpnI/XbaI site and inserted into the vector pGem3 for in vitro translation. For in vitro translation, the AR dimerization mutant AR/R581D (gift of D. Pearce) (27) was subcloned from pCMV5 into pGem4. SRC-1 was in vitro transcribed/translated from the pCR3.1 vector (gift from B. O'Malley). GST fusion proteins of SRC-1 NIDs were made by digesting the pCR3.1 SRC-1 construct with XcmI/BamHI and AvaI/XbaI. Fragments encoding NID 1-3 (amino acids 568-954; 1.1 kb) and NID 4 (amino acids 1139-1441; 1.2 kb) were subcloned in the GST expression vector pGEX3.

Oligonucleotide Primers-- Fragments from the Slp enhancer were generated by polymerase chain reaction, using the primer sequences below. The oligo 1 (T) and (B) primers were used to produce a 70-bp DNA fragment containing the octamer-like binding site (FPIV) and HRE-1 and -3 (15) (see Fig. 1C). Oligo 1 (T) and (B) are complementary over 15 bases at their 3' ends; there is 16 bp between HRE-1 and -3. Primers that introduced linker scanning mutations into oligo 1 (oligos 5-8) have been described previously (3, 16). In oligo 5 the octamer-like binding site has been disrupted (italicized sequence), whereas in oligo 6 this site has been converted to the Oct-1 consensus sequence. Oligos 7 and 8 were used to mutate HRE-1 and HRE-3, respectively. Oligo 2 and oligo 3 contain the binding sites for Oct-1 or receptor, respectively.


GCCCCTGAA<B><IT>AGCTCGAGCT</IT></B>

<AR><R><C></C><C><UP>                   FPIV      HRE-1</UP></C></R><R><C><UP>oligo 1 </UP>(<UP>T</UP>)<UP>:</UP></C><C><UP>5′-CTCCTGAACCTGCTT</UP><UNL><UP><B>ATGTAATT</B></UP></UNL><UP>ATC</UP><UNL><UP><B>TGTTCT</B></UP></UNL><UP>GTGGTCAGCCAGT-3′</UP></C></R><R><C><UP>oligo 1 </UP>(<UP>B</UP>)<UP>:</UP></C><C><UP>5′-GCCCCT</UP><UNL><UP><B>GAAACA</B>GCC<B>TGTTCT</B></UP></UNL><UP>GAGAACTGGCTGACCACAG-3′</UP></C></R><R><C></C><C><UP>              HRE-3</UP></C></R><R><C><UP>oligo 5 </UP>(<UP>T</UP>)<UP>:</UP></C><C><UP>5′-CTCCTGAACCTGCTT<B>GCTCGAGCA</B>TCTGTTCTGTGGTCAGCCAGT-3′</UP></C></R><R><C><UP>oligo 6 </UP>(<UP>T</UP>)<UP>:</UP></C><C><UP>5′-CTCCTGAACCTGCTT<B>ATGCAAATA</B>TCTGTTCTGTGGTCAGCCAGT-3′</UP></C></R><R><C><UP>oligo 7 </UP>(<UP>T</UP>)<UP>:</UP></C><C><UP>5′-CTCCTGAACCTGCTTATGTAA<B>TGCTCGAGC</B>CTGTGGTCAGCCAGT-3′</UP></C></R><R><C><UP>oligo 8 </UP>(<UP>B</UP>)<UP>:</UP></C><C><UP>5′-GCCCCTGAA<B>AGCTCGAGCT</B>CTGAGAACTGGCTGACCACAG-3′</UP></C></R><R><C></C><C><UP>                 FPIV</UP></C></R><R><C><UP>oligo 2:</UP></C><C><UP>5′-CTGAACCTGCTT</UP><UNL><UP><B>ATGTAATT</B></UP></UNL><UP>ATCTGTTC-3′</UP></C></R><R><C><UP>oligo 3:</UP></C><C><UP>5′-CTC</UP><UNL><UP><B>AGAACA</B>GGC<B>TGTTTC</B></UP></UNL><UP>AGGGG-3′</UP></C></R><R><C></C><C><UP>           HRE-3</UP></C></R></AR>

Cells and Transfection-- COS-7 cells were transiently transfected with pCMV5 AR or GR DNA (10 µg/10 cm2 plate) using the calcium phosphate method as previously described (28). The DNA precipitates were left on the cells for 8 h, followed by a glycerol shock with 15% glycerol in 1× HBS buffer (0.3 M NaCl, 50 mM HEPES, 1 mM Na2HPO4, pH 7.05). After rinsing twice with phosphate-buffered saline, cells were incubated in Dulbecco's modified Eagle's medium, 8% charcoal-stripped fetal bovine serum containing either 10-7 M dihydrotestosterone (DHT) or 10-6 M dexamethasone (Dex), as appropriate for 2 days prior to harvesting.

Whole Cell Extract Preparation-- Whole cell extracts were prepared from transfected cells as described (29) with some modifications. Cell pellets were resuspended in 1 ml of buffer A (supplemented with 10-10 M DHT, 10-10 M Dex, and protease inhibitors), homogenized by 20 strokes of a B pestle in a Dounce homogenizer, and then centrifuged at 10,000 rpm for 10 min (4 °C). The supernatant was collected, and the protein concentration was determined by the Bradford method (Bio-Rad protein assay kit) using bovine serum albumin as standard. Quality of extracts was assessed by Western blot analysis for integrity of receptors.

Western Blots-- For analysis of transfected cells, 5-10 µg of whole cell extracts were resuspended in an equal volume of SDS sample buffer and boiled for 5 min. Samples were electrophoresed on 7.5-8% SDS-PAGE gels. Proteins were transferred to a nitrocellulose membrane using a semi-dry blotter (Buchler Instruments) at room temperature (3 mA/cm2). Nonspecific sites were blocked for 30 min with 5% nonfat dry milk in Tween 20/Tris-buffered saline (TBS, 1% Tween 20, 10 mM Tris, pH 7, 5 mM NaCl). Receptors and Oct-1 were revealed using antibodies specific for these proteins (diluted in 5% nonfat dry milk, TBS), and a horseradish peroxidase-conjugated secondary antibody (1:2000, ECL). The anti-AR rabbit polyclonal antiserum used for Western blots was raised against a GST fusion protein including amino acid residues 133-334 of mAR. GR and Oct-1 were detected with mouse monoclonal antibodies (FIGR2 for GR, a generous gift of W. Pratt, and 5G5 for Oct-1, kindly provided by N. Segil). The bands were visualized with the ECL chemiluminescence kit (Amersham Pharmacia Biotech).

Coimmunoprecipitation Assays-- Coimmunoprecipitation experiments were performed essentially as described by Kutoh et al. (23). The lysates were precleared with protein A-Sepharose for 30 min and then incubated with either an anti-AR rabbit polyclonal antibody directed against a peptide sequence between base 245 and 263 of mAR, or with anti-GR mouse monoclonal antibody FIGR2, for 1 h at 4 °C. The antigen-antibody complexes were collected by the addition of protein A-Sepharose, and pellets were washed in 20 mM sodium phosphate buffer and extracted with 25 µl of SDS sample buffer. The eluted samples were analyzed on 7.5-8% SDS-PAGE gels and subsequently blotted to nitrocellulose membranes. Western blots were performed with anti-Oct-1 or receptor antibodies as described above.

In some experiments the precipitated complexes were treated with micrococcal nuclease (MNase) prior to elution (30). In assays where DNA was added to the reaction, lysates were treated with 4 units of MNase and 2 mM CaCl2 at 25 °C for 5 min (31), followed by centrifugation at 10,000 rpm for 1 min (4 °C). The supernatant was collected and 4-8 mM EGTA was added to inhibit the nuclease activity. Before immunoprecipitation, these MNase-treated extracts were incubated in the presence or absence of 30-100 ng of specific DNA or poly(dI/dC), for 15-30 min at 4 °C.

GST Pull-down Assays-- GST expression vectors were transformed into Escherichia coli DH5alpha cells and induced with 0.2 mM isopropyl-beta -D-thiogalactopyranoside. The GST-fused proteins were purified by glutathione affinity chromatography (32), and the relative amount of bound fusion protein was determined by Coomassie Blue staining of SDS-PAGE gels. The receptors were translated in vitro using the coupled transcription-translation TNT reticulocyte lysate system (Promega) in the presence of [35S]methionine, using the manufacturer's protocol. Following translation, appropriate hormone was added to each reaction (100 nM DHT for AR, 100 nM Dex for GR). Binding of in vitro translated receptors to isolated GST-fused proteins was performed essentially as described by Préfontaine et al. (21). Equal amounts (50 × 103 cpm) of labeled proteins were incubated with 0.5 µg of immobilized GST fusion proteins in 200 µl binding buffer (supplemented with protease inhibitors) for 6-8 h at 4 °C. The precipitate was washed 5 times with 1 ml of binding buffer. Retained proteins were eluted in SDS sample buffer, electrophoresed, and analyzed by autoradiography (Autofluor; National Diagnostics). For GST pull downs of both SRC-1 and AR simultaneously, equal amounts of labeled proteins (25 × 103 cpm) were incubated with 0.5 µg of immobilized GST-Oct-1 as described above. Retained proteins were analyzed by autoradiography as before.

Limited Proteolytic Digestion-- Limited proteolytic digestion of translated [35S]methionine-labeled receptors (AR and GR) and chimeras (GAA and aGAA) with 20 µg/ml trypsin was carried out essentially as described by Allan et al. (33). Before proteolytic digestion, receptors were hormone-treated (100 nM DHT or Dex, as appropriate, for 10 min at room temperature) and then incubated in the presence or absence of 100 ng of oligo 1 (30 min on ice). 100 ng of nonspecific competitor, poly(dI/dC), was added to control samples. The difference in protease sensitivity among the receptors was quantified using a densitometer and scanning software (NIH Image 1.6) for which the results were plotted as previously described (34).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Binding to DNA Enhances Interaction of AR, but Not GR, with Oct-1-- In vitro binding studies and analysis in transfection of linker scan mutations of the Slp enhancer support a role for Oct-1 in androgen-specific activation (17). To confirm this, the ability of AR to associate with Oct-1 in vivo was analyzed by coimmunopreciptitation with receptors expressed in COS-7 cells by transient transfection (Fig. 1). AR or GR were precipitated from whole cell lysates with receptor-specific antibodies, and immunoprecipitated complexes were resolved by electrophoresis, and the presence of associated Oct-1 was revealed by Western blot. The specificity of the antibodies was demonstrated by immunoblotting the transfected COS-7 cell lysates (Fig. 1A). In the presence of AR or GR, Oct-1 coprecipitated with receptor antibodies but not with preimmune serum (Fig. 1B). Interaction of GR with Oct-1 was readily detected in the cell extract but was less evident for AR. Reprobing the Western filters with receptor antisera suggested that the differences in coprecipitated Oct-1 levels were not due to gross differences in levels of receptor expression (Fig. 1B). Thus, Oct-1 detectably interacts with both receptors in solution but apparently forms a more stable or higher affinity complex with GR than with AR.



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Fig. 1.   DNA binding enhances interaction of Oct-1 with AR but not GR. A, AR and GR were transiently expressed in COS-7 cells and detected by immunoblotting using 5 µg of whole cell extracts to confirm efficient receptor expression. Endogenous levels of Oct-1 (right panel of each pair) were detected by reprobing the filter with Oct-1 antibody and appear comparable in COS-7 and CV-1 cells, before or after transfection. LNCaP and HTC cell extracts were used as positive controls for the expression of AR and GR, respectively. Arrows show location of full-length receptors and Oct-1. B, the ability of Oct-1 to interact with receptors was tested by coimmunoprecipitation from transfected COS-7 cell extracts with antisera (alpha -IP) specific for AR (ar), GR (gr), or preimmune serum (pre). Mock-transfected cell extracts (receptor -, last lane) were immunoprecipitated with anti-AR to reveal specificity of complex formation. Presence of Oct-1 in the complex was detected by Western blot; Oct-1 coprecipitated efficiently with GR but much less so with AR. Membranes were reprobed with anti-AR or anti-GR to confirm receptor in the immunoprecipitation complex (lower panels). C, coimmunoprecipitation of Oct-1 with receptor was tested in the absence (-) or presence (+) of 30 ng of a fragment (oligo 1) of the Slp enhancer (diagrammed above), containing receptor- (HRE) and Oct-1 (FPIV)-binding sites, as shown. The sequence of the fragment is given under "Materials and Methods." The 120-bp androgen-specific Slp enhancer resides 2 kb upstream of the start site of transcription; diamonds represent CBFalpha 1 sites also critical for hormonal response. Reactions without specific oligos contained 30 ng of poly(dI/dC). Oct-1 precipitation with AR was markedly increased by the presence of specific DNA. In the lower panel, oligos encompassing just the Oct-1- (oligo 2) or receptor (oligo 3)-binding sites were added together or singly to coimmunoprecipitation reactions, in comparison to the intact enhancer fragment (oligo 1). Only when both DNA-binding sites were present, whether in one or separate oligos, was protein interaction notably enhanced. Specific DNAs (or poly(dI/dC) in the first sample) totaled 30 ng. Levels of binding were compared with 10% of the input lysate (5 µg) in both panels. All panels in this figure are representative of at least six independent experiments.

Since AR and Oct-1 presumably interact functionally when bound to DNA, we asked whether adding their respective binding sites would allow greater complex formation (Fig. 1C). Coimmunoprecipitation of Oct-1 with AR antibody was enhanced 2-3-fold by including a 70-bp fragment from the Slp enhancer (oligo 1) containing binding sites for Oct-1 (within FPIV) and the receptors (HRE-1 and HRE-3). HRE-3 is a consensus binding site, whereas HRE-1 is a half-site which AR, but not GR, appears to utilize in vivo (16). The DNA-mediated increase in interaction with Oct-1 was not observed with GR. Enhancement of the Oct-1 signal was not due to increased AR levels in the immune complex (see Fig. 2). This result suggested that binding to DNA enhanced the AR, but not GR, interaction with Oct-1.



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Fig. 2.   AR, but not GR, interaction with Oct-1 is sensitive to treatments that disrupt DNA. In the upper panel, coimmunoprecipitation of Oct-1 with AR from transfected COS-7 cells was compared in the presence (+) or absence (-) of oligo 1, as in Fig. 1, with the addition of 50 µg/ml EtBr where indicated to test specific dependence on DNA structural integrity. This treatment abolished the ability of Oct-1 to coprecipitate with AR. In the lower panels, lysates were treated with 4 units of MNase prior to immunoprecipitation with anti-AR (middle panels) or anti-GR (lower panels), to degrade endogenous DNA. After MNase treatment, lysates were incubated with 4 mM EGTA to inactivate the enzyme, with or without oligo 1 as indicated. The ability of Oct-1 to coprecipitate with AR was abrogated by MNase and restored by subsequent addition of oligo 1, whereas coprecipitation of Oct-1 with GR was unaffected by MNase and not enhanced by addition of DNA. Uniform presence of receptors in the complexes was shown by reprobing the membranes with anti-AR or anti-GR (lower panel of pair). Panels are representative of at least eight independent experiments.

To distinguish whether binding to DNA promoted direct protein-protein contact or simply tethered both factors via the nucleic acid, coimmunoprecipitations were performed with separate oligonucleotides containing the binding site for Oct-1 (oligo 2) or receptor (oligo 3). AR-Oct-1 interaction was strengthened even when both binding sites were on separate molecules (Fig. 1C). Oligo 2 alone somewhat enhanced interaction, perhaps due to AR recognition of the partial HRE-1 included in its sequence (5 of 6 half-site bps). However, neither binding site alone increased the association between AR and Oct-1 as dramatically as the two together. Therefore, the DNA-dependent increase in AR-Oct-1 interaction involves protein-protein contacts enhanced when both factors are DNA-bound.

AR Interaction with Oct-1 Depends on Sequence-specific DNA Binding of Both Proteins-- To confirm the DNA dependence of AR-Oct-1 association, cell lysates were treated prior to immunoprecipitation with reagents that disrupt DNA-protein interaction, by either distorting DNA structure by intercalation (EtBr) or by degrading nucleic acid (MNase), as described previously (30). Addition of 50 µg/ml EtBr reduced AR interaction with Oct-1 (Fig. 2) but did not affect Oct-1 interaction with GR (see below). This suggested that the small amount of Oct-1 coprecipitated with AR from lysates without added oligonucleotides (see Fig. 1B) might be due to residual cellular DNA. To eliminate this DNA, cell lysates were treated with MNase, which was subsequently inactivated with EGTA. Similarly to EtBr, MNase abrogated interaction of Oct-1 with AR but not GR (Fig. 2). Furthermore, the association between AR and Oct-1 in MNase-treated extracts could be restored by the addition of the enhancer DNA, oligo 1. In neither case was the amount of receptor precipitated affected. DNA-dependent AR-Oct-1 interaction was also observed using CV-1 (kidney) and LNCaP (prostate) cell lysates, confirming the generality of the DNA effect.2

To validate further the DNA-mediated increase in AR-Oct-1 interaction, coimmunoprecipitations were performed with mutant enhancer fragments (Fig. 3). Oligonucleotides 5, 7, and 8 contain mutations that disrupt binding of Oct-1 (oligo 5) or receptors (oligo 7 and 8) to the enhancer. In oligo 6, the octamer-like site within the FPIV region was converted to the consensus Oct-1 sequence. These mutations impair androgen response in transient transfection assays (16). Mutations that disrupt binding of either Oct-1 (oligo 5) or AR (oligo 7 and 8) to the enhancer only weakly restored interaction between AR and Oct-1 in MNase-treated COS-7 lysates (Fig. 3). However, oligo 6, which increases Oct-1 binding to the enhancer (17), rescued AR-Oct-1 interaction to nearly the same extent as the wild type sequence. Thus, maximal DNA-dependent interaction of AR-Oct-1 requires both AR and Oct-1 to be specifically bound to their cognate elements.



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Fig. 3.   AR-Oct-1 interaction is most efficient when both proteins are DNA-bound. Diagrammed relative to the enhancer fragment (oligo 1) are mutant oligos designed to alter binding of Oct-1 or receptor. The octamer-like site within the FPIV region was mutated to impair binding (oligo 5) or changed to match the Oct-1 consensus element (oligo 6). Oligos 7 and 8 contain mutations in the AR-binding sites of HRE-1 and -3. Oct-1 coimmunoprecipitation with AR was compared in MNase-treated extract (except for untreated extract in the 1st lane) as in Fig. 2, with addition of oligos as indicated above each lane. Oligos 1 and 6, which have functional Oct-1 and HRE sites, led to efficient coprecipitation. Mutation of either HRE (oligo 7 or 8) was sufficient to decrease AR-Oct-1 interaction. 20 µg of lysate from COS-7 cells transfected with AR was used per sample. This gel is representative of six independent experiments.

Oct-1 Contacts the DBD of AR but Requires AR N/C Interaction for DNA-dependent Enhancement-- To identify the receptor domain(s) involved in the Oct-1 interaction and determine whether this was sufficient to communicate DNA binding, GST pull-down assays were performed with in vitro translated AR fragments (Fig. 4). Full-length receptors and fragments containing the LBD were treated following in vitro translation with 100 nM of the appropriate hormone. Interaction of full-length AR with GST-Oct-1 was specific since GST alone did not retain the receptors nor did GST-Oct-1 retain luciferase (Fig. 4A, upper panel). To confirm that the influence of DNA binding could be detected in this approach, the pull-down experiment was performed with full-length AR and GR in the presence and absence of EtBr or MNase (Fig. 4A, lower panel). Interaction between AR and GST-Oct-1 showed similar sensitivity to EtBr and MNase as in the immunoprecipitation assays, and the interaction could be rescued after MNase treatment by addition of enhancer DNA. Neither MNase nor EtBr (not shown) treatments affected interaction of GR with Oct-1. In this experiment, the source of "contaminating" DNA was apparently AR's own cDNA used as the template for transcription/translation (data not shown). HREs within the coding sequence of AR have been shown to be involved in AR autoregulation (35). Thus, DNA binding stabilized the AR-Oct-1 interaction, regardless of the source of the receptor or the influence of diverse cellular proteins.



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Fig. 4.   Interaction of in vitro translated AR with GST-Oct-1 is sensitive to EtBr and MNase treatment. A, 50 × 103 cpm of in vitro translated AR or luciferase were incubated with 0.5 µg of GST or GST-Oct-1 in the presence (+) or absence (-) of 50 µg/ml EtBr. The labeled proteins were detected by autoradiography and arrows indicate their expected migration. AR was retrieved only by GST-Oct-1 in the absence of EtBr; nonspecific retrieval of luciferase was not seen. In the lower panel, reticulocyte lysates with in vitro translated AR or GR were treated with 4 units of MNase prior to incubation with GST (1st two lanes) or GST-Oct-1 (next 6 lanes). After MNase treatment, lysates were incubated with 4 mM EGTA in the presence or absence of oligo 1 as indicated. AR, but not GR, binding to GST-Oct-1 was severely decreased by MNase treatment but was restored by addition of oligo 1. Levels of binding were compared with 10% of the total input cpm. B, 50 × 103 cpm of in vitro translated AR were incubated with 0.5 µg of GST, GST-Brn-1 (Brn), or GST-Oct-1 (Oct) in the presence (+) or absence (-) of 50 µg of EtBr. Brn-1, unlike Oct-1, showed no dependence on DNA for interaction with AR. Levels of binding were compared with 10% of the total input cpm. All experiments were performed a minimum of three times.

To determine whether DNA dependence in AR-factor interactions was due to an intrinsic instability of AR or to a particular association with POU factors, we compared interaction with Brain-1 (Brn-1), a POU protein expressed in kidney as well as neural tissue (36). In pull-down assays, in vitro translated AR was retrieved with either full-length Oct-1 or Brn-1 fused to GST (Fig. 4B). Whereas both GST-Oct-1 and GST-Brn-1 interacted with AR, AR-Brn-1 association did not require DNA binding (of either protein), as it was not sensitive to EtBr. Thus DNA dependence is not a general feature of AR interaction with other proteins.

Previous studies have shown that GR-Oct-1 interaction is mediated through the receptor DNA binding domain (DBD) (21). For this reason, the interaction of the AR DBD with GST-Oct-1, and its DNA dependence, was examined in GST pull-down assays (Fig. 5). Interaction of the AR DBD with Oct-1 was insensitive to EtBr. Although MNase treatment reduced association of Oct-1 and the AR DBD somewhat, addition of the enhancer fragment did not increase interaction above that level. In general, protein-protein interactions that were sensitive to EtBr and that could be rescued after MNase treatment by the addition of DNA were considered DNA-dependent. Thus, the AR DBD was sufficient for Oct-1 interaction but was not sufficient for enhanced interaction in the presence of DNA.



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Fig. 5.   Interaction of AR N and C termini is required for DNA-dependent AR-Oct-1 interaction. A, 50 × 103 cpm of in vitro translated AR DBD (see "Materials and Methods") were incubated with 0.5 µg of GST-Oct-1 (or GST alone in the 1st lane) in the presence (+) or absence (-) of 50 µg/ml EtBr or 4 units of MNase. After MNase treatment and inactivation, oligo 1 was added as indicated. Input lane contains 5% of the amount of in vitro translated AR DBD used. The labeled proteins were detected by autoradiography. Interaction of the AR DBD with GST-Oct-1 showed no effect of EtBr, MNase treatment, or added DNA. B, 50 × 103 cpm of in vitro translated chimeric receptors AGA and GAG, which are full-length AR and GR in which the DBDs have been swapped, were tested for interaction with 0.5 µg of GST-Oct-1 in the presence (+) or absence (-) of 4 units of MNase. After MNase treatment, oligo 1 was added as indicated. AGA but not GAG was sensitive to MNase in interaction with Oct-1 and recovered upon addition of DNA. In the lower panel, the chimeras GAA, which has the GR N terminus fused to the AR DBD and LBD, and aGAA, which has the first 37 amino acids of AR fused to GAA, were retrieved with GST-Oct-1 as described above in the presence or absence of MNase or EtBr, and the labeled proteins were detected by autoradiography. aGAA, but not GAA, was sensitive to MNase in its interaction with Oct-1 but recovered upon addition of DNA. C, the mutant AR R518D, which fails to dimerize, was in vitro translated and retrieved with GST-Oct-1 in the presence (+) or absence (-) of 50 µg/ml EtBr. Compared with similar counts/min of AR in the 1st lane, AR R518D interacts with Oct-1 less efficiently but is not dependent on DNA. The input lane contains 10% of the amount of in vitro translated AR R518D used. This experiment is representative of three independent assays.

To define the domains involved in communicating the DNA-bound state of AR, AR and GR chimeras were tested for interaction with Oct-1 in the presence and absence of DNA. These chimeras have been described previously in functional studies of the Slp enhancer (7). In the GAG and AGA chimeras, the AR and GR DBDs have been swapped. The region of the DBD exchanged in the chimeras includes the residues Cys-500 and Leu-501 of the GR DBD that are required for GR-Oct-1 interaction (21). Both GAG and AGA were retained by GST-Oct-1; however AGA, but not GAG, was sensitive to MNase (Fig. 5B). This confirmed that the DBD identity was insufficient for communicating the effect of DNA binding. We reasoned that the N and C termini of AR might be involved, as N/C interaction is fundamental to numerous AR functions (37-39). To confirm this, receptor chimeras GAA and aGAA were tested for DNA-dependent interaction with GST-Oct-1. In GAA, the N terminus of GR is fused to the AR DBD and LBD. In aGAA, the first 37 amino acids of AR are fused to the translation start site of GAA. This most N-terminal region of AR contains one of the two prominent sites for interaction with the LBD of AR (40). In functional studies with the Slp enhancer, aGAA conferred activation half that of AR levels, whereas GAA behaved similar to GR and failed to transactivate the androgen-specific target (7). In the GST pull-down assays, interaction of aGAA, but not GAA, was sensitive to EtBr and MNase (Fig. 5B), suggesting that in fact AR N/C interaction was required for DNA-dependence.

Since the interaction between the AR N terminus and LBD is required for dimerization (39), we tested AR-Oct-1 DNA-dependent interaction with an AR mutant unable to form dimers (AR/R581D) (41). Oct-1 interaction with AR monomers was weak and insensitive to EtBr (Fig. 5C). Thus, DNA-dependent AR-Oct-1 interaction required both the N terminus and AR LBD and was influenced by AR dimerization upon DNA binding.

AR, but Not GR, Is More Sensitive to Proteolysis When Bound to DNA-- Enhancement of AR-Oct-1 interaction in the presence of DNA suggested that binding to DNA induced a conformational change required for AR, but not GR, to interact with Oct-1. To test such an allosteric effect of DNA, limited proteolytic digestion of AR and GR was compared in the presence and absence of oligo 1 (Fig. 6). Proteolytic analysis has been used to contrast the conformation of unliganded receptor LBDs to LBDs occupied by agonist or antagonist (33, 42). In vitro translated AR or GR bound to hormone (100 nM DHT or Dex) was digested with 20 µg/ml trypsin for 1-10 min, with or without oligo 1. Trypsinization produced two major AR bands of 35 and 30 kDa and one band of 35 kDa from GR (Fig. 6). Previous studies have shown that these fragments derive from the LBDs of the receptors and are stabilized by ligand (42, 43). The size of receptor fragments did not vary in the presence of DNA (Fig. 6). However, the extent or rate of proteolytic digestion differed between free versus DNA-bound receptors. Autoradiograms from three independent experiments with each receptor were scanned, and the density of the major LBD fragment at each time point was determined relative to the density at 1 min without DNA; these values are plotted in the graphs to the right in Fig. 6. Both the 35- and 30-kDa fragments of AR were more readily cleaved by trypsin upon DNA binding. In contrast, the 35-kDa band of GR was, if anything, more resistant to further digestion when GR was DNA-bound. The effect of DNA binding on AR conformation was relatively subtle compared with that produced by ligand initially but was consistently demonstrable in these assays.



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Fig. 6.   Conformational change upon DNA binding is apparent for AR but not GR. 50 × 103 cpm of in vitro translated AR or GR were digested with 20 µg/ml of trypsin for 0-10 min in the presence (+) or absence (-) of 50 ng of oligo 1. 100 nM DHT (AR) or dex (GR) was added to receptors prior to protease digestion. Reactions without specific oligos (-) contained 50 ng of poly(dI/dC). The trypsin-resistant fragments of AR and GR were separated on 12.5% SDS-polyacrylamide gels and detected by autoradiography. Arrows indicate receptor fragments whose densities were quantified for the graphs to the right by scanning with NIH image version 1.6. Points on the graph represent the averaged density value from three independent assays relative to the density of the AR 35-kDa protease fragment in the absence of DNA at 1 min of digestion. Open circles are + DNA, black circles without DNA. * indicates p < 0.05, by the Student's t test. AR but not GR shows a statistically significant increased sensitivity to protease in the presence of DNA.

To examine the role of AR N/C interaction in the DNA-dependent conformational change, trypsinization assays were performed with the GAA and aGAA chimeras, as above (Fig. 7). The proteolytic pattern of the GAA and aGAA chimeras was similar to the AR. However, the rate of digestion of GAA was more similar to GR than to AR, although aGAA, like AR, showed more sensitivity to trypsin when DNA-bound. Therefore, binding to the Slp enhancer caused a detectable change in conformation of the liganded AR, but not GR, LBD, which was in part dependent on N/C interaction.



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Fig. 7.   Conformational change of receptor upon DNA binding involves N/C interaction. 50 × 103 cpm of in vitro translated GAA, the chimeric receptor with the N terminus of GR replacing that of AR, and aGAA, containing the first 37 amino acids of AR fused to the N terminus of GAA, were treated with 100 nM DHT and digested with 20 µg/ml trypsin in the presence or absence of 50 ng of oligo 1 for the amount of time indicated, as in Fig. 6. Resistant fragments of the receptor chimeras were detected by autoradiography following PAGE; arrows indicate the fragments whose densities were quantified for the graphs to the right, as in Fig. 6. * indicates p < 0.05 by the Student's t test. aGAA, but not GAA, shows greater sensitivity to trypsin in the presence of DNA.

AR Interaction with the Coactivator SRC-1 Is Enhanced When Both AR and Oct-1 Are DNA-bound-- That a conformational change in the liganded AR LBD occurred upon DNA binding led us to conjecture that the functional outcome might be to reposition the AF-2 surface in such a way as to increase interaction with coactivators. We therefore tested the influence of DNA on interaction of AR with steroid receptor coactivator 1 (SRC-1), one of the p160 family whose members enhance hormone response by direct interaction with ligand-occupied nuclear receptors (11). The receptor LBDs interact with three nuclear receptor interaction domains (NID 1-3) of the p160s via LXXLL motifs (44). SRC-1 is unusual in having a fourth NID (NID 4) close to the C terminus (see Fig. 8) that is involved in facilitating AR N/C interaction and in enhancing AR activation (45, 46).



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Fig. 8.   Interaction of NID 4, but not NID 1-3, of SRC-1 with AR is increased in the presence of DNA. Shown at top is a schematic representation of AR AF-1 and -2 regions and their corresponding nuclear interaction domains (NID) in SRC-1. In vitro translated AR or GR was retrieved with 0.5 µg of GST-NID 4 (middle panel) or GST-NID 1-3 (lower panel) in the presence (+) or absence (-) of 4 units of MNase. Oligo 1 was added after MNase treatment as indicated. Levels of binding were compared with 10% of the total input counts/min. In all cases, MNase treatment reduced interaction of receptors with SRC-1 subfragments; however, AR interaction with NID 4 was partially recovered by addition of DNA.

GST fusion proteins containing NID 1-3 or NID 4 of SRC-1 were interacted with liganded receptors (Fig. 8). In vitro translated AR association with GST-NID 4 was enhanced in MNase-treated lysates by addition of oligo 1 (Fig. 8). However, association of AR with the NID 1-3 region was not sensitive to such treatment. GR interacted similarly with both NID 1-3 and NID 4 regardless of the presence or absence of DNA.

Finally, we asked whether intact SRC-1 could be recruited to the AR-Oct-1 complex in a DNA-dependent manner, by testing the ability of GST-Oct-1 to simultaneously retrieve AR and SRC-1 (Fig. 9). GST-Oct-1 was able to interact with SRC-1 even in the absence of AR to some extent and appeared to enhance interaction with AR when both were present (compare AR in 2nd and 4th lanes of top panel, Fig. 9). The SRC-1, AR, and Oct-1 ternary complex was sensitive to both EtBr and MNase (Fig. 9). The multiprotein complex could be rescued after MNase treatment, either by addition of the enhancer fragment or by oligos 2 and 3 added together (Fig. 9, lower panel). Mutant versions of oligo 1 incapable of binding AR or Oct-1 (oligos 5 and 7), or oligo 2 or 3 individually, supported less interaction of SRC-1 and AR with GST-Oct-1. Thus, recruitment of SRC-1 to the multiprotein complex was more efficient when both AR and Oct-1 were DNA-bound. Collectively, these results suggest that binding to the Slp enhancer causes a conformational change in both AR and Oct-1 that promotes protein-protein interaction and facilitates recruitment of the coactivator SRC-1 to the ternary complex. In this manner, the arrangement of the binding sites, rather than the specificity of the individual factors, may contribute to differential hormonal response.



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Fig. 9.   Recruitment of full-length SRC-1 to the ternary complex is enhanced when both AR and Oct-1 are DNA-bound. AR and SRC-1 were in vitro translated and tested singly and together for interaction with 0.5 µg of GST or GST-Oct-1 in the presence or absence of 50 µg/ml EtBr or 4 units of MNase (indicated below the panel), as in Fig. 4. Levels of binding were compared with 20% of the total input counts/min (50 × 103). The upper panel is without added DNA. In the lower panel, MNase was inactivated with EGTA and the oligos shown in Fig. 3 added as indicated below each lane.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have contrasted the interaction of androgen and glucocorticoid receptors with Oct-1 to determine whether the differential interplay of generally versatile proteins can confer selective gene regulation. Association of Oct-1 in cellular extracts with AR is weak relative to GR but is markedly enhanced by inclusion of DNA fragments from the androgen-specific Slp enhancer. This enhancer contains intertwined binding sites for Oct-1 and receptor (16, 17). The DNA-dependent AR-Oct-1 association requires that both proteins are DNA-bound and that the N and C termini of AR are present to communicate nucleic acid contact at the DBD. Both these features suggest enhanced protein-protein interaction is due to conformational changes imposed by the DNA rather than (or in addition to) DNA-directed presentation of particular pre-existing protein facades. This is supported by data from proteolytic digestions that reveal DNA-mediated changes in the conformation of the liganded AR LBD, again dependent on AR N/C interaction. A functional outcome of the altered conformation is to enhance recruitment of the coactivator SRC-1, via its NID 4 domain. Strikingly, SRC-1 detects the DNA-bound state of Oct-1 as well as AR. Thus the coactivator may more efficiently integrate into a functional transcription complex, directed by the precise array of enhancer binding sites (see model in Fig. 10).



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Fig. 10.   Model for DNA-dependent protein-protein interactions. A, AR, Oct-1, and SRC-1 can interact in solution but with low affinity or stability. B, binding to cognate sites within the enhancer induces conformational changes in both AR and Oct-1 that favor their interaction and facilitates recruitment of the coactivator SRC-1 to promote transcription. One SRC-1 molecule is shown for simplicity although the stoichiometry remains to be determined.

The interactions of AR and GR with Oct-1 differ qualitatively, in that association with GR, but not AR, is readily detectable in the absence of DNA. Previous studies demonstrated direct interaction between GR and Oct-1 via their DNA binding domains in the absence of DNA (21). This interaction in solution underlies glucocorticoid-induced repression of Oct-1 activation of the histone H2b promoter (23). Functional interaction between GR and Oct-1 also occurs on DNA, where the arrangement of the respective protein-binding sites dictates enhancement or repression of transcription. For example, on the MMTV promoter, hormonal induction is mediated by cooperative binding of GR and Oct-1 to adjacent sites (21). In contrast, repression of gonadotropin-releasing hormone transcription occurs by tethering of GR to DNA-bound Oct-1 (47). AR, like GR, cooperates with Oct-1 on the MMTV promoter, through multiple clustered HREs (19). AR-Oct-1 association on the Slp enhancer also requires both factors to be DNA-bound. The functional relevance of this is inferred since mutations that disrupt the AR-Oct-1 association in vitro also reduce androgen response of the enhancer in transfection. Synergism between transcription factors dictated by a precise array of binding sites has been shown for numerous promoters (9). In the case of FGF4, gene activation requires interaction between SOX 2 and Oct-3 that is dictated by the spatial arrangement of their cognate elements (48). In the case of the Slp enhancer, the DNA dependence of the AR-Oct-1 interaction, coupled with the particular array of consensus and nonconsensus binding sites, may accentuate the ability of AR, but not GR, to activate this element.

The DBD is a general prerequisite for DNA-dependent effects, but it is interaction of the AR N and C termini that transmits the status of DNA binding to enhance association with Oct-1. This is most apparent from experiments with AR/GR chimeric receptors, where only those with both the N and C termini of AR (AGA and aGAA) show sensitivity to EtBr or MNase, regardless of the identity of the DBD. The N/C interaction promotes dimerization, ligand binding, and functional synergism between AF-1 and AF-2 domains for PR and ER, as well as AR (37, 39, 49). On the Slp enhancer, the N/C interaction facilitates cooperative binding of AR, but not GR, thus enhancing use of nonconsensus elements (7). Recently, N/C interaction was shown to utilize two N-terminal sequences: 23FXXLF27, which contacts primarily the AF-2 domain, and 433WXXLF437, which recognizes a distinct region of the AR LBD (40). The FXXLF motif at least appears critical for DNA-dependent association of AR with Oct-1, since the first 37 amino acids of AR, including this sequence, confer this ability on GAA when added to the N terminus (aGAA). Conformational analysis of the liganded receptors by proteolytic digestion reveals a subtle change in the AR, but not GR, LBD in the presence of DNA. Previous studies of GR bound to HRE sequences also reported absence of detectable changes in the GR LBD (43). However, not all conformational changes are revealed by this assay, since DNA-bound ER shows slower ligand dissociation but no difference in sensitivity to trypsin (50). The greater sensitivity to protease of aGAA compared with GAA supports the idea that conformational differences of the AR LBD are due to N/C interaction. Thus, DNA-dependent association of AR with Oct-1 is due to DNA-mediated changes in the LBD that requires N/C interaction to perceive modifications at the DBD surface. Conformational changes induced upon DNA binding have also been reported for other members of the nuclear receptor family. For example, binding to DR4 elements induces conformational changes of retinoid X receptor beta  in the thyroid receptor/retinoid X receptor beta  heterodimer that modify triiodothyronine-mediated transcription (34).

A functional outcome of the DNA-induced repositioning of the AR LBD appears to be increased recruitment of coactivators to the complex. SRC-1, a prototypic coactivator, interacts with the AF-2 region within the LBD by a central nuclear interaction domain that contains three LXXLL motifs (NID 1-3) or NR boxes (51). Unlike other p160s, SRC-1 contains an extra NR box (NID 4) that interacts with the AF-1 domain (52). The interaction of AR with the SRC-1, however, is unusual in comparison to that of other receptors, in that the AF-1 of the AR interacts with NID 1-3, whereas AF-2 contacts the NID 4 region (53, 54). Furthermore, SRC-1 enhances AR activation by facilitating N/C interaction. Additional reports indicate the SRC-1 interaction with AR is mediated by the N terminus and the DBD, whereas the AF-2 appears to be primarily involved in the N/C interaction (55, 56). Our results confirm that NID 4 interacts with the LBD of the AR. Furthermore, interaction with NID 4, but not with NID 1-3, is influenced by the DNA binding of the AR, since association is abrogated by EtBr treatment (not shown) and restored by addition of DNA following MNase treatment. Thus, the repositioning of either AF-2 or AF-1, or both, upon binding DNA seems to facilitate recruitment of SRC-1 in a manner somewhat particular to AR compared with other receptors. This unusual interaction may come into play in some cases of ligand-independent AR activity, such as in advanced prostate cancer.

In physical interaction assays with full-length SRC-1, we were surprised to find that Oct-1 as well as AR associates directly with the coactivator. Moreover, the formation of the multiprotein complex is enhanced by the presence of DNA. This suggests that the functional relevance of DNA dependence for both partners in the AR-Oct-1 interaction is to increase SRC-1 recruitment. Direct interaction between SRC-1 and Oct-1 is not entirely unprecedented since AP-1 and NF-kappa B, which also functionally interact with nuclear receptors, also recruit this coactivator (57, 58). In addition, the POU factor Pit-1 interacts with the coactivator CBP/p300, which is itself recruited by SRC-1 (59). Furthermore, it was recently reported that a DNA-dependent retinoic acid receptor/retinoid X receptor interaction increases SRC-1 recruitment in a ligand-dependent manner (60). This study also demonstrated that retinoic acid receptor binding to a DR5 retinoic acid response element facilitates recruitment of the corepressor N-CoR in coimmunoprecipitation assays. The recently solved crystal structure of the peroxisome proliferator receptor-gamma LBD shows a single SRC-1 molecule can associate with a receptor dimer (61). The stoichiometry of the multiprotein complex formed by AR, SRC-1, and Oct-1 in the presence of DNA remains to be determined.

Similar to the interaction between AR and Oct-1, the association of SRC-1 into the multiprotein complex relies on DNA elements and is disrupted by the same mutations in these elements that disrupt AR or Oct-1 binding. Furthermore, the binding sites for AR and Oct-1 need not be contiguous for interaction with SRC-1, suggesting that the coactivator is sensitive to contact between the transcription factors, as well as their DNA-bound state (see Fig. 10). Direct contact between Oct-1 and AR, together with the need for both factors to be DNA bound, suggests SRC-1 is cooperatively recruited to the multiprotein complex. Ultimately, it is the sequence and arrangement of binding sites in the Slp enhancer that promotes a selective interaction between Oct-1 and AR, rather than GR. That is, the nonconsensus Oct-1 binding site overlaps a half-site HRE (HRE-1) shown in functional analysis to be utilized by AR but not by GR (7). Binding of Oct-1 and AR to this region induces a conformational change required for protein contacts. This in turn facilitates cooperative recruitment of SRC-1 to a ternary complex that interfaces efficiently with the transcription machinery (see model in Fig. 10). Collectively, these results emphasize the context-dependent nature of transcriptional specificity, where subtle preferences in DNA-binding sites, intrinsic differences in the factors, and differential interactions with other nuclear proteins sum to near-absolute selectivity in vivo.


    ACKNOWLEDGEMENTS

We thank our generous colleagues for the following reagents: Neil Segil for the Oct-1 antibody, 5G5, and the GST-Oct-1 plasmid; Bill Pratt for GR antibody, FIGR; Scott Young for the GST-Brn-1 plasmid; Dave Pearce for the AR mutant R581D; and Bert O'Malley for SRC-1 plasmid. We thank Alessandra Tovaglieri for help with the Brn-1 experiment. Michele Brogley and Elizabeth Hughes provided excellent technical assistance. Members of the laboratory, particularly Arno Scheller and Chris Krebs, provided invaluable advice throughout. We thank Ron Koenig for many helpful suggestions.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK-56356 (to D. M. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by training grants to the Reproductive Sciences Program and the Cell and Molecular Biology Program.

§ To whom correspondence should be addressed. Tel.: 734-764-4563; Fax: 734-763-3784; E-mail: drobins@umich.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008689200

2 M. I. Gonzalez and D. M. Robins, unpublished data.


    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; GR, glucocorticoid receptor; GST, glutathione S-transferase; bp, base pair; kb, kilobase pair; HRE, hormone response element; MNase, micrococcal nuclease; MMTV, mouse mammary tumor virus; oligo, oligonucleotide; DHT, dihydrotestosterone; Dex, dexamethasone; PAGE, polyacrylamide gel electrophoresis; LBD, ligand binding domain; DBD, DNA binding domain.


    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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