AR Suppresses Transcription of the {alpha} Glycoprotein Hormone Subunit Gene Through Protein-Protein Interactions with cJun and Activation Transcription Factor 2

Joan S. Jorgensen and John H. Nilson

Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106-4965

Address all correspondence and requests for reprints to: Dr. John Nilson, Department of Pharmacology, Case Western Reserve University School of Medicine, W319, 2109 Adelbert Road, Cleveland, Ohio 44106-4965.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previously, we reported that the AR directly suppressed transcription of the {alpha} glycoprotein hormone subunit ({alpha}GSU) gene in a ligand-dependent fashion while ER had no effect. Mutagenesis studies of the {alpha}GSU promoter indicated that two elements were required for AR-mediated suppression: the {alpha} basal element and tandem cAMP response elements (CREs). Because several members of the bZip family of transcriptional proteins can bind the CREs, we used several functional assays to determine whether AR interacts selectively with cJun, activation transcription factor 2 (ATF2), or CRE binding protein (CREB). When tested by cotransfection with AR, cJun and ATF2 specifically rescued androgen-mediated suppression of the {alpha}GSU-reporter construct in a gonadotrope-derived cell line. In contrast, cotransfected CREB displayed no activity in this rescue assay. In fact, overexpression of CREB alone diminished activity of the {alpha}GSU promoter, suggesting that the transcriptional activity normally conferred by the tandem CREs in gonadotropes requires their occupancy by cJun/ATF2 heterodimers. Binding assays carried out with a glutathione-S-transferase-AR fusion protein indicated that the receptor itself also displayed a clear preference for binding cJun and ATF2. Furthermore, we ruled out the possibility that AR suppressed activity of the {alpha}GSU promoter by reducing synthesis of these bZip proteins. Additional experiments suggested that phosphorylation of AR or histone acetylation are unlikely requirements for AR suppression of {alpha}GSU promoter activity. Thus, our data suggest that AR suppresses activity of the {alpha}GSU promoter through direct protein-protein interactions with cJun and ATF2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
REPRODUCTIVE FITNESS RELIES on complex regulation of many hormones that collectively define the hypothalamic-pituitary-gonadal axis. Pulsatile release of GnRH from the hypothalamus stimulates the synthesis and secretion of LH and FSH in gonadotropes of the pituitary (1). Upon secretion, LH and FSH stimulate synthesis of the gonadal steroids, estrogen and androgen (1). These steroids provide negative feedback for gonadotropin production via their respective receptors in both the pituitary and hypothalamus. Here, and in the companion paper (2), we focus our studies on the genes encoding LH and address the mechanism by which gonadal steroids regulate their transcription directly in the pituitary.

LH is a heterodimeric glycoprotein hormone consisting of an {alpha}-subunit ({alpha}GSU) common to all glycoprotein hormones, and a unique ß-subunit (3). The genes that encode these subunits reside on separate chromosomes and are controlled by entirely different 5'-flanking regions (4). Although common hormones regulate {alpha}GSU and LHß subunit gene expression, we postulate that different mechanisms are used. In this paper we will focus on the mechanisms underlying androgen-dependent suppression of {alpha}GSU promoter activity whereas the companion paper (2) will focus on LHß.

We have previously shown, using transient transfection assays in {alpha}T3–1 cells, that androgens can directly suppress {alpha}GSU gene activity while estrogens have no effect (5). Studies with various AR mutants identified the DNA-binding domain (DBD) and adjoining hinge region as the minimal domains necessary and sufficient for suppression of the {alpha}GSU promoter (6). However, in the context of the entire receptor, both the DBD and ligand-binding domain (LBD) are required for AR-mediated suppression of the {alpha}GSU gene, highlighting the necessity of its ligand (6). While a high-affinity binding element for AR is present in the proximal 111 bp of the {alpha}GSU promoter, mutation of this element had no effect on AR-mediated suppression (6). Instead, block mutation studies indicated that two regulatory elements, the tandem cAMP regulatory elements (CREs) and the {alpha} basal element ({alpha}BE), which are critical for targeting expression of the {alpha}GSU gene to gonadotropes, are each also essential for androgen-mediated suppression (6). Together, these data suggest that AR may exert its effect by binding directly to the proteins that occupy one or both of these sites.

Because the identity of proteins that bind {alpha}BE are unknown (7), we have concentrated on those that can bind the tandem CREs. Several members of the bZip family of transcription factors bind the tandem CREs in the {alpha}GSU promoter, including CRE binding protein (CREB), CRE modulator (CREM), activating transcription factors 1 and 2 (ATF1, ATF2), and cJun (8). Here we investigate whether specific members of the bZip protein family can be implicated in AR suppression of {alpha}GSU promoter activity. In addition, a number of direct and indirect mechanisms for transcriptional repression by nuclear receptors (NR) have been described (9, 10). Therefore, we also determined whether additional indirect mechanisms were contributing to androgen-dependent suppression of {alpha}GSU promoter activity. Assessment included changes in critical protein expression, phosphorylation status of the AR-DBD, and alterations in histone acetyltransferase (HAT) activity.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional Evidence for a Preferential Role of cJun and ATF2 in AR-Mediated Suppression of {alpha}GSU Promoter Activity
Although the tandem CREs play a critical role in AR-mediated suppression of {alpha}GSU promoter activity, it remains unclear whether its functional activity can be linked directly to bZip proteins known to bind canonical CREs. We reasoned that if AR attenuates activity of the {alpha}GSU promoter by interfering with the action of bZip family members, then their overexpression should rescue the promoter from the negative effect of androgens. Therefore, we tested this possibility by performing cotransfection assays with a constant amount of an expression vector encoding AR and increasing amounts of expression vectors encoding either CREB, cJun, or ATF2. The reporter vector contained 1500 bp of {alpha}GSU 5'-flanking sequence linked to luciferase cDNA. All transfections were carried out in {alpha}T3–1 cells incubated with 100 nM dihydrotestosterone (DHT) (5, 6).

While cJun and ATF2 had little effect on basal promoter activity when transfected in the absence of AR, both bZip proteins rescued the reporter construct from AR-mediated suppression even when cotransfected at a 1:1 ratio (Fig. 1Go). Addition of both cJun and ATF2 together resulted in the same {alpha}GSU promoter activity as each alone, indicating that the endogenous proteins are not limiting (data not shown). In contrast, overexpression of CREB alone markedly attenuated activity of the {alpha}GSU promoter. This negative effect of CREB appears specific for the {alpha}GSU promoter since its overexpression did not compromise activity of rous sarcoma virus (RSV), SV40, or LHß promoters (data not shown). More importantly, overexpression of CREB failed to rescue the {alpha}GSU promoter from the suppressive effect of AR, suggesting that not all bZip family members are functionally equivalent. In this regard, overexpression of constitutively active SF-1 (SF-1{Delta}LBD) (11), SF-1, Egr-1, or Pitx1 also failed to rescue the {alpha}GSU promoter from androgen-negative regulation (data not shown) providing further support for specific roles of cJun and ATF2. Importantly, no significant difference in promoter activity was found when cDNA from individual expression vectors was increased from 60 to 600 ng/well (Fig. 1Go). This suggests that rescue activity is caused by specific interactions with AR, and is not due to an increase in the amount of transfected cDNA representing the transcription factors.



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Figure 1. cJun and ATF2 Rescue {alpha}GSU Promoter Suppression by AR. CREB Suppresses {alpha}GSU Gene Activity on Its Own and, in the Presence of AR, Abrogates {alpha}GSU Gene Activity

The (-1500/+45 bp) human {alpha}GSU promoter linked to luciferase was cotransfected with a control expression plasmid (CMV5GH, white bar), wild-type AR (hatched bar), or the indicated transcription factors (X). cJun (black bars), ATF2 (light gray bars), and CREB (dark gray bars) expression vectors were added in increasing DNA concentrations (as indicated) in the presence of a constant amount of AR plasmid DNA. Transfections were performed in {alpha}T3–1 cells maintained in charcoal-stripped media with 100 nM DHT. Luciferase activity of each experiment was normalized by measuring RSV- or SV40- (for ATF2 coexpression) ß-galactosidase activity to control for transfection efficiency. Data represent the luciferase/ß-galactosidase activity of each assay normalized to luciferase/ß-galactosidase of the full-length {alpha}GSU promoter in the presence of CMVGH. Transfections were performed a minimum of three times, and the error bars represent the SEM of these experiments.

 
ATF2 Specifically Binds to AR-DBD and Confers Specificity to cJun
Although the above data provide evidence for a functional role of cJun and ATF2, it remains unclear whether they act by binding directly to AR. Therefore, we assessed this possibility by testing whether cJun and ATF2 can bind to GST fusion proteins containing native or mutant DBDs of AR. The AR mutation, C576A, occurs in the proximal zinc finger and renders the protein incapable of activating an androgen response element (ARE)-containing promoter (MMTV), or suppressing the {alpha}GSU promoter (6). As shown in Fig. 2Go, cJun bound to glutathione-S-transferase (GST) fusion proteins containing both the native and mutant DBD of AR. Since the C576A mutation abolishes the suppressive effect of AR, these data suggest that homodimers of cJun lack functional specificity. In contrast, ATF2 bound selectively only to the GST protein containing the native AR DBD. In addition, when incubated in the presence of cJun, ATF2 conferred specific binding of both transcription factors only to the wild-type AR-DBD. Moreover, the specific binding properties of cJun and ATF2 contrast even further with CREB, which bound nonspecifically to GST alone and the two GST fusion proteins. Collectively, these data complement the transient transfection data and suggest that AR suppresses activity of the {alpha}GSU promoter by binding to heterodimers of cJun and ATF2 that occupy the tandem CREs.



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Figure 2. ATF2 Specifically Binds Wild-Type AR-DBD and Confers Specific Binding for cJun When Incubated with cJun and AR-DBD

Pull-down assays were performed with bacterially produced GST fusion proteins (GST-AR DBD, GST-AR DBD-C576A, GST control) and in vitro transcribed and translated [35S] methionine-labeled CREB, cJun, ATF2, and combined cJun/ATF2. Interacting proteins were separated on 10% SDS-PAGE, transferred to 3 M paper (Whatman, Clifton, NJ) and visualized by autoradiography. Interactions are compared with 20% labeled input for each protein. Binding assays were repeated a minimum of three times. A cartoon depicting bZip family member proteins bound to the tandem CRE sequences is shown above the GST pull-down data.

 
Previously, we reported that AR suppresses transcription of the {alpha}GSU gene in gonadotrope-derived cells and in transgenic mice (5, 6, 12). Furthermore, we identified the tandem CREs as targets for AR (6). We also determined that a large subset of bZip family members bind the CREs in gonadotropes, including CREB, CREM, ATF1, ATF2, and cJun (8). Here, we demonstrate that of the bZip proteins tested, only cJun and ATF2 can directly interact with AR to rescue suppression of {alpha}GSU promoter activity. The selective functional activity displayed by cJun and ATF2 toward AR, when interacting with the tandem CREs of the {alpha}GSU promoter, is not without precedent. Indeed, cJun has been shown to bind the DBDs of both AR (13) and GR (14). In this regard, Kallio and colleagues (15) also showed that AR inhibited binding of cJun to AP-1 elements. As discussed below, this selective response with cJun and ATF2 implies that only a limited number of CRE-binding proteins can mediate the suppressive effects of AR when bound to the tandem CREs of the {alpha}GSU promoter in gonadotropes.

We established the specificity of interactions between AR and cJun/ATF2 through the use of a mutant AR harboring an alanine substitution for cysteine in the first zinc finger region (C576A) of the DBD. This mutant AR was shown previously to be unable to suppress activity of the {alpha}GSU promoter, suggesting that the cysteine substitution alters a critical protein-protein interaction (6). This notion is supported by other studies showing that mutations in the GR-DBD (C500Y, L501P) abrogated octamer factor binding and their recruitment to DNA (16). Another level of functional specificity is achieved by the binding of cJun/ATF2, since cJun alone bound both the wild-type and mutant AR-DBD while ATF2 bound only to the wild-type construct. Interestingly, when ATF2 and cJun were added together, cJun no longer bound the mutant AR (Fig. 2Go). Together, these data suggest that the cJun/ATF2 heterodimers expose a specific protein interface necessary for mediating the suppressive effects of AR. Alternatively, the loss of binding between cJun and the AR mutant (C576A) could be due to ATF2 physically pulling cJun from its interaction with C576A because of the high affinity between the heterodimer pair. Importantly, these data identify a new binding partner for AR, namely ATF2.

We found it noteworthy that overexpression of CREB alone repressed activity of the {alpha}GSU promoter in a gonadotrope-derived cell line, while overexpression of cJun or ATF2 alone had little effect on transcription (Fig. 1Go). These findings contrast with earlier work indicating that overexpression of cJun in a cell line derived from trophoblasts repressed activity of the {alpha}GSU promoter through its tandem CREs (17). Together these observations suggest that the activity of individual CRE-binding proteins may vary between cell types. For example, in an earlier study, we replaced the tandem CREs with AP-1 elements that bind only cJun-containing heterodimers. The activity of this mutant {alpha}GSU promoter was severely abrogated when examined in trophoblasts, but not in gonadotropes (18). This differential activity of the {alpha}GSU promoter is further supported by the fact that most other nonprimates harbor a single variant CRE that binds only cJun/ATF2 (8, 19). Interestingly, these nonprimates express their {alpha}GSU gene in gonadotropes, but fail to express it in trophoblasts (19, 20, 21). For these reasons, we focused on cJun, ATF2, and CREB rather than CREM or ATF1. Thus, there appears to be a strong correlation between the binding of heterodimers that contain cJun and gonadotrope-specific expression of the {alpha}GSU gene. Since androgens negatively regulate expression of {alpha}GSU in all mammals, our data suggest that cJun is a critical target of AR and that its heterodimer partner, ATF2, adds yet another level of selectivity.

The suppression of {alpha}GSU transcription by CREB suggests that it competes with endogenous cJun/ATF2 for CRE binding and inhibits gene activity. Interestingly, the addition of both CREB and AR completely abrogated {alpha}GSU promoter activity (Fig. 1Go). AR has no effect on CREB expression as tested by immunoblot assay in androgen- treated {alpha}T3–1 cells (data not shown). Perhaps the loss in {alpha}GSU promoter activity occurs as a result of CREB and AR competing for additional adaptor proteins such as CREB-binding protein (CBP), or because CREB inhibits cJun and ATF2 from binding the CREs along with AR binding the remaining heterodimers, or a combination of these events. The addition of CBP in transient transfection assays had no effect on AR suppression of {alpha}GSU promoter activity (data not shown). However, cotransfection with both CREB and CBP may be what is required to relieve the {alpha}GSU promoter from the suppressive effects of AR.

AR-Mediated Suppression of {alpha}GSU Promoter Activity Does Not Depend On Altered Protein Concentrations
While assessing AR protein-protein interactions, we evaluated whether additional indirect mechanisms were contributing to the suppression of the {alpha}GSU promoter. For example, complete negative regulation by androgens could require a concerted set of transcriptional and posttranscriptional mechanisms including altered expression of critical factors. Whole-cell lysates, from {alpha}T3–1 cells transiently transfected with AR, were prepared at specific time points after treatment. For AR measurements, lysates were prepared after 1 h, based on reports of ligand-induced degradation of ER (22), and after 24 h, since all transfection assays were harvested at this time point. Expression of AR appeared to be stabilized in the presence of ligand (Fig. 3AGo). Western blot analyses of cJun, ATF2, and CREB, however, did not indicate any change in protein expression after overexpression of AR (data not shown).



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Figure 3. AR Suppression of the {alpha}GSU Promoter Is Not Caused by Indirect Mechanisms

A, Western blot analysis of transfected AR: An expression vector for AR (CMVhAR) was transfected into {alpha}T3–1 cells and treated with either EtOH (-) or DHT (+). All cells were treated with 100 nM GnRH. Western blot analysis of AR was performed on 40 µg whole-cell lysates at the indicated time points. Western blot analysis of endogenous p53: {alpha}T3–1 cells were transfected with CMVhAR and treated with 100 nM DHT for 24 h. Half of the cells were also treated for the last 8 h with 25 µM MG132. Western blot analysis of p53 was performed on 100 µg whole-cell lysates. Transient transfection assays were performed in {alpha}T3–1 cells that were cotransfected with (-1,500 to +45 bp) human {alpha}GSU promoter linked to luciferase, SV40-ß-galactosidase, and expression vectors for hAR (CMV5hAR). Cells were maintained in charcoal stripped media containing 100 nM DHT for 24 h, and 0 [dimethylsulfoxide (DMSO) vehicle] or 25 µM proteasome inhibitor, MG132, for the final 8 h. Luciferase activity of each construct was normalized to ß-galactosidase activity to control for transfection efficiency. Luciferase activity of the {alpha}GSU promoter was set to 1 in the absence of DHT; activity in the presence of DHT was reported as a percent of control activity. There was no significant difference in suppression between the two treatments (a: P < 0.41). B, {alpha}T3–1 cells were cotransfected with (-1,500/+45 bp) human {alpha}GSU promoter linked to luciferase, RSV-ß-galactosidase, and expression vectors for either GH (CMV5GH, Control, shaded bars) or AR (CMV5hAR, black bars). Cells were maintained in charcoal-stripped media containing 100 nM DHT and 0 (100% EtOH, vehicle), 0.01, 0.05, or 0.1 µM HDAC inhibitor, TSA, for 24 h. Luciferase activity of each construct was normalized to ß-galactosidase activity to control for transfection efficiency. Data represent the luciferase/ß-galactosidase activity of each treatment normalized to luciferase/ß-galactosidase of the full-length {alpha}GSU promoter in the presence of CMVGH. Transfections were performed a minimum of three times, and the error bars represent SE of these combined experiments. TK promoter activity was measured by luciferase activity in the presence of similar concentrations of TSA as a measure of HDAC activity (inset). Each treatment point is compared with that of TK promoter in the presence of vehicle (0). C, {alpha}T3–1 cells were cotransfected with (-1,500/+45 bp) human {alpha}GSU promoter linked to luciferase, RSV-ß-galactosidase, and expression vectors for either GH (Control), wild-type AR (AR), mutant AR-S81,94A (S81, 94A), or mutant AR-S650A (S650A). Cells were maintained in charcoal- stripped media containing 100 nM DHT for 24 h. Luciferase activity of each construct was normalized to ß-galactosidase activity to control for transfection efficiency. Data represent the luciferase/ß-galactosidase activity of each treatment normalized to luciferase/ß-galactosidase of the full-length {alpha}GSU promoter in the presence of CMVGH. Transfections were done a minimum of three times, and the error bars represent SE of these combined experiments.

 
Steadily emerging evidence suggests that the effectiveness of steroids may be limited by their ability to down-regulate their cognate receptors through the proteasome pathway (22, 23, 24, 25). We reasoned that if DHT stimulates ubiquitination of AR, marking it for degradation by proteasomes, then pharmacological blockade of the proteasome pathway should potentiate the negative regulatory effect of the steroid on the activity of the {alpha}GSU promoter. In addition, based on recent reports of GR inducing mRNAs of the proteasome pathway (26, 27), we investigated whether AR may stimulate degradation of other critical proteins. To test these possibilities, we performed transient transfection assays in the presence of MG132, an inhibitor of the proteasome degradation pathway. A concentration of 25 µM MG132 was determined to be sufficient for blocking proteasome activity based on stabilization of p53 (Fig. 3AGo), a protein particularly sensitive to degradation by the proteasome pathway (28). To account for hormone-independent effects of MG132, we transfected cells both in the presence and absence of DHT. Addition of MG132 had no significant impact on the suppressive effect of DHT, supporting previous evidence that the levels of AR were unchanged (Fig. 3AGo). Together, these data suggest that AR mediates the suppressive effect of DHT on the {alpha}GSU promoter independently of the proteasome degradation pathway.

AR-Mediated Suppression of the {alpha}GSU Promoter Does Not Require Histone Deacetylase (HDAC) Activity
Histone deacetylation has been associated with repressor activity of nuclear receptors as chromatin returns to a highly packaged, transcriptionally inert state (29, 30, 31). A number of the transcriptional proteins that regulate activity of the {alpha}GSU promoter are also known to affect the acetylation status of histones. For example, ATF2 has intrinsic HAT activity that specifically acetylates histones H2B and H4 in vitro (32). The middle portion of ATF2 (residues 112–350) has significant similarity to the HAT domain of p300/CBP-associated factor (PCAF), particularly motif A, which binds acetyl coenzyme A (32). Interestingly, motif A of the HAT domain was also found to be responsible for stimulation of CRE-dependent transcription (32). In addition, p300/CBP has been shown to either stimulate or repress promoter activity by acetylating non-histone proteins, including transcription factors (33, 34, 35, 36, 37). Finally, AR is also a target for acetylation, with lysine residues within the carboxy terminus affected by either trichostatin A (TSA) or the HAT-containing transcriptional coactivators p300 and PCAF. This acetylation may be functionally significant, depending on the cell type, given the recent demonstration that this posttranscriptional modification was important for hormone-dependent transactivation in prostate cell lines (38).

Nuclear receptors such as retinoic acid and thyroid hormone receptors function as potent transcriptional repressors by interacting with corepressors that recruit HDAC complexes to the promoter (39). To address whether histone deacetylation was associated with AR-mediated suppression of the {alpha}GSU promoter, transient transfection assays were performed in the presence of increasing amounts of TSA, a specific inhibitor of class I and II HDAC enzymes. Specific members of class I and II HDAC enzymes are known to interact with the corepressor SMRT (signal mediator and regulator of transcription) (40). A reporter vector containing the minimal thymidine kinase (TK) promoter was used as a positive control; its activity was stimulated by increasing concentrations of TSA treatment (inset, Fig. 3BGo) (41, 42). In contrast, TSA had no impact on the activity of the {alpha}GSU promoter, either in the presence or absence of AR (Fig. 3BGo). While we acknowledge that we have not tested for class III HDAC enzyme activity, our data suggest that class I and II HDAC enzymes have no ability to alter the transcriptional properties of the {alpha}GSU promoter in {alpha}T3–1 cells. This makes it less likely that AR interacts with a nuclear corepressor such as SMRT to suppress gonadotropin gene expression in these cells.

Phosphorylation Status of the AR-DBD Does Not Determine Its Ability to Suppress the {alpha}GSU Promoter
Phosphorylation of AR has been suggested to be important for two steps of receptor activation: acquisition of the ability to bind ligand and enhancement of DNA binding and subsequent transactivation (43). Indeed, mutation of serine to alanine in the hinge region (S650A) resulted in decreased phosphorylation and transactivation (44).1 To determine whether this also holds for transrepression, we examined activity of the {alpha}GSU promoter in transient transfection assays using two AR mutants. One carries a double mutation in the amino-terminal domain, AR S81, 94A, whereas the other harbors the mutation in the hinge region described above (AR S650A). While specific serine residues (S650) in the AR-DBD and hinge region have been shown to be required for AR activation (43, 44), we did not find this residue or others in the amino terminus (81, 94) to be important for AR-mediated suppression of {alpha}GSU (Fig. 3CGo). As no other phosphorylation sites have been detected in the AR-DBD-hinge region (554–660) (43, 44), these data suggest that the phosphorylation status of AR is not important for its repressive effect on the {alpha}GSU promoter.

In summary, we propose a model whereby AR suppresses {alpha}GSU promoter activity through direct protein-protein interactions with a specific subset of CRE-binding transcription proteins, including cJun and ATF2 (Fig. 4Go). Because AR suppression requires functional CRE sequences (6), we conclude that the protein-protein interactions occur within the promoter sequences. While cJun and ATF2 play critical roles in mediating the suppressive effects of androgens, we have also shown in previous studies (6) that essential contributions also come from an adjacent regulatory element, {alpha}BE. These combined data suggest that shared adaptor proteins may be involved in transducing the signal from the proteins that bind {alpha}BE and the CREs. We are currently identifying proteins that bind {alpha}BE and predict that they will probably form a higher order complex with cJun and ATF2. While AR may potentially interact with proteins that bind {alpha}BE or those involved in bridging {alpha}BE and the CREs, our data suggest that AR-mediated suppression of {alpha}GSU promoter activity requires at least the selective participation of cJun and ATF2. We suggest that AR interacts with cJun and ATF2 via its DBD leaving other regions such as the LBD free to interact with other adaptor proteins. These interactions most likely disrupt the synergy that is required between proteins that bind {alpha}BE and cJun/ATF2 on the CREs and consequently attenuate activity of the {alpha}GSU promoter.



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Figure 4. AR-DBD Interacts with the cJun/ATF2 Heterodimer and Interrupts Critical Communication Between Proteins That Bind the Synergistic Pentameric Complex of {alpha}GSU Binding Elements Causing Repression of Gene Activity

Because AR suppression requires functional CRE and {alpha}BE sequences, we propose that AR interactions occur within the promoter sequences. We suggest that AR selectively interacts with at least cJun and ATF2 via its DBD leaving other regions, such as the LBD, free to interact with other adaptor proteins. These interactions interrupt communication between transcription factors and the initiation complex to suppress gene activity.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
DHT, E2, TSA, isopropyl ß-D-thiogalactopyranoside, GnRH, Igepal, phenylmethyl sulfonylfluoride (PMSF), pepstatin A, leupeptin, and aprotinin were purchased from Sigma (St. Louis, MO). MG132 (Z-Leu-Leu-Leu-H) was purchased from Peptides International, Peptide Institute, Inc. (Osaka, Japan). Radiolabeled methionine was purchased from Dupont-NEN Life Science Products (Boston, MA). DNA-modifying enzymes and restriction enzymes were purchased from either Roche Molecular Biochemicals (Indianapolis, IN) or Life Technologies, Inc. (Gaithersburg, MD).

DNA
All plasmid DNAs were prepared from overnight bacterial cultures using QIAGEN DNA plasmid columns according to manufacturer’s protocol (QIAGEN, Chatsworth, CA). The human {alpha}GSU promoter construct has been described previously (7, 8, 45). The wild-type human AR (hAR) expression vector consists of the full-length AR cDNA fused to the cytomegalovirus (CMV) promoter (46). CMVGH (47) was described previously and has been used as a control expression vector that encodes a protein unrelated to gonadotrope activity. AR mutants S81, 94A, and S650A were generously provided by Elizabeth Wilson (University of North Carolina, Chapel Hill, NC) (44). CMVCREB was constructed by inserting the EcoRI/XhoI fragment of CREB{Delta} into pcDNA3 (Invitrogen, San Diego, CA) (48). pCMV2-cJun was kindly supplied by Paul Dobner (University of Massachusetts Medical Center, Worcester, MA) (49). pCMV2-cJun was digested with EcoRI, treated with calf alkaline phosphatase, and ligated into pGEM-4Z (Promega Corp., Madison, WI) for TnT reactions. ATF2 (50) was constructed by inserting the full-length 1,900-bp BamHI fragment from RSV-ATF2, kindly donated by Michael Green (University of Massachusetts Medical Center, Worcester, MA), into CMV5 and pcDNA3 expression vectors. GST-hAR-DBD encoding amino acids 554–644 of hAR inserted into pGEX-5X-1 (Amersham Pharmacia Biotech, Uppsala, Sweden) was generously provided by Drs. Olli Janne and Jorma Palvimo (University of Helsinki, Helsinki, Finland) (15). GST-hAR-DBD-C576A was made by inserting the PCR fragment containing residues 554–644 of full-length mutant AR-C576A (46) (PCR primers 5' with BamHI linker 5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with EcoRI linker 5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3') into pGEX-2T (Amersham Pharmacia Biotech).

Cell Culture and Transient Transfections
{alpha}T3–1 cells were maintained in high-glucose DMEM supplemented with 5% FBS, 5% horse serum, penicillin, and streptomycin (Life Technologies, Inc.). Twenty-four hours before transfection, 180,000 cells were plated per 35-mm well in six-well plates. Cells were transfected with the indicated DNAs using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer’s guidelines. Reporter constructs (luciferase, 1.25 µg/well) were cotransfected with expression vectors as indicated. The amount of transfected cDNAs was kept constant in the dose-response transfections by adding empty CMV expression vector (CMV5). The lipofectamine/DNA solution was replaced with complete medium containing charcoal-stripped serum along with various treatments after 12–16 h. Treatments included 100 nM DHT, 100% EtOH vehicle, MG132 (as indicated), or TSA (as indicated). Cells were harvested 24 h later using 150 µl of reporter lysis buffer (Promega Corp., Madison, WI). Luciferase activity was quantified by luminescence using 15 µl lysate and 100 µl luciferase assay reagent (Promega Corp.). ß-Galactosidase activity was quantified also by luminescence using the Galacto-light assay system (Tropix, Bedford, MA). The values were averaged over a minimum of three independent experiments.

Western Blot Analysis
Cells were rinsed twice with ice-cold PBS solution and harvested from the plates, and then resuspended in cell lysis buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl, pH 8, with protease inhibitors PMSF, aprotinin, and leupeptin) and incubated on ice with frequent vortexing for 30 min. The cell debris was pelleted and the supernatant was analyzed for protein content by Bradford analysis (Bio-Rad Protein Assay, Bio-Rad Laboratories, Inc. Hercules, CA). Lysate (30–40 µg) was resolved on an SDS-PAGE gel using a 4% stacking gel and a 10% separating gel as described elsewhere (7). Proteins were then transferred to a nitrocellulose membrane (Protran, Schleicher & Schuell, Inc., Keene, NH) and rinsed in a solution of PBS-Tween (0.1% Tween-20 in PBS). The membrane was blocked in PBS-Tween with 2.5% dry milk at 4 C overnight, and then incubated with primary antibodies, as indicated, in PBS-Tween-2.5% dry milk for 2–3 h at room temperature. The membrane was rinsed three times in PBS-Tween, and then incubated with PBS-Tween plus 2.5% dry milk containing the secondary antibody for 1–2 h at room temperature. After three 15-min rinses in PBS-Tween, the antibody-labeled proteins were visualized by chemiluminescence using the Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products, Boston, MA).

Antibodies
The following antibodies were used. Primary: p53, AR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); secondary: antirabbit horseradish peroxidase (Santa Cruz Biotechnology, Inc., or Amersham Pharmacia Biotech).

Purification of GST Fusion Constructs and GST-Pull-Down Assays
Empty GST construct (pGEX-5X-1), GST-hAR-DBD, or GST-hAR-DBD-C576A was transformed into the DH5{alpha} strain of Escherichia coli. A single colony was inoculated into 2 ml LB + ampicillin (100 µg/ml) and incubated in a 37 C shaker for 5 h. This inoculation was then diluted 1:15 in fresh LB-amp broth and incubated at 37 C overnight. Sixteen hours later, a further dilution (1:100) was incubated for 3 h at 37 C. Isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 1 mM, and culture was grown for another 4 h at 37 C. Bacteria were pelleted at 7500 x g for 10 min at 4 C and then frozen overnight at -80 C. The pellet was resuspended in 3 ml NET (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, PMSF, pepstatin A, leupeptin, and aprotinin protease inhibitor solution) with 1 mg/ml lysozyme added. The pellet was vortexed frequently during the 15- to 30-min incubation. The cells were disrupted by one freeze-thaw cycle, a 30 sec sonication, and two additional freeze-thaw cycles. The suspension was centrifuged at 35,000 rpm for 1 h at 4 C, and the supernatant was aliquoted and stored at -80 C.

GST-protein extracts (75 µl) were incubated with 25 µl Glutathione Sepharose Beads (Amersham Pharmacia Biotech) for at least 2.5 h at 4 C and then washed twice with 1 ml NENT buffer (20 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, 0.5% dry milk, and the protease inhibitor solution) and twice with 1 ml binding buffer (20 mM HEPES, pH 7.9, 10% glycerol, 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 1 mM EDTA, and the protease inhibitor solution). The beads were resuspended in 200 µl binding buffer and incubated with 4–6 µl in vitro translated products at 4 C overnight. Indicated expression vectors were used to make [35S] methionine-labeled in vitro translated products with TnT Coupled Reticulocyte Lysate reaction system according to manufacturer’s instructions (Promega Corp.). The matrix was washed three times with 1 ml NENT buffer, and then twice with binding buffer and resuspended in 20 µl elution buffer (3 mg/ml glutathione in 50 mM Tris-Cl, pH 7.5). After a 10-min incubation at room temperature, the suspension was centrifuged, and 18 µl of the eluant were loaded onto a 10% SDS-PAGE gel for analysis. GST-hAR-DBD, GST-hAR-DBD-C576A, or GST-bound radiolabeled protein products were visualized after exposure to film for 5 d (Biomax MR, Eastman Kodak Co., Rochester, NY).

Statistical Analysis
Differences in luciferase activity compared with control were analyzed by a two-tailed Student’s t test (Fig. 3BGo).


    ACKNOWLEDGMENTS
 
The authors wish to thank Drs. Monica Montano, Paul MacDonald, and Amy Wilson for critical evaluation of this manuscript. We would also like to thank Helai Mohammad for the CMVCREB plasmid.


    FOOTNOTES
 
This work was supported by NIH Grants R01-DK-28559 (J.H.N.) and K08-DK-02600 (J.S.J.).

Abbreviations: ATF, Activation transcription factor; AR, androgen receptor; ARE, androgen response element; {alpha}BE, {alpha} basal element; CRE, cAMP response element; CBP, CREB binding protein; CMV, cytomegalovirus; CREB, CRE binding protein; CREM, CRE modulator; DBD, DNA-binding protein; DHT, dihydrotestosterone; ER, estrogen receptor; hAR, human AR; HAT, histone acetyltransferase; HDAC, histone deacetylase; LBD, ligand-binding domain; PMSF, phenylmethylsulfonyl fluoride; RSV, rous sarcoma virus; TK, thymidine kinase; TSA, trichostatin A.

1 Full-length AR numbering is based on 919 residues. Back

Received for publication March 13, 2001. Accepted for publication May 14, 2001.


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 RESULTS AND DISCUSSION
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