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Molecular Endocrinology 20 (9): 2052-2061
Copyright © 2006 by The Endocrine Society

The Role of the General Transcription Factor IIF in Androgen Receptor-Dependent Transcription

M. Ansar Choudhry1, Andrew Ball1 and Iain J. McEwan

School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Dr. Iain J. McEwan, School of Medical Sciences, Institute of Medical Sciences Building, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom. E-mail: iain.mcewan{at}abdn.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) is a member of the steroid receptor subfamily of nuclear receptors and is important for normal male sexual differentiation and fertility. The major transactivation function of the AR, termed activation function 1 (AF1), is modular in structure and has been mapped to the N terminus of the protein. To understand better the mechanisms whereby the AR activates transcription, we have established a novel cell-free transcription assay. This is based on the use of a dual reporter gene template, containing promoter proximal and distal G-less cassettes, which result in different size transcripts that can be easily detected and quantified. The promoter proximal transcript gives an indication of transcription initiation and promoter escape, whereas the relative levels of the distal transcript indicate elongation efficiency. The AR-AF1-Lex protein enhanced production of both transcripts whereas, in the absence of DNA binding, the AF1 domain squelched both initiation and elongation. Mutations in the transactivation domain that impaired transactivation and/or binding of the general transcription factor IIF (TFIIF) were found to reduce the ability of AR-AF1 to squelch transcription. Addition of recombinant TFIIF reversed squelching of the promoter-proximal but not the -distal G-less transcript, whereas addition of TATA-binding protein failed to reverse squelching of either transcript. Taken together, these results demonstrate that the AR N-terminal transactivation function, AF1, has the potential to regulate transcription at both the level of initiation and elongation, and that interactions with TFIIF are important during preinitiation complex assembly/open complex formation and/or promoter escape.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a member of the nuclear receptor superfamily and is responsible for regulating androgen-responsive genes important for controlling male sexual differentiation and fertility (1, 2, 3, 4). The receptor functions as a ligand-activated transcription factor, and the major transactivation function has been mapped to the structurally distinct amino-terminal domain (NTD). The importance of sequences between amino acids 142 and 485 for gene activation has been highlighted for both the human and the rat AR, by deletion analysis (5, 6, 7), use of fusion proteins (8, 9, 10), and by point mutagenesis (11, 12).

We have previously identified and characterized the interaction between the AR-activation function 1 (AF1) and the general transcription factor TFIIF (9, 10). TFIIF is a {alpha}2ß2 heterotetrameric component of the general transcriptional apparatus and has been found to act at multiple steps during the transcription cycle. During transcription initiation TFIIF plays an important role in assembly and stability of the preinitiation complex (PIC), open complex formation, and promoter escape. Subsequently, TFIIF enhances transcription elongation by preventing pausing by the RNA polymerase II (RNAPII) enzyme (reviewed in Refs. 13 and 14). In protein-protein binding studies, the large subunit of TFIIF, termed RAP74 (RNA polymerase II-associating protein 74), was identified as a target for the amino-terminal transactivation function of the AR (amino acids 142–485) and was shown to specifically reverse AR-dependent squelching of basal transcription (9). More recently we have mapped the AR-binding site to the N- and C-terminal domains of RAP74 and have identified point mutations that selectively disrupt interactions with TFIIF, but not binding to the coactivator protein SRC-1a (10, 12). Significantly, binding of TFIIF (RAP74) induces folding of the AR-AF1 domain to a more protease-resistant conformation (15) and results in an increase in {alpha}-helical structure (16).

Whereas considerable progress in identifying androgen-regulated genes and protein partners for the receptor has been seen in recent years, the mechanism(s) of AR-dependent transcription remain largely unknown. To study the role of the AR during transcription initiation and elongation and to further investigate the role of TFIIF in AR-dependent transcription, we have used an elegant in vitro transcription method developed by Lee and Greenleaf (17), in which dual G-less cassettes are differentially positioned with respect to the promoter. The promoter-proximal, small G-less cassette measures initiation, promoter escape, and early elongation, whereas the large, promoter distal G-less cassette provides a measure of elongation efficiency. This system has allowed the role of the AR to be investigated at different steps in the transcription cycle, through the ability of the AR-transactivation function to activate or squelch basal transcription. The AR polypeptide (amino acids 142–485) squelched transcription of both G-less cassettes in a concentration-dependent manner. This is consistent with receptor-dependent regulation of transcription at the levels of PIC assembly, promoter escape, and elongation. Mutations within the AR:AF1 domain that are impaired for TFIIF binding and/or activation failed to squelch basal transcription. Significantly, recombinant TFIIF added back to the system selectively rescued transcription of the promoter-proximal G-less cassette. These results strongly suggest that AR-TFIIF interactions are important during the initiation steps of the transcription cycle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The AR-AF1 Transactivation Function Regulates Both Transcriptional Initiation and Elongation
Although a dramatic increase in the number of factors that interact with steroid receptors has been seen in recent years, including the AR (reviewed in Ref. 18), the mechanistic details of receptor-dependent gene regulation remain largely undetermined. In the present study, we have used a cell-free transcription assay together with a dual reporter gene template to determine the role of the AR at different steps of the transcription cycle. The structure of the AR is shown schematically in Fig. 1AGo, with the portion of the N terminus corresponding to the transactivation function, AR-AF1, highlighted. This region of the protein was expressed in bacteria as described and used in in vitro experiments to measure the impact of the AR N terminus on transcription initiation and elongation. Figure 1BGo shows the partially purified proteins used in the present study.


Figure 1
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  		Fig. 1. Expression of Recombinant Proteins in E. coli

A, Schematic representation of the human AR showing the functional and structural domain organization. Recombinant proteins representing the AR-AF1 transactivation domain, alone and as a Lex DNA-binding domain fusion protein, are also shown. B, Sodium dodecyl sulfate-polyacrylamide gel showing purified recombinant proteins: AR-AF1 and AR-AF1-Lex, and the basal transcription factors TFIIF and TBP. DBD, DNA-binding domain; LBD, ligand-binding domain; IIF, transcription factor IIF.

 
The reconstitution of AR-AF1 activity in vitro provides an important system for investigating the mechanism of receptor-dependent transactivation. Figure 2AGo shows a schematic diagram of the two reporter gene constructs, pSLG407 and pSLG-4xLex, containing dual G-less cassette reporter genes driven by the adenovirus major late (AdML) promoter. The promoter-proximal and -distal G-less cassettes are separated by 368 bp and give rise to short (85 nucleotides) and long (377 nucleotides) RNA transcripts, respectively. Production of the short transcript indicates early transcription initiation events, whereas production of the long transcript has been correlated with the phosphorylation status of the C-terminal domain (CTD) of the large subunit of RNA polymerase II and therefore serves as a measure of elongation efficiency (17). The pSLG-4xLex template has had the upstream stimulatory factor binding site (UAS) at –50 within the AdML promoter replaced by 4x tandem LexA-binding sites. Transcription in HeLa nuclear extracts was template- and activator-dependent, with maximum stimulation of both the short and long transcripts observed between 0.1–10 nM of recombinant AR-AF1-Lex protein (Fig. 2Go, B and C). At protein concentrations above 2 µM, the activator protein, AR-AF1-Lex, resulted in self-squelching (inhibition) of transcription (Fig. 2Go, B and C). This phenomenon has been observed previously for other activators, most notably VP16, and with the isolated AR-AF1 domain and a single reporter gene (Ref. 9 and references therein). Under these conditions, a consideration of the calculated molar ratio of long G-less transcript (LGT) to short G-less transcript (SGT) does not show any dramatic effect on elongation rate relative to initiation. To examine the role of RNA polymerase II and phosphorylation of the CTD of the large subunit in the activation by AR-AF1, the inhibitors {alpha}-amanitin and 5, 6-dichlorobenzimadazole riboside (DRB) were used. Addition of {alpha}-amanitin at 25 µg/ ml dramatically inhibited transcription demonstrating the involvement of RNA polymerase II (Fig. 3AGo). The kinase inhibitor, DRB, resulted in a concentration dependent inhibition of transcription, reducing the levels of both transcripts, but having a greater impact on production of the long transcript (Fig. 3Go). At 50 µM of DRB, transcription past +867 was inhibited 92%, compared with 73% past +124. A greater effect on the production of the long transcript is also revealed by a reduction in the molar ratio of the LGT:SGT, from 0.38 to 0.12, which would be consistent with the elongating form of the RNA polymerase being hyperphosphorylated on the CTD of the large subunit. Taken together, these results demonstrate that AR-AF1 is capable of activating transcription by RNA polymerase II under cell-free conditions, when tethered to the promoter via a heterologous DNA-binding domain.


Figure 2
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  		Fig. 2. Activation of Transcription in Vitro by AR-AF1

A, Schematic representation of the dual G-less cassette-bearing templates, pSLG407 and pSLG-4Lex, used in the in vitro transcription assays. The promoter-proximal and -distal G-less reporter genes are separated by 368 bp, and resolution of the transcripts after RNaseT1 digestion results in transcripts of 85 and 377 nucleotides, respectively. Transcription is driven by the AdML promoter (pSLG407) or 4xdouble LexA response elements upstream of the TATA box (pSLG-4xLex). B, A representative gel showing activation of transcription from the pSLG-4Lex template in response to increasing concentration of AR-AF1-Lex (1 pM to 10 µM). LGTs and SGTs are indicated. C, Quantification of the short and long transcripts from the gel shown in panel B. The results shown are representative of six experiments showing similar trends.

 

Figure 3
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  		Fig. 3. Involvement of RNA Polymerase II and Phosphorylation of the Large Subunit CTD in Receptor-Dependent Transcription in Vitro

A, Production of both the short and long transcripts was essentially completely inhibited by 25 µg/ ml {alpha}-amanitin. B, Increasing concentrations of the kinase inhibitor DRB resulted in a dose-dependent inhibition of both the short and long transcripts. The percent inhibition of each transcript is indicated above each bar. The results shown are representative of four experiments showing similar trends.

 
The ability of an activator protein to inhibit or squelch transcription at elevated concentrations has proved a useful means of investigating protein-protein interactions and transcription regulation (see Ref. 9 and references therein). Using the pSLG 407 template, the levels of transcription in HeLa nuclear extracts were quantified and normalized using the basal signal obtained with no recombinant proteins added to the transcription system. Addition of increasing amounts of the AR-AF1 polypeptide, in the absence of a DNA-binding domain, resulted in marked squelching of both the short and long transcripts. Figure 4Go shows there is a dose-dependent inhibition of both transcripts with increasing amounts of AR-AF1 (0.8–5 µM). To demonstrate the specificity of squelching and to relate this activity to the ability of AR-AF1 to activate transcription, we used a series of point mutations in AF1 previously shown to impair activation in vivo (M1–M4) and/or disrupt protein-protein interactions with the general transcription factor TFIIF (M3 and M6) (Fig. 5AGo; and Refs. 10 and 12). Figure 5Go (panels B and C) shows that mutations in highly conserved hydrophobic amino acids (M3, M4) or a six-amino acid repeat sequence (M6) failed to squelch basal transcription from the AdML promoter. The mutant polypeptide M2 had variable activity but showed a general reduction in ability to squelch. In contrast, the mutations present in M1, which have a more modest affect on transactivation in vivo (12), do not prevent squelching, as this polypeptide was as effective as the wild-type protein. Together these data suggest that the squelching of basal transcription is a specific phenomenon relevant to the ability of the AR to activate transcription, as mutations that impair activation also impact upon the ability of the isolated AR-AF1 domain to squelch transcription.


Figure 4
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  		Fig. 4. AR-AF1 Squelches Basal Transcription through Protein-Protein Interactions

A, Squelching of elongation and initiation by AR-AF1. A representative gel is shown of squelching of basal transcription, from template pSLG407, by increasing concentrations of the isolated AR-AF1 domain (0.8 to 5 µM) in the presence of 75 µg of HeLa nuclear extracts. The SGTs and LGTs are labeled. B, Plot of relative transcriptional activity against amount of AR-AF1 polypeptide added. The data are from three independent experiments, and the intensities of the long and short G-less transcripts have been normalized against values obtained from the basal reaction (no AR-AF1 added) (mean ± SD).

 

Figure 5
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  		Fig. 5. Point Mutations in AR-AF1 that Impair Function Are Defective in Squelching

A, Schematic representation of the point mutations introduced into the AF1 domain. Mutations M1–M4 represent alanine substitutions in highly conserved hydrophobic residues and have been characterized previously (12 ). M6 represents the mutation of serine 159 and 162 to alanine in a six-amino repeat sequence (42 ). The predicted secondary structure for the AR-AF1 is shown above a schematic of the polypeptide domain; the long bars represent {alpha}-helix and the short bars represent ß-strand. A representative Coomassie blue-stained gel of wild-type and mutant AR-AF1 polypeptides (2–6 µg of protein per lane) is shown. B, Represented transcription assay showing the effects of adding 100 pmol (3.3 µM) AR-AF1 wild-type and mutant polypeptides (M1–M4 and M6) on basal transcription. M1–M4 and M6 are as described in panel A. C, Relative transcription levels of the short and long transcripts from template pSLG407 from the representative experiment in panel B. Similar trends were observed in up to five independent experiments. WT, Wild type.

 
TFIIF Specifically Rescues AR-AF1-Dependent Squelching of Transcription Initiation
Previously, we have demonstrated an interaction between the N terminus of the AR and the large subunit of the general transcription factor TFIIF (9, 10). Therefore, the effect of adding recombinant holo-TFIIF on AR-dependent squelching of the promoter-proximal and -distal G-less cassettes was tested. Figure 6AGo shows representative results, with the first two lanes showing the basal response of the system in the absence of exogenous recombinant proteins: both transcripts are clearly observed. As expected, addition of 100 pmol AR-AF1 (3.3 µM) led to squelching of both the short and long transcripts (Fig. 6AGo, lanes 3 and 4). Addition of 10 and 20 pmol TFIIF in the presence of 100 pmol AR-AF1, failed to rescue transcription past +847 (long transcript), but significantly increased transcription of the short G-less cassette (Fig. 6AGo, lanes 5–8). Figure 6BGo shows the quantitative analysis of the data from three independent experiments. These results strongly support a mechanism whereby AR-AF1 squelching of initiation and/or promoter escape is mediated via interactions with TFIIF. These findings, together with the results using mutant AF1 polypeptides, demonstrate that squelching of transcription is the result of selective action of AR-AF1 on the basal transcription machinery.


Figure 6
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  		Fig. 6. TFIIF Rescues AF1-Dependent Squelching of Transcription Initiation

A, Reversal of AR-AF1-mediated squelching by TFIIF. A representative gel is shown of the reversal of squelching experiments, using 50 µg of total HeLa nuclear extract protein per reaction. Reactions were carried out in duplicate, and additions of 10 pmol of AR-AF1 (3.3 µM) and/or TFIIF (10 or 20 pmol) into the system are indicated below each pair of lanes. The SGTs and LGTs are labeled. B, Plot of relative transcriptional activity in the absence or presence of added recombinant proteins. The data are from three independent experiments (mean ± SD).

 
The ability of TFIIF to selectively reverse AR-AF1 squelching of early transcriptional events strongly suggest this is a specific response and not simply a reflection of TFIIF being limited in the assay. However, to test this further, we used another of the general transcription factors, the TATA-binding protein (TBP). TBP has previously been shown to be a target for a number of cellular and viral activators, including members of the nuclear receptor superfamily, and has been shown to reverse squelching by c-myc (19) and VP16 (9) under cell-free conditions. However, addition of partially purified human (h)TBP failed to significantly reverse AR-dependent squelching of either transcript (Fig. 7Go).


Figure 7
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  		Fig. 7. Recombinant TBP Fails to Reverse AF-1-Dependent Squelching

Basal transcription was squelched by the addition of 100 pmol AR-AF1 (3.3 µM) to 50 µg of total HeLa nuclear extract protein per reaction. Addition of 10 or 20 pmol of hTBP had no significant affects on the levels of either the short or long transcripts in the presence of AR-AF1. Reactions were carried out in duplicate and the SGTs and LGTs were quantified. The data from four independent experiments have been plotted as shown (mean ± SD).

 
Importantly, addition of recombinant TBP or TFIIF, in the absence of AR-AF1, to HeLa nuclear extracts failed to stimulate basal levels of transcription of either the short or the long transcript and, instead, both factors showed variable degrees of squelching (data not shown). This further argues that the rescue of transcription initiation, in the presence of AR-AF1, is not simply a reflection of TFIIF having a global stimulatory action on transcription in this system. Taken together, the above results support a model whereby AR interactions with TFIIF are specific and important for one or more of the early events in transcription initiation, but subsequent actions of the receptor on the elongating RNA polymerase II may be independent of TFIIF.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using a novel dual reporter gene construct in vitro, we have shown that the AR-AF1 domain can regulate both transcription initiation and elongation. When targeted to the promoter via a heterologous DNA-binding domain, AR-AF1 increased the levels of both a promoter-proximal (short) and a promoter-distal (long) transcript. A similar activation response is observed with the glucocorticoid receptor AF1 domain (termed {tau}1) for the concentration range 9 nM to 1 µM, with maximum stimulation at 75–100 nM concentration and self-squelching of activation at 1 µM (Choudhry, M. A., and I. J. McEwan, unpublished observations). In the absence of a DNA-binding domain, the AR-AF1 domain squelched basal transcription. This activity was specific because mutations that impair transactivation and/or protein-protein interactions failed to squelch transcription. The ability of the isolated AR-transactivation function to squelch basal transcription was used to investigate protein-protein interactions important for receptor-dependent transcription. Addition of recombinant holo-TFIIF reversed squelching of the promoter-proximal, but not the promoter-distal transcript. Taken together, these results suggest that the AR is capable of regulating early events in the transcription cycle, PIC formation, initiation, and/or promoter escape, at least in part, through interactions with TFIIF. Interestingly, under conditions where the promoter proximal transcript was unaffected, the AR-AF1 domain selectively squelched the promoter-distal G-less reporter gene (Ball, A., and I. J. McEwan, unpublished observations), supporting an additional role for the AR in regulating elongation efficiency by the RNA polymerase II complex. However, addition of TFIIF failed to rescue transcription past +867. This might suggest that AR-AF1 targets a different component of the RNA polymerase II elongating complex. Indeed, a recent report showed an interaction between the receptor and the second largest subunit of the polymerase enzyme (20). Alternatively, the failure of recombinant TFIIF may indicate AF-1-dependent sequelching of elongation is more complex than simply sequestering a single factor necessary for transcription. Further studies will be needed to addresses these issues. There is now considerable evidence that both viral and cellular activators act at multiple steps during the transcription reaction (see Refs. 21, 22, 23, 24, 25, 26). On the basis of the classification system proposed by Blau et al. (23), the AR would join p53, E2F, and VP16 as a class type IIB activator, acting at both initiation steps and during elongation.

TFIIF is a heterotetramer consisting of RNA polymerase II-associating proteins (RAP) 30 and 74, which interact through a novel triple barrel fold (27). TFIIF is involved in multiple steps during initiation, including stabilizing the binding of RNA polymerase II, open complex formation, and the synthesis of the first phosphodiester bond of the new transcript, and is part of the elongation complex, where it reduces pausing by the RNA polymerase (28, 29, 30, 31). In an elegant series of cross-linking studies, TFIIF was shown to mediate bending of the promoter DNA around the PIC, and this may be important for open complex formation by allowing access for TFIIH helicase activity (32). The present assay does not permit us to identify which of these activities of TFIIF is regulated by the AR during transcription activation. However, in the present in vitro transcription system, TFIIF may be more limiting for initiation and early promoter escape but not for elongation per se. This can be seen by the affect of TFIIF on transcription in the absence of the AR polypeptide, where addition of recombinant holo-TFIIF significantly squelched the production of the long transcript, but had a more modest effect on the levels of the short transcript. In contrast, addition of TBP under these conditions squelched both the short and long transcription, suggesting it is not limiting in our assay (Ball, A., and I. J. McEwan, unpublished observations). Thus, the AR may act to recruit TFIIF to the PIC and early elongating complex. Alternatively, because the AR-binding sites within RAP74 map to regions involved in protein-protein and/or protein-DNA interactions (10), it is tempting to speculate that the receptor may compete for TFIIF interactions with components of the PIC (29, 33, 34, 35) and/or DNA (32) and thus lead to release of the RNA polymerase during initiation. Interestingly, the C terminus of RAP74 (amino acids 436–517) also interacts with the phosphatase FCP1, which removes phosphate groups from the CTD of the largest subunit of RNA polymerase II (36, 37, 38). Thus, the binding of AR-AF1 may act to prevent recruitment of the phosphatase and dephosphorylation of the RNA polymerase II large subunit CTD. Further experiments will be required to distinguish between these possibilities.

The multisubunit general transcription factor TFIIH has previously been implicated in activator-dependent regulation of elongation (39). Recently, an interaction between the cyclin-activated kinase (CAK) subunits of TFIIH and the AR-NTD has been reported (40). Previously, it has been shown that TFIIH CAK phosphorylates the CTD of the large subunit of RNA polymerase II (39) and, interestingly, the retinoic acid (41) and the estrogen receptors (42). Thus, it will be interesting to determine whether CAK enhancement of AR activity is the result of direct phosphorylation of the receptor or the CTD of the large subunit of RNA polymerase II. Recently, Chang and co-workers (43) provided evidence that androgen treatment could enhance elongation of the prostate-specific antigen (PSA) gene and showed an interaction between the AR-NTD and the positive elongation factor b (P-TEFb). Similarly, the cellular activators nuclear factor-{kappa}B (21) and c-myc (25) were shown to enhance transcription elongation via interactions with P-TEFb. P-TEFb consists of cdk9 and cyclin T subunits, which also act to phosphorylate the CTD of the large subunit of RNA polymerase II (44). Hyperphosphorylation of the CTD is critical for the transition of the polymerase enzyme to the elongation-competent form; thus the actions of TFIIH and P-TEFb are likely to be during the early steps of the transcription cycle (Refs. 13 and 14 and references therein).

Recently, a number of groups have characterized the binding of the AR to the promoter and enhancer sequences of the PSA gene using chromatin immunoprecipitation assays. These studies demonstrate the recruitment of the receptor and coactivator proteins [i.e. transcriptional intermediary factor 2, cAMP response element binding protein (CREB)-binding protein], together with the RNA polymerase to the enhancer and/or promoter sequences in a hormone-dependent manner. Interestingly, Kang et al. (45) demonstrated clear cycling of the receptor, coactivators, and polymerase at the PSA promoter. Subsequent studies from this group identified changes in histone modifications (acetylation, methylation, and phosphorylation) in response to AR recruitment and coactivator binding (45). Although results from the different groups have emphasized some variation in the loading of the AR at the enhancer and promoter regions, they have allowed two models to be proposed for the possible mechanism of action of the receptor. In the first there is recruitment of the receptor and the transcription machinery to both the promoter and the enhancer with looping of the DNA in between (see Ref. 46). An alternative model involves loading of the receptor and RNA polymerase primarily at the enhancer with sliding of the enzyme to the promoter (see Ref. 47). These models need not be mutually exclusive. However, Louie et al. (47) show convincingly, through sequential chromatin immunoprecipitation assays, that whereas the polymerase is found at both the promoter and the enhancer, the AR is predominantly at the enhancer. Furthermore, the occupancy of the promoter sequences by the polymerase appears to be dependent upon phosphorylation of the CTD of the RNA polymerase because blocking phosphorylation reduced the enzyme at the promoter and increased binding to the enhancer (47). Taken together, these studies provide an important series of snapshots at an endogenous promoter but do not directly address the mechanism of receptor-dependent regulation of initiation and/or elongation.

We have previously reported that the AR-AF1 domains lack stable secondary structure, but become more structured in the presence of the chemical chaperone, trimethyl amine-N-oxide or upon binding of RAP74, the large subunit of TFIIF (15). We then mapped the AR-interacting domain to the NTDs and CTDs of RAP74 (10). Using Fourier-transformed infrared spectroscopy, it could be demonstrated that the RAP-74-CTD induced a significant increase in {alpha}-helix structure within the AR-AF1 domain, which further enhanced interactions with a member of the p160 family of coactivators (16). In the present study, we have described a novel use of a dual-reporter gene cell-free transcription assay, which provides a useful model system in which to dissect the function of the AR during transcription initiation and elongation. Using this system we could show that interactions with the general transcription factor TFIIF are important for AR-dependent regulation of one or more steps during transcription initiation, involving PIC assembly and/or promoter clearance. Furthermore, using the ability of the isolated AR-AF1 domain to squelch transcription, it was possible to identify a distinct role for the receptor during transcription elongation. Thus, interactions with the TFIIF are important for functional folding of the AR-AF1 domain and its ability to regulate transcription initiation. Further studies will be necessary to 1) determine which function(s) of TFIIF are regulated by the AR and 2) identify the target(s) for the AR in the elongation complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Reporter Constructs
Expression plasmids pET-23d/RAP74 1–517 and pETRAP30, encoding the large and small subunit of TFIIF, respectively, were kindly provided by Dr. Z. F. Burton (Michigan State University, East Lansing, MI). Recombinant RAP74 has a C-terminal hexahistidine tag to aid with purification. The hTBP expression plasmid, phTFIID was a gift from Dr. A. Berk (University of California, Los Angeles, CA). The dual reporter gene template, pSLG407, used in the in vitro transcription reactions, was a kind gift from Dr. A. Greenleaf (Duke University, Durham, NC). PSLG407-Lex was created by digesting pSLG407 with PmlI restriction enzyme, which was then blunt ended by incubating with deoxynucleotide triphosphates and Klenow fragment (DNA polymerase; Roche, Indianapolis, IN), dephosphorylated with shrimp alkaline phosphatase (Roche), and gel purified. The LexA DNA response element (highlighted in bold) double-stranded oligonucleotides, 5'-ctgaacctgtatgtacatacagagatctcgggggatccctgtatgtacatacagcctgca-3' (upper strand), were polymerized four times and gel purified. The LexA DNA response elements (4x) fragment was then cloned into the linear vector using the Liga-Fast kit (Promega Corp., Madison, WI) and a DNA ratio of vector-insert of 2:1. A positive clone was identified and confirmed by sequencing and used in subsequent transcription assays.

Purification of Recombinant Proteins
The AR amino-terminal transactivation function, amino acids 142–485 (Fig. 1AGo), alone or fused to the LexA DNA-binding domain, together with RAP74 were expressed in bacteria by the addition of 1 mM isopropyl-ß-D-thiogalactopyranoside and purified as described previously (9, 10), with the addition that after freeze thawing and incubation with 0.1 mg/ml lysozyme, the cells were sonicated using a VibraCell probe sonicator (Sonics and Materials, Inc., Newtown, CT). The soluble recombinant proteins were purified from the supernatant by metal ion chelation chromatography using either QIAGEN nitrilotriacetic acid (QIAGEN, Chatsworth, CA), or CLONTECH Talon resins (CLONTECH Laboratories, Inc., Palo Alto, CA) and dialyzed against 20 mM HEPES, 5% glycerol, 100 mM KCl, and 1 mM dithiothreitol (DTT), pH 7.9 (Fig. 1BGo). Protein concentration was measured by the method of Bradford (48) and proteins were aliquoted and snap frozen in liquid nitrogen before use.

The hTBP was expressed as above and partially purified by a series of ammonium sulfate cuts, from 0–30%, 30–60%, and 60–90% saturation. The precipitated proteins were resuspended in the HEPES dialysis buffer and extensively dialyzed to remove ammonium sulfate. The 0–30% cut was found to be enriched for hTBP (Fig. 1BGo).

RAP30 was expressed in BLR(DE3) cells and isolated from inclusion bodies. The cells were disrupted by exposure to 6 M urea, and the debris was removed by centrifugation to give a supernatant highly enriched for RAP30.

Refolding of holo-TFIIF
TFIIF was reconstituted by refolding the RAP30 and RAP74 subunits together as described elsewhere (see Ref. 29). Briefly, approximately equimolar amounts of the two components were mixed in the presence of 6 M urea, and the urea was gradually dialyzed from the system. The refolded proteins were subject to metal ion chelation chromatography, and eluted proteins were dialyzed against 20 mM HEPES, 5% glycerol, 100 mM sodium acetate, 1 mM DTT (pH 7.9) and analyzed by SDS-PAGE (Fig. 1BGo). Because RAP30 lacks a polyhistidine tag, any RAP30 co-purifying with RAP74 is assumed to be present as a result of cofolding of the two subunits to give intact holo-TFIIF. Protein concentration was measured by the method of Bradford (48) and aliquots were snap frozen using liquid nitrogen and stored at –80 C until use.

In Vitro Transcription Reactions
Transcription from the pSLG407 and pSLG-4xLex templates was performed using a HeLa nuclear extract-based transcription system. HeLa nuclear extract (HNE) was obtained commercially from Computer Cell Culture Centre (Seneffe, Belgium). Briefly, exogenous proteins, as indicated, were preincubated with 50 or 75 µg HeLa nuclear extract and 100 ng of template DNA on ice for 25 min before addition of the reaction mixture containing 12 mM HEPES-KOH (pH 7.9), 6 mM MgCl2, 12% (vol/vol) glycerol, 60 mM KCl, 0.1 mM EDTA, 0.4 units Prime RNAse inhibitor (Eppendorf), 1 mM DTT, and 0.4 or 0.75 mM of each of the following: ATP, CTP, GTP, 4 µM UTP, 0.185 MBq {alpha}-32P-UTP, and 4 mM phosphoenol pyruvate. Final reaction volume was 30 µl. The reaction was incubated at 30 C for 60–90 min and arrested by the addition of 200 µl 10 mM Tris, 300 mM NaCl, 5 mM EDTA (pH 7.5), and 2 mIU of RNAse T1. After treatment with Proteinase K (400 µg/ml) the RNase T1-resistant transcripts were recovered and resolved on a 10% acrylamide/7 M urea sequencing gel. For activation reactions, increasing amounts of AR-AF1-Lex were added, whereas squelching was achieved with the AR-AF1 polypeptide alone. The gel was analyzed by autoradiography, and the levels of each transcript were quantified using a Fuji BioImager 2000 phosphor imager (Fuji, Stamford, CT) and AIDA2 software package (Isotopenmessgerate GmbH, Straubenhardt, Germany). The molar ratio of the LGT:SGT was calculated based on the number of uridines incorporated into each transcript: 24 and 138 in the short and long transcripts, respectively.

In the reversal of squelching experiments, the concentration of AR-AF1 used was chosen to give 60–80% inhibition of basal transcription, and the recombinant general transcription factors were added at 10 or 20 pmol.


   ACKNOWLEDGMENTS
 
We thank Drs. A. Berk (University of California, Los Angeles, CA), A. O. Brinkmann (Erasmus University, Rotterdam, The Netherlands), Z. Burton (Michigan State University, East Lansing, MI), and A. Greenleaf (Duke University, Durham, NC) for generously providing plasmid constructs used in this study.


   FOOTNOTES
 
Present Address for A.B.: School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, Scotland, United Kingdom.

This work was supported by the Biotechnology and Biological Science Research Council, Grants 1/C10407 and 1/C18001.

First Published Online April 27, 2006

1 M.A.C. and A.B. contributed equally to this work and should be considered joint first authors. Back

Abbreviations: AdML promoter, Adenovirus major late promoter; AF1, activation function 1; AR, androgen receptor; CAK, cyclin-activated kinase; CTD, C-terminal domain; DRB, 5,6-dichlorobenzimadazole riboside; DTT, dithiothreitol; LGT, long G-less transcript; NTD, N-terminal domain; PIC, preinitiation complex; PSA, prostate-specific antigen; P-TEFb, positive elongation factor b; RAP, RNA polymerase II-associating protein; SGT, short G-less transcript; TBP, TATA-binding protein; TFIIF, transcription factor IIF.

Received for publication December 2, 2005. Accepted for publication April 17, 2006.


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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