Secretoglobin 2A1 Is under Selective Androgen Control Mediated by a Peculiar Binding Site for Sp Family Transcription Factors

Fei Xiao, Andreas Mirwald, Maria Papaioannou, Aria Baniahmad and Jörg Klug

Institut für Anatomie und Zellbiologie (F.X., J.K.), Justus-Liebig-Universität Giessen, D-35385 Giessen, Germany; Institut für Molekularbiologie und Tumorforschung (A.M.), Philipps-Universität Marburg, D-35033 Marburg, Germany; and Institut für Humangenetik und Anthropologie (M.P., A.B.), Klinikum der Friedrich-Schiller-Universität Jena, 07740 Jena, Germany

Address all correspondence and requests for reprints to: Dr. Jörg Klug, Justus-Liebig-Universität Giessen, Institut für Anatomie und Zellbiologie, Aulweg 123, D-35385 Giessen, Germany. E-mail: joerg.klug{at}anatomie.med.uni-giessen.de.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears and is expressed in the prostate. Here we show that SCGB 2A1 is under androgen control in the androgen-responsive prostatic cell line LNCaP and can be induced more than 20-fold by dihydrotestosterone. Only 6 h after androgen treatment, a strong DNase I-hypersensitive site is induced in the proximal promoter within chromatin. Within the boundaries of this DNase I-hypersensitive site a minimal 32-bp peculiar dimeric inverted repeat variant GC box (dim-IR-GA box) was found to confer androgen but not glucocorticoid responsiveness in gene transfer experiments. Mutations of both GA boxes that abolish binding of Sp1 and Sp3 also abrogate the androgen response. In an EMSA the DNA binding domain of the androgen receptor (AR) was not able to bind directly to the dim-IR-GA box. However, AR is functionally required for the hormone response because induction can be inhibited with the nonsteroidal antagonist bicalutamide. Chromatin immunoprecipitation experiments demonstrated that AR is recruited to the proximal promoter 10 min after androgen treatment. Therefore we propose that SCGB 2A1 represents a new class of androgen target genes that are purely under indirect AR control mediated by DNA-bound Sp factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANDROGENS, MOST NOTABLY testosterone and dihydrotestosterone (DHT), have numerous clinical actions in the developing embryo as well as in the pubertal and adult male (1). Androgen action is mediated by the androgen receptor (AR), a ligand-dependent transcription factor, belonging to the steroid hormone receptor branch of the superfamily of nuclear receptors. These are highly modular, intracellular key regulators equipped with a variable N-terminal transactivation domain, DNA-binding domain (DBD), hinge region, and C-terminal ligand-binding domain. Within its N-terminal transactivation domain the AR possesses a potent ligand-independent transcription activation function (AF)1, and within its ligand-binding domain a weaker ligand-dependent AF2. Whereas corepressor proteins bind to the aporeceptor, docking conditions are changed to enable coactivator binding when an agonistic hormone is bound and, thus, an intramolecular interaction of AF1 and AF2 is enabled (2). The liganded receptor binds to androgen response elements (AREs) of androgen-regulated target genes as a homodimer and thus activates transcription of ARE-containing genes.

In the last few years it became evident that the AR molecule, like all steroid hormone receptors, serves as a docking station for a plethora of coregulators (3). The nature of coregulator binding surfaces and thus the elicited molecular switch can be modified by ligand binding, phosphorylation (4), acetylation, and sumoylation (5, 6). All coregulators serve to modify the transactivation potential of DNA-bound receptor. Contrary to these genomic actions of AR function, fast nongenomic actions (7) and cross talk of AR signaling with other intracellular signaling pathways (8) have been observed. Cross talk affects cell growth in general, but no individual target gene could be identified yet. Therefore, our picture of androgen action has gained an order of complexity that has not been anticipated before.

Some androgen-responsive genes belong to a family of small, secreted, and dimeric proteins that are expressed in various epithelial tissues and are now called secretoglobins (SCGBs) (9) (www.gene.ucl.ac.uk/nomenclature/genefamily/scgb.html). Human SCGB 2A1, also known as lipophilin C (10), lacryglobin (11), and mammaglobin B (12), is a small protein that forms heterodimers with SCGB 1D1 (lipophilin A) in tears (10). SCGB 2A1 is expressed in salivary gland, mammary gland, and uterus (12) as well as in thymus, prostate, testis, kidney, trachea, and ovary (13). It displays highest homology to rat prostatic binding protein component 3 (C3), which has a similar expression pattern and has been found in the prostate (14, 15) and in salivary and lacrimal glands (16). All three rat prostatic binding protein components are androgen responsive (17), but only in the C3 gene could a functional ARE be identified in the first intron (18).

Here we show that SCGB 2A1 is under selective androgen control in the prostate cell line LNCaP. Mapping of DNase I-hypersensitive sites (DHSs) and deletion analysis of the SCGB 2A1 promoter identified a 32-nucleotide dimeric binding site for Sp family transcription factors (dim-IR-GA box) that is conferring the androgen response. We provide evidence that AR is functionally required for hormone induction but does not bind directly to the dim-IR-GA box. Instead, androgen control is indirectly mediated by a complex binding site for Sp transcription factors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of SCGB 2A1 in LNCaP Cells Is Androgen Dependent
Because SCGB 2A1 expression had been reported in the prostate (13) and could be localized to the glandular epithelium (Schwall, L., K.-H. Schäfer, A. Schulz, A. Meinhardt, and J. Klug, manuscript in preparation) expression was analyzed in the androgen-responsive prostate cell line LNCaP (19). Total RNA was prepared from LNCaP cells grown in the absence of the androgen DHT, or after treating them with 10–8 M DHT for different time intervals, and analyzed by Northern blotting (Fig. 1Go). In the absence of DHT, basal expression of SCGB 2A1 in LNCaP cells is low. After 6 h of DHT treatment, SCGB 2A1 is maximally induced and expression remains high for at least another 18 h. Under these conditions SCGB 2A1 mRNA is not detectable in total RNA from AR-negative HeLa cells. Quantification of hybridization signals with a PhosphorImager and normalization for 18S rRNA expression yielded a 21-fold induction of expression in LNCaP cells at the 6 h time point.



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Fig. 1. Northern Blot Analysis of SCGB 2A1 Expression in LNCaP and HeLa Cells

Total RNA was extracted from LNCaP and HeLa cells using Trizol (Invitrogen, Karlsruhe, Germany). LNCaP cells were grown either in the absence of DHT or treated with 10–8 M DHT for up to 24 h before RNA preparation. RPMI medium was supplemented with charcoal-treated fetal calf serum. RNA samples (30 µg each) were denatured by glyoxal treatment (59 ), separated on a 1.5% agarose gel, and transferred to a positively charged nylon membrane (60 ). Northern hybridizations were performed with SCGB 2A1 cDNA labeled with [{alpha}-32P]dCTP using the Megaprime DNA labeling kit (Amersham Biosciences) in 6x standard sodium citrate, 2x Denhardt’s reagent, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA. The washed membrane was exposed to a phosphor image screen for 24 h and analyzed on a Fuji phosphor imager FLA3000G (Raytest, Straubenhardt, Germany). Subsequently, the blot was stripped and rehybridized with the 32P-labeled 18S rRNA probe 5'-ACGGTATCTGATCGTCTTCGAACC-3' as loading control.

 
Chromatin Is Altered in the Proximal SCGB 2A1 Promoter after Hormone Treatment
For the identification of promoter elements functionally relevant for the androgen-dependent expression of SCGB 2A1 in LNCaP cells, DHSs were mapped. DHSs represent local perturbations of the nucleosome array in nuclei rendering DNA at those sites accessible to DNA-binding regulatory factors or nucleases.

Nuclei were isolated from LNCaP cells grown in the presence or absence of 10–8 M DHT. Nuclei from HeLa cells and naked DNA were used as controls. Nuclei were treated with increasing amounts of DNase I. For naked DNA one tenth of the amounts used for nuclei was employed. Purified DNA was digested with the restriction enzyme XbaI, generating an 11-kb genomic fragment spanning from –3 kb to +8 kb of the SCGB 2A1 gene as indicated in Fig. 2BGo. Restricted DNA samples were separated on an agarose gel and analyzed by Southern blotting with a 380-bp probe located at the 5'-end of the XbaI fragment, from –2455 to –2176 upstream of the transcription start site (Fig. 2Go).



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Fig. 2. Mapping of DHSs in the SCGB 2A1 Gene

A, Southern blot analysis of XbaI-digested genomic DNA from DNase I-treated samples. Nuclei were isolated from LNCaP cells treated without DHT (lanes 1 and 2) or with DHT for 1 h (lanes 3 and 4), 6 h (lanes 5 and 6), or 24 h (lanes 7 and 8), respectively. Naked DNA (lanes 9, 12, and 13) and HeLa nuclei (lanes 10 and 11) were used as negative controls. Nuclei were incubated with increasing concentrations of DNase I as indicated. The Southern blot was hybridized with a PCR-amplified probe located at the 5'-end of the XbaI restriction fragment. The intact 10-kb XbaI fragment, a nonspecific hypersensitive site (NS) present in all samples and the LNCaP-specific hypersensitive site (HS) are indicated. B, Schematic representation of the SCGB 2A1 gene. Black bars represent the three exons; a horizontal arrow indicates the position and length of the hybridization probe. The position of the specific HS is indicated by a vertical arrow.

 
As shown in Fig. 2AGo, one prominent hypersensitive site was detected in the proximal promoter of the SCGB 2A1 gene at approximately –100 bp. This DHS was hormone dependent because it only appeared in samples from LNCaP cells that were treated for 6 or 24 h with DHT, but was absent in samples from untreated cells or cells that were treated with DHT for only 1 h. The DHS also appeared to be specific, because it could not be detected in naked DNA and in nuclei from HeLa cells. In another experiment (data not shown), DNA purified from DNase I-treated nuclei was digested with XhoI, producing a restriction fragment of about 10 kb from +1.7 kb upstream of the SCGB 2A1 gene start point of transcription to some 8 kb downstream. The hybridization probe was placed at the 3'-end of this fragment to detect the DHS from downstream. Both mapping results confirmed a strong DHS in the proximal promoter region of the SCGB 2A1 gene after androgen treatment.

The Proximal Promoter (–136/+50) of the SCGB 2A1 Gene Is Sufficient for Both Constitutive and Androgen-Induced Expression
The mapping of a DHS in the SCGB 2A1 gene indicated that only the gene’s promoter is responsible for the regulation by androgens. To confirm this result and to more precisely delineate the androgen-responsive region, promoter deletions were cloned in front of the luciferase reporter gene (Fig. 3Go, upper panel). The different promoter fragments were amplified from a human P1 clone that was shown to contain at least three genes of the SCGB gene cluster, among them SCGB 2A1 [(20), SCGB 2A1 is called LGB in this reference]. Transient transfections into LNCaP cells were performed by the calcium phosphate DNA coprecipitation method. For all constructs the basal level of transcription is low and varies only by a factor of 2 (Fig. 3Go, upper panel). Upon addition of the androgen DHT, the longer promoter constructs with 5'-endpoints from –1476 to –575 were induced approximately 11-fold. When the region between –575 and –382 is deleted, transcription is induced by a factor of 23, indicating the loss of a negative element. Deleting further down to –136 bp reduces the induction level to 15-fold, suggesting that one or more positive acting elements are eliminated. When proximal promoter sequences from –136 to –53 bp were removed, hormone regulation was abolished.



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Fig. 3. Transient Transfection of LNCaP Cells with Reporter Gene Constructs Containing SCGB 2A1 Promoter Fragments

On the left-hand side the 5'-promoter deletions of the SCGB 2A1 gene that were linked to the luciferase reporter gene (Luc, upper part), or to the luciferase gene under control of the HSV tk promoter (tk-Luc, lower part) are schematically shown. The numbers indicate start and end point of the promoter fragments relative to the transcription start point (according to GenBank accession no. NT_033903). All constructs were sequenced from the luciferase gene into the insert for verification of at least some 400 bp. The graphs on the right-hand side show the results of transient transfection assays. LNCaP cells were transiently transfected with 5 µg of the indicated construct and cotransfected with 0.5 µg pRSV-ß-gal as internal standard. The cells were treated with 10–8 M DHT alone or together with 5 x 10–6 M bicalutamide as indicated. Cell extracts were prepared and luciferase activity was determined. Values were normalized for ß-gal activity and protein concentration as described in Materials and Methods. The luciferase activity of the (–53/+50)-Luc construct (~8000 RLU in a typical assay) in the absence of DHT was set to 1-fold.

 
The Androgen Response Requires a Dimeric Inverted Repeat Type GA Box
To test whether the identified hormone-responsive region can be functionally transferred to a heterologous promoter, the SCGB 2A1 promoter fragment starting at –136 and terminating at –53 bp was cloned in front of the Herpes simplex virus (HSV) thymidine kinase (tk) promoter directing expression of a reporter gene (HSV-tk-Luc). The tk promoter used terminated at –90 bp and contained one GC box in front of the tk TATA box. Surprisingly, this construct was not androgen responsive in LNCaP cells (Fig. 3Go, lower panel) like another construct containing a longer piece of the SCGB 2A1 promoter (–382/–53), indicating that the androgen-responsive region was no longer intact or could not work together with a heterologous promoter. Therefore, in another set of constructs the 3'-end of the promoter pieces was shifted to –28 bp, immediately upstream of the TATA box. By inclusion of 25 more bp at the 3'-end, androgen responsiveness could be fully restored in both constructs (–136/–28)-tk-Luc and (–382/–28)-tk-Luc. By setting the deletion endpoint at –53 the androgen-responsive region had been destroyed, apparently. Close inspection of the sequence around –50 bp revealed a peculiar dimeric inverted repeat type GA box (dim-IR-GA box; see Fig. 6BGo). Even the minimal 32-bp promoter piece –59/–28 containing only the dim-IR-GA box is sufficient to confer strong androgen responsiveness to the tk promoter [(–59/–28)-tk-Luc]. The level of induction of the tk constructs by DHT is comparable to the induction level of the SCGB 2A1-Luc constructs. The tk promoter itself is induced 2- to 3-fold by DHT.



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Fig. 6. Binding of NF1, NF-Y, and Sp Factors Is Functionally Important for SCGB 2A1 Promoter Activity and Androgen Induction

A, A schematic representation of the SCGB 2A1 reporter constructs used is shown in the middle. The NF1 binding site is symbolized by a solid circle, the NF-Y binding site by a solid square, and the distal and proximal GA boxes of the dim-IR-GA box are indicated by bold arrows. Mutations in binding sites are indicated by vertical bars. The sequence of the mutation introduced is shown on the left-hand side. The effect of each mutation on DNA binding of the cognate factor, as measured by EMSAs, is indicated: –DNA binding is abrogated; + DNA binding is not significantly affected; +/– DNA binding is moderately affected. On the right-hand side the cognate luciferase assay results are shown. LNCaP cells were transiently transfected with 5 µg of the indicated construct and cotransfected with 0.5 µg of pRSV-ß-gal as internal standard. The cells were treated with 10–8 M DHT as indicated. The assay results were normalized for ß-gal activity and protein amount as described in Materials and Methods. The activation was calculated based on the luciferase activity of the (–53/+50)-Luc construct set to 1 in the absence of DHT as in Fig. 3BGo. B, Sequence of the dim-IR-GA box. A distal and a proximal GA box are oriented as an inverted repeat with four intervening nucleotides. The two half-sites containing the GA boxes are 12 bp long (indicated by arrows) and slightly exceed the length of a GC box (9 bp, each of three zinc fingers contacting 3 nucleotides). Both repeats are almost identical with two substitutions only but still conserve the type of base pair (GC and AT). A consensus GC box and consensus ARE are shown for comparison. Palindromic regions are highlighted with a black background. Numbers in panels A and B refer to the position of the start point of transcription. wt, Wild-type.

 
The dim-IR-GA Box of the SCGB 2A1 Promoter Is a Binding Site for Sp Family Proteins
Each half-site of the GC-rich dim-IR-GA box contains the motif GGGAGG reminiscent of a classical GC box (Fig. 6BGo). Because some 18% of mammalian biologically active Sp1 binding sites are GA boxes, i.e. contain an A instead of the C in the GC box (see Sp1 binding site matrix of public Transfac 6.0 database at www.gene-regulation.com), we investigated in an EMSA whether Sp family proteins can bind to the dim-IR-GA box in vitro (Fig. 4AGo). With nuclear extracts from HeLa or LNCaP cells grown in the presence or absence of DHT, the typical protein-DNA complex pattern of the Sp family, consisting of three major bands, appears when either a consensus GC box (lanes 10–13) (21) or the SCGB 2A1 dim-IR-GA box (lanes 1–4) is used as a probe. When an oligonucleotide probe of random but defined sequence is used in a 50-fold molar excess, no competition is observed (lanes 5 and 14) with either probe. However, the unlabeled GC box fully competes with the formation of Sp-DNA complexes on the dim-IR-GA box (lane 6), and the unlabeled dim-IR-GA box strongly competes with the formation of Sp-DNA complexes on the GC box (lane 15). Direct competitor comparisons (data not shown) indicated that the affinity of Sp factors to the dim-IR-GA box is lower than to the consensus GC box.



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Fig. 4. Identification of Binding Sites for Sp Transcription Factors, NF-Y and NF1, by EMSA

A, Indicated nuclear extracts were incubated with the SCGB 2A1 dim-IR-GA box (lanes 2–9) and a canonical GC box probe (lanes 11–18). Competitions with oligonucleotides were performed as indicated. "Random" is an oligonucleotide of random sequence as shown in panel D. Anti-Sp1 and/or -Sp3 antibodies were used in lanes 7–9 and 16–18 as indicated. Sp factor-DNA complexes and supershifts are indicated on the right. B, Similar EMSA as in panel A but using an NF-Y box probe (from –100 to –124 within FP II; see Fig. 5CGo) and the well-described Y box of the major histocompatibility complex class II (61 ). Competitions were performed with unlabeled probes, random oligo, and a Y box mutant as shown in panel D. Moreover, a supershift was performed (lanes 9 and 18) using a polyclonal antibody against one of the three NF-Y subunits (CBF-A, Santa Cruz, sc-7711). Serum of a healthy rabbit ("Preimmune") was used as negative control. An NF1 probe (from –163 to –179 within FP I; see Fig. 5CGo) was used in the EMSA in panel C. In lane 9, 25 ng of a truncated NF1 protein containing the DBD was used. As competitors, unlabeled probe, random oligo, an NF1-binding site mutant, and an NF1 consensus binding site as shown in panel D were used. D, Sequences of all probes and competitors employed. The two GA boxes within the dim-IR-GA box are highlighted in bold and the dim-IR-GA box, as well as the GC box within the oligonucleotides, is highlighted in gray. In all other oligonucleotides, binding site core sequences are highlighted in gray. Ab, Antibody; Cons, consensus; mut, mutation.

 
The use of antibodies directed against the two most common ubiquitous Sp factors, Sp1 and Sp3, unequivocally showed that the observed DNA-protein complexes do contain the two proteins. Rabbit anti-Sp1 antiserum is able to supershift the upper complex band of the Sp1/Sp3-DNA double band (lanes 7 and 16). Likewise, rabbit anti-Sp3 antiserum was able to supershift the lower complex band of that double band (lanes 8 and 17) as well as a complex band of lower mobility. The latter one is due to short Sp3 isoforms that originate from internal start codons (22). Both antisera together supershift all observed major complex bands (lanes 9 and 18). All supershifts are more complete with the dim-IR-GA-box than with the consensus GC box, demonstrating again that the affinity of Sp1 and Sp3 for the dim-IR-GA box is lower than for the consensus GC box.

Binding of Sp Family Proteins Is Functionally Required for the Androgen Response whereas Nuclear Factor 1 (NF1) Participates in the Androgen Response and Nuclear Factor Y (NF-Y) Is Important for Basal Activity
The transfection experiments with promoter deletion/reporter gene constructs (Fig. 3Go) showed that in the proximal promoter of the SCGB 2A1 gene at least two binding sites for positively acting transcription factors are located. To identify their positions and cognate regulatory factor candidates, DNase I footprinting experiments were performed with nuclear extracts from LNCaP cells grown in the presence or absence of DHT and from HeLa cells. Both strands of the –382/+50 XhoI/KpnI promoter fragment were 3'-end labeled before DNase I footprinting reactions were set up (Fig. 5Go, A and B). In the lower strand (Fig. 5AGo) two strong footprints (FPs) (from –186 to –155 = FP I and –136 to –109 = FP II) were detected (Fig. 5CGo provides a sequence overlay). The cognate binding proteins are of a ubiquitous nature because the same FPs are produced with HeLa nuclear extract. Another weak but LNCaP-specific FP starts at position –36 and ends at an upstream position that could not be clearly identified (somewhere before –76 = FP III). This weak FP overlaps with the previously identified dim-IR-GA box (Fig. 6BGo).



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Fig. 5. DNase I Footprinting of the –382/+50 SCGB 2A1 Promoter Fragment

Lower (A) and upper strand (B) of the –382/+50 XhoI/KpnI promoter fragment were 3'-end labeled at the upstream and downstream end, respectively. In lanes 1 and 10 of panel A and lane 1 of panel B, Maxam-Gilbert G-reactions of the same fragment were applied. Nuclear extract (30 µg per reaction) was used from cells as indicated (lanes 4–9, panel A; and lanes 4–12, panel B). No extract was used for samples 2 and 3 in panels A and B (naked DNA). For each extract two different amounts of DNase I were used as shown above each lane. FPs are indicated on the right, and the numbers refer to the distance from the start site of transcription. The asterisk indicates a hypersensitive site. C, FP I, FP II, and FP III (see text) are marked along the SCGB 2A1 promoter sequence. NF1 and NF-Y binding site core sequences are printed in bold as well as the two GA boxes of the dim-IR-GA box. NE, Nuclear extract.

 
FP I and FP II were confirmed on the upper strand (Fig. 5BGo) (from –178 to –156 = FP I and –129 to –104 = FP II). In the region of the dim-IR-GA box (–59 to –28) again no clear FP could be detected, but in the vicinity of position –21 within the TATA box an LNCaP-specific hypersensitive site is apparent (shown by an asterisk). None of the FPs was androgen dependent. The footprinted region I is missing in the (–136/+50)-Luc reporter construct that showed an approximately one third lower activity upon hormone induction than the (–382/+50)-Luc construct that includes this element (Fig. 3Go).

A search for transcription factor binding sites in the SCGB 2A1 promotor (http://transfac.gbf.de/TRANSFAC/ and http://www.gene-regulation.com) clearly revealed the transcription factor NF-Y as the prime candidate being responsible for FP II that contains a canonical CCAAT box. FP I contains TTGGA in its core (lower strand), reminiscent of the nuclear factor 1 core sequence TTGGC. EMSA experiments confirmed that NF-Y (Fig. 4BGo) and NF1 (Fig. 4CGo) can bind to the identified CCAAT box and the TTGGA element, respectively, in vitro.

To demonstrate that the binding of Sp family factors, NF1 and NF-Y, is also functionally relevant, mutations that abrogated or impaired (distal and proximal GA box mutants) DNA binding (Fig. 5Go) were introduced into each factor-binding site within the context of the promoter extending from –382 to +50 (Fig. 6AGo) and tested in transfections with LNCaP cells.

Mutation of the distal GA box, which has a moderate effect on Sp factor binding (data not shown), has a dramatic effect on hormone-induced transcription of the reporter gene (Fig. 6AGo), leading to a 75% decrease in expression (down to 6-fold induction upon addition of hormone). Mutation of the proximal GA box, which has virtually no effect on Sp factor binding, still leads to a moderate decrease in DHT-induced expression of the reporter (down by 55%, 11-fold activation remaining). When both GA boxes are mutated, which leads to abrogation of Sp factor binding, the hormone response of the mutated reporter is almost abolished (2-fold activation left). All mutants have only a weak effect on the basal expression level, with the double mutant showing the largest effect (50% decrease). Therefore, binding of Sp factors to the dim-IR-GA box is functionally required for mediating the androgen response of the SCGB 2A1 gene.

For NF1 the proximal half-site of the identified binding site was mutated from TTGGA to TTTTA in the context of the promoter terminating at –382. Abrogation of NF1 binding leads to a 40% decrease in induced transcription and to a slight increase in basal transcription (Fig. 6AGo). Thus, NF1 seems to aid the hormone response. The activities of the –136/+50 wild-type promoter and the –382/+50 promoter containing the NF1 point mutations at around position –175 are very similar. This indicates that the SCGB 2A1 promoter from –136 up to –382 does contain only one functionally important positive element, which is the NF1 binding site.

NF-Y binding was abolished when the CCAAT core sequence was mutated to CTCAT. Introduction of these two point mutations into the SCGB 2A1 promoter terminating at –136 leads to an approximately 30% decrease in reporter gene activity under basal conditions (no hormone added) as well as under induced conditions (DHT added). The same result was obtained when the mutations were introduced into the promoter terminating at –382 (Fig. 6AGo). Therefore, NF-Y is required for basal promoter activity but not for hormone induction.

The AR Does Not Recognize the dim-IR-GA Box Directly but Is Recruited to the Promoter and Functionally Required
The nonsteroidal AR antagonist bicalutamide (Casodex, AstraZeneca, Wedel, Germany), was used to explore the role of AR in hormone induction. A more than 500-fold molar excess (5 x 10–6 M) of bicalutamide over DHT was used to antagonize androgen action in transient transfections. The results (Fig. 3Go, upper panel) show that induction by DHT of the (–136/+50)-Luc and (–382/+50)-Luc constructs in the presence of bicalutamide is abolished, indicating that AR is required for the androgen response.

Next we tested by EMSAs whether AR can bind directly to the dim-IR-GA box. As receptor source we used a fragment of the rat AR (amino acids 533–637) containing the DBD that was expressed in Escherichia coli. Its ability to specifically interact with functional AREs is well documented (23). As positive ARE control we used a probe containing the intronic ARE of the rat PBP C3(1) gene (24). Recombinant AR-DBD formed a single dominant complex band (Fig. 7AGo, lane 2) that could be strongly competed already by adding an approximately 10-fold molar excess of unlabeled C3(1)-ARE (lane 3), whereas the dim-IR-GA box or an oligonucleotide of random sequence did not compete even in approximately 100-fold molar excess (lanes 5–6 and 7–8, respectively).



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Fig. 7. AR Cannot Bind to the dim-IR-GA Box but Is Recruited to the SCGB 2A1 Promoter

A and B, for EMSA 200 ng of AR-DBD was incubated with a probe containing the intronic ARE of the C3(1 ) gene (lanes 1–8) and the SCGB 2A1 dim-IR-GA box (lane 9). Competitions with unlabeled oligonucleotides were performed as indicated (some 10-fold molar excess in lanes 3, 5, and 7; some 100-fold molar excess in lanes 4, 6, and 8). The AR-DBD/DNA complex band is indicated on the right. Panel A shows a 2-h phosphor image, and panel B shows an 8-d phosphor image exposure of the same gel. C, Sequences of oligonucleotides used. The two GA boxes within the dim-IR-GA box are highlighted in bold, and the dim-IR-GA box, as well as the half-sites within the C3(1 ) ARE, is highlighted in gray. D, Fragmented chromatin of LNCaP cells was immunoprecipitated with AR antibody and the SCGB 2A1 promoter region from (–123) to (+65) (dim-IR-GA) or a 207-bp fragment from the far upstream promoter of the human telomerase reverse transcriptase gene (negative control) were amplified. Immunoprecipitation was performed at the given time points after hormone treatment with R1881. IP, Immunoprecipitation; Neg., negative.

 
More importantly, the AR-DBD did not form any detectable complex with the labeled dim-IR-GA box probe (lane 9). A long time phosphor image exposure also did not produce a visible band (Fig. 7BGo).

Although not binding directly to the ARE, we could show, by chromatin immunoprecipitation (ChIP) experiments followed by PCR, that 10 min after androgen induction the AR is recruited to the SCGB 2A1 promoter region from (–123) to (+65) centered around the dim-IR-GA box (Fig. 7DGo). Repeating the ChIP experiment at different time points after androgen induction revealed that AR recruitment is weak 30 min after hormone treatment but is restored again after 50 min.

Furthermore we reasoned that if a functional, but up to now unrecognized, ARE is still present in the dim-IR-GA box this element should also confer glucocorticoid responsiveness. Therefore the (–382/+50)-Luc and (–59/+28)-tk-Luc constructs were transfected into LNCaP cells and induced with DHT or the glucocorticoid dexamethasone (Fig. 8Go). As positive control, the promoter of the prostate-specific antigen (PSA) was used that contains a very strong androgen and glucocorticoid response unit (25). Because the LNCaP cells do not contain endogenous glucocorticoid receptor (GR), an expression construct for GR was cotransfected. As Fig. 8Go shows, the PSA-Luc construct responded to androgens with a 230-fold increase in transcription of the reporter gene. With dexamethasone the induction was still 110-fold. This represents the typical response pattern of a steroid hormone response unit and also demonstrated that the GR expression construct was able to express functional GR. Contrary to the results obtained with the PSA-Luc construct the SCGB 2A1 constructs (–382/+50)-Luc and (–59/+28)-tk-Luc did not respond at all to dexamethasone whereas they did respond to the androgen DHT as shown before. Thus, the dim-IR-GA box selectively responds to androgens.



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Fig. 8. The SCGB 2A1 Promoter Does Not Respond to Glucocorticoids

Five micrograms of SCGB 2A1 (–382/+50)-Luc (–59/–28)-tk-Luc or PSA-Luc construct were transfected into LNCaP cells. pRSV-GR (0.5 µg) for ectopical expression of GR were cotransfected as indicated. pRSV-ß-gal (0.5 µg) were always cotransfected as internal standard. The cells were treated with 10–8 M DHT or 10–8 M DEX as indicated at the bottom. The fold induction (plus and minus hormone) or activation of luciferase activity is shown after normalization for ß-gal activity and amount of protein. The activity of the (–382/+50)-Luc construct in the absence of hormone was set to 1. DEX, Dexamethasone.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results presented in this study show that the SCGB 2A1 gene is under selective androgen control that is mediated by Sp family transcription factors. Within 10 kb containing and surrounding the SCGB 2A1 gene, one prominent androgen-dependent DHS was mapped in the proximal promoter. A time course experiment revealed that it takes 6 h of hormone treatment until this site becomes maximally accessible for DNase I. Contrary to this observation, steroid hormone induction of DHSs in other promoters has been described to occur in minutes. Zaret and Yamamoto (26) reported as early as two decades ago that a DHS is induced in a derivative of the mouse mammary tumor virus promoter within minutes of dexamethasone treatment. Likewise, DHSs were detected in rat tryptophan oxygenase and tyrosine aminotransferase genes after 1 h of dexamethasone treatment (27). Therefore, the delayed appearance of the DHS in the SCGB 2A1 promoter led us to hypothesize that its androgen response could be mediated by the AR through a new or modified mechanism.

Within the boundaries of the mapped DHS, an unusual functional binding site for Sp family transcription factors, the dim-IR-GA box, could be identified. Because the magnitude of induction by DHT of 1) SCGB 2A1 mRNA in LNCaP cells, 2) of a transfected full promoter-reporter gene construct, and 3) of a dim-IR-GA box-tk reporter gene construct is comparable, this peculiar element accounts for full androgen regulation of the gene. The dim-IR-GA box is an imperfect palindrome with two 12-bp half-sites separated by 4 bp (Fig. 6BGo). The sequence of both half-sites deviates in only two positions and is very GC rich. Its GC content and the GGACCC core motif of each half-site immediately suggested that both half-sites are variant GC boxes (GA boxes) and, thus, binding sites for Sp family transcription factors (21).

All members of the Sp family exhibit very similar structural features (21, 28). Their highly conserved 81-amino acid DBD contains three C2H2-type zinc fingers close to the C terminus and glutamine-rich activation domains adjacent to serine/threonine stretches in their N-terminal regions. Due to the high conservation of the DBD Sp1, Sp3 and Sp4 recognize a GC box with virtually the same affinity (29, 30). Each zinc finger of Sp1 recognizes a DNA triplet via specific interactions with a recognition helix. Some 18% of mammalian biologically active Sp1 binding sites are GA boxes, i.e. contain an A instead of the C in the GC box. Functional GA boxes like in the SCGB 2A1 promoter are, for example, described in the promoter of the rat {alpha}2A-adrenergic receptor gene (31) and in the promoter of the GH gene (32).

The dim-IR-GA box contains the three consecutive DNA triplets TGG GAG GGA in the distal GA box, and AGG GAG GCA in the proximal GA box. Each of the three triplets in the distal GC box is a predicted preferred one, whereas only the triplet in position 2 of the proximal GC box is the preferred GAG (33). The other two triplets are similar to preferred ones but not identical. Therefore, the distal GA box is a predicted high-affinity Sp binding site, and the proximal site can be expected to show only low affinity. This prediction is in agreement with the results of transfection experiments confirming that the distal GA box is functionally more important for mediating the androgen response than the proximal GA box.

Several studies have shown that Sp binding sites are implicated in estrogen induction of the cathepsin D, hsp27, and uteroglobin genes (34, 35, 36) as well as in glucocorticoid induction of the ubiquitin gene (37) and androgen induction of the gene encoding mouse vas deferens protein (38). In all these examples a hormone response element forms a composite unit or element with a GC box. Therefore, we were also trying to identify an ARE or cryptic ARE within or close to the dim-IR-GA box. Due to the high GC content of this region, sequence comparisons with classical or androgen-selective direct repeat AREs (24, 39) did not yield any candidate element. More importantly we could not show direct binding of the AR-DBD to the dim-IR-GA box in an EMSA, whereas binding to the intronic ARE of the rat PBP C3(1) gene (24) was strong and specific. An 8-d phosphor image exposure could not reveal any spurious binding of AR-DBD to the labeled dim-IR-GA box.

We also reasoned that if a functional, but hidden, ARE is present in the dim-IR-GA box, it should confer glucocorticoid responsiveness. Even androgen-selective ADR3 elements still allow glucocorticoid induction in the 2- to 7-fold range (24, 39). To test for the presence of such a cryptic ARE, androgen and glucocorticoid responses of the SCGB 2A1 and PSA promoters were compared in LNCaP cells. The PSA promoter contains two functionally active androgen response regions, an enhancer region at –4.2 kb, and a promoter region in the proximal 400 bp (40, 41). The latter provides only a low level of androgen regulation (4- to 6-fold induction) dependent on the presence of two AREs, ARE-I at –170 bp and ARE-II at –394 bp. ARE-III, presumably together with other still unknown elements in the enhancer, is responsible for full androgen regulation (some 3000-fold induction). Those multiple AREs have been shown to be responsive to androgens and glucocorticoids alike (25). Because LNCaP cells do not express the GR (42), it had to be ectopically expressed from a cotransfected GR expression construct. Contrary to the PSA promoter that strongly responded to the androgen DHT and the glucocorticoid dexamethasone, exemplifying the archetype of a genomic response, neither the SCGB 2A1 promoter nor the isolated dim-IR-GA box in front of the tk promoter responded to dexamethasone at all. Therefore, delayed appearance of the androgen-inducible DHS, together with the absence of a DNA binding site for the AR-DBD within the dim-IR-GA box and unresponsiveness of the promoter to glucocorticoids, led us to conclude that the androgen response, in the case of the SCGB 2A1 promoter, is not mediated by direct DNA binding.

Next we asked whether the AR is indirectly involved in the hormone response. Therefore, the nonsteroidal AR antagonist bicalutamide, which does not affect AR DNA binding (43), was used to explore the role of AR in hormone induction. Because an excess of bicalutamide was able to abolish induction by DHT of the (–136/+50)-Luc and (–382/+50)-Luc constructs in transfection experiments, we concluded that AR is functionally required. This was further substantiated by ChIP experiments showing that AR is tethered to the proximal SCGB 2A1 promoter 10 min after hormone treatment. Compared with the PSA enhancer (Baniahmad, A., manuscript in preparation), the amplified fragment band of the SCGB2A1 promoter is much weaker, indicating that less AR is recruited. This could explain why the hormone response of the PSA promoter is 2 orders of magnitude higher than the SCGB 2A1 response.

An indirect androgen response that is mediated by Sp factors can be explained by a plethora of mechanisms. By using a mammalian one-hybrid assay and coimmunoprecipitation experiments, a direct interaction of AR and Sp1 has been implicated in the moderate 6-fold androgen induction of the p21 gene (44) that contains a classical ARE in addition to six GC boxes. Contrary to this report, Sp1 could not be detected in a general AR interaction screen based on specific binding of cytoplasmic and nuclear proteins to immobilized AR (Bevan, C. L., unpublished observation). Other mechanisms include a hormone-dependent bridging protein between AR and Sp factors like small nuclear RING finger protein (45) and androgen induction of the Src/Shc/Erk pathway (8) that could impinge on Sp transactivation potential (46). Moreover, posttranslational modifications of Sp1/Sp3 like acetylation (47) and SUMOylation (48, 49) could be under hormonal control and thereby regulate the transcriptional activity of Sp1 or Sp3.

DNase I footprinting, sequence analysis, EMSAs, and transfection experiments revealed that, in addition to Sp factors, ubiquitous transcription factors NF1 and NF-Y are required for expression of SCGB 2A1. A typical NF1 binding site was identified at approximately –167 and a CAAT or Y box at approximately –110. Transfection of reporter gene constructs with binding site mutants that were shown in EMSAs to abrogate DNA binding proved that NF-Y is functionally important for basal promoter activity whereas NF1 also participates in mediating the androgen response. This is in agreement with numerous results indicating that NF-Y has a crucial role in establishing proper promoter occupancy, whereas in the case of NF1 a functional synergism with steroid hormone receptors has been suggested (50, 51).

The results presented in this study identify the SCGB 2A1 gene as a representative of a new class of androgen target genes in the prostate that are selectively under androgenic control. Androgen induction requires DNA-bound Sp family transcription factors as well as AR that is not directly bound to an ARE. Therefore, Sp factors could be involved in the hormone response of a larger fraction of steroid hormone-responsive genes than previously recognized. Future investigations on the mechanism might identify new target molecules for intervention therapy of androgen-dependent prostate cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Constructs
SCGB 2A1 promoter deletions were cloned into the luciferase reporter vector pGAW (52). SCGB 2A1 promoter fragments were amplified from P1 clone ICRFP700J1347Q6 that contains four SCGB genes (20). The upstream PCR primer defined the upstream promoter truncation point and provided an XhoI site for cloning (see Table 1Go). The downstream primer contained the SCGB 2A1 promoter sequence from +50 to +32 and a KpnI site (underlined) for cloning (5'-GGCGGTACCTGTCTGTGTTCAGTCGTGC-3'). The PCR was carried out for 30 cycles at 94 C for 40 sec, 57 C for 40 sec, and 72 C for 30 sec, followed by a final extension at 72 C for 5 min. Amplified PCR fragments were purified with the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany), restricted with XhoI and KpnI, and ligated into pGAW. The sequences of all constructs were checked (Seqlab, Göttingen, Germany) across the promoter/luciferase border using Glprimer2 (Promega, Mannheim, Germany). Plasmid DNA for transfections was prepared from transformed E. coli DH5{alpha} using Nucleobond PC 500 columns (Macherey & Nagel, Düren, Germany).


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Table 1. Oligonucleotides Used for 5'-Promoter Deletion Constructs, Mapping of DHSs, Mutagenesis, and ChIP Assay PCR

 
Some promoter fragments were cloned into a pGL3-basic reporter vector containing the HSV-tk gene promoter from (–90) to (+51). Fragments were amplified by PCR using a 5'-truncation primer and a downstream primer containing SCGB 2A1 sequences up to (–53) or (–28) and terminating with an XhoI site for subcloning the fragment immediately upstream of the tk promoter.

The oligonucleotides used for PCR are listed in Table 1Go. Oligonucleotides were either synthesized on an Applied Biosystems 380A oligonucleotide synthesizer or were supplied by MWG-Biotech (Ebersberg, Germany).

PCR-Mediated Mutagenesis
Point mutations were introduced at specific sites by producing two overlapping mutant fragments via PCR (left and right arms) that were then joined together by PCR-directed homologous recombination (53).

In a first PCR the left arm was generated from one of the wild-type promoter deletion constructs. The primers used were the upstream truncation primer containing an XhoI site and a downstream primer containing the desired mutation(s) (see Table 1Go).

The right arm was also generated by PCR from the same wild-type promoter construct. The upstream primer was complementary to the mutation primer used for the left arm, and the downstream primer was the downstream primer used to generate all promoter deletion constructs containing a KpnI site (see above). Therefore, left and right arms were partially overlapping.

Left and right arms were agarose gel purified with the GFX PCR DNA and gel band purification kit (Amersham Biosciences, Freiburg, Germany) and used as templates (~1 ng each) to create the chimeric full-length mutation fragment by PCR-directed homologous recombination (53). For the recombination PCR, the upstream primer for generating the left arm was used as upstream primer, and the downstream primer used for generating the right arm was used as downstream primer. All PCRs were carried out with PfuTurbo polymerase for 30 cycles with 30 sec at 94 C, 40 sec at 58 C, and 30 sec at 72 C, followed by a final extension at 72 C for 7 min. The amplified PCR fragments were purified with the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany), restricted with XhoI and KpnI, followed by gel purification using the GFX PCR DNA and gel band purification kit (Amersham Biosciences) and subcloning into pGAW (see above).

EMSAs
The sequences of oligonucleotides used for EMSAs are provided in Results. A pair of complementary single-stranded oligonucleotides (200 ng/µl each) was heated to 95 C for 5 min in 100 µl of annealing buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 30 mM KCl) in a water bath and cooled to room temperature over 2 h. Most double-stranded oligonucleotides had 4-bp 5'-protruding ends. Double-stranded DNA (50 ng) was labeled by filling in the ends with [{alpha}-32P]dCTP and Klenow fragment. Labeled double-stranded probes were purified over Nick columns (Amersham Biosciences) according to the instructions provided by the manufacturer or by electrophoresis through a 12% native polyacrylamide gel followed by elution.

For EMSAs 3 µg nuclear extract or 200 ng of AR-DBD were preincubated with 0.5 µg unspecific competitor polydeoxyinosinic deoxcytidylic acid in 1x binding buffer (10 mM HEPES-KOH, pH 7.9; 8.5% glycerol; 50 mM KCl; 0.1 mM EDTA; and 0.2 mM dithiothreitol) or 1x binding buffer containing 10 mM MgCl2, respectively, for 10 min on ice. Nuclear extracts of LNCaP and HeLa cells were prepared according to Andrews and Faller (54), and rat AR-DBD was prepared from pRIT2TAR exactly as described elsewhere (23). Protein concentrations were determined by the dye binding assay (55). Subsequently, 2 x 104 cpm of labeled double-stranded oligonucleotide were added to yield a final volume of 20 µl, and incubation was continued at room temperature for another 15 min. The competition experiments were performed by adding 50 ng of unlabeled oligonucleotide (~100-fold molar excess) together with labeled probe. Samples were run on a 4% native polyacrylamide EMSA gel (acrylamide to bisacrylamide ratio, 39:1) in 0.5x Tris-buffered EDTA buffer at 150 V and 20 mA for 2 h. The gel was dried for 1 h at 80 C under vacuum in a gel dryer (Bio-Rad, München, Germany) and exposed to a phosphor image screen for 2 h and analyzed on a Fuji PhosphorImager FLA3000G (Raytest, Straubenhardt, Germany).

Mapping of DHSs
Mapping was performed according to Cockerill (56). Briefly, 4 x 107 LNCaP or HeLa cells were harvested into ice-cold PBS and washed twice with 25 ml of ice-cold PBS without calcium and magnesium salts. Cells were spun down by centrifugation at 1000 x g for 5 min at room temperature and resuspended in 5 ml cell lysis buffer (60 mM KCl; 15 mM MgCl2; 10 mM Tris-HCl, pH 7.4; 300 mM sucrose; 0.1 mM EGTA; and 0.1 mM Pefabloc). The suspension was homogenized by squeezing the lysed cells five times out of a 10-ml pipette held hard on the bottom of a 50-ml centrifuge tube. Incubation was continued on ice until more than 80% of cells were lysed as determined by trypan blue staining. The cell lysate was adjusted to 30 ml with cell lysis buffer. The nuclei were spun down at 1500 rpm at room temperature for 5 min and resuspended in 1 ml nuclei digestion buffer (60 mM KCl; 15 mM NaCl; 5 mM MgCl2; 10 mM Tris-HCl, pH 7.4; 300 mM sucrose; 0.1 mM EGTA).

Aliquots (250 µl) of resuspended nuclei were transferred to a series of 2-ml tubes and digested with DNase I (2, 4, and 6 µg/ml) for 3 min at 22 C in a waterbath by adding 2.5 µl 100 mM CaCl2. Reactions were stopped by adding 250 µl proteinase K digestion buffer [100 mM Tris-HCl, pH 8.0; 40 mM EDTA; 2% sodium dodecyl sulfate (SDS); 0.2 mg/ml proteinase K], and incubation was continued for 16 h at 50 C with rotation. After three extractions with phenol/chloroform, DNA was ethanol precipitated and dissolved in 200 µl of TE by rotation overnight.

Cell Culture and Transfections
HeLa and LNCaP cells were cultured as monolayers in DMEM or RPMI medium, respectively, supplemented with 10% charcoal-treated fetal calf serum and antibiotics and grown at 37 C in 5% CO2. In the case of LNCaP cells, RPMI medium was changed for DMEM 1 h before transfection.

Cells were transfected by the calcium-phosphate method according to Sambrook and Russell (57) using 5 µg of construct DNA and 0.5 µg pCH110 (Amersham Biosciences) expressing ß-galactosidase as internal standard per 6-cm plate. The DNA-phosphate coprecipitate was added to cells exactly 1 min after the addition of HEPES-buffered saline (58). Chloroquine (0.1 mM) was used as transfection facilitator. DNA-phosphate coprecipitate and chloroquine were exchanged for 15% glycerol in 1x HEPES-buffered saline after 4 h and for 2 min. Incubation was continued in medium with or without hormones for 48 h. Sample relative light unit (RLU) values were normalized for protein concentration, and reagent background RLUs were subtracted. ß-Galactosidase results were used to normalize for plate-to-plate variations in transfection efficiency.

ChIP Assay and PCR
LNCaP cells were grown in RPMI-medium 1640 supplemented with 5% (vol/vol) charcoal-treated fetal bovine serum. After 3 d of cultivation cells were treated with 10–8 M R1881 (PerkinElmer, Rodgau, Germany) for the indicated time. Nuclear proteins were cross-linked to DNA by adding formaldehyde directly to the medium to a final concentration of 1% at 37 C for 10 min. Cross-linking within the cells was stopped by adding glycine to a final concentration of 0.125 M and incubating at room temperature for 5 min on a rocking platform. Cells were then rinsed twice with ice-cold PBS and collected into ice-cold PBS supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). After centrifugation, the cell pellets were resuspended in lysis buffer (1% SDS; 10 mM EDTA; 50 mM Tris-HCl, pH 8.0) and protease inhibitors. The lysates were sonicated 10 times for 10 sec on ice and at 10% of maximum power (Branson W-250/W) to yield DNA fragments of 1000 bp in length. After centrifugation, supernatants were collected and diluted in ChIP dilution buffer (0.01% Triton X-100; 2 mM EDTA; 150 mM NaCl; 20 mM Tris-HCl, pH 8.0) followed by preclearing with 30 µl of salmon sperm DNA/protein A agarose 50% slurry (Upstate Biotechnology, Inc., Lake Placid, NY) for 1 h at 4 C with agitation. Immunoprecipitation with the rabbit anti-AR antibody (Upstate Biotechnology) was performed at 4 C overnight. The immunocomplexes were collected with 30 µl of salmon sperm DNA/protein A agarose 50% slurry for another 2 h with rotation at 4 C. Agarose beads were pelleted by centrifugation and washed sequentially for 10 min each with 1 ml low-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8; and 150 mM NaCl), high-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8; and 500 mM NaCl) and LiCl wash buffer (0.25 M LiCl; 1% Nonidet P-40; 1% deoxycholate; 1 mM EDTA; and 10 mM Tris-HCl, pH 8). Finally, beads were washed twice with TE8. Immunocomplexes were eluted from the beads by adding freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) twice. Eluates were pooled and cross-links were reversed by adding NaCl to final concentration of 200 mM and heated at 65 C overnight. Remaining proteins and RNA were digested by adding proteinase K (final concentration, 40 µg/ml) and RNase A (20 µg/ml), respectively, and incubating at 55 C for 3 h. The DNA fragments were purified with a DNA purification kit (QIAquick PCR Purification Kit; QIAGEN, Hilden, Germany).

For PCR, 2 µl of 50 µl obtained after DNA purification were used. Cycling conditions were as follows: initial denaturation at 94 C for 3 min, followed by 33 cycles of denaturation at 94 C for 45 sec, annealing at 62 C for 30 sec, elongation at 72 C for 90 sec, and one final elongation at 72 C for 10 min. PCR products were separated by electrophoresis through 2.0% agarose supplemented with 0.2 µg/ml ethidium bromide. Primer sequences are provided in Table 1Go.


    ACKNOWLEDGMENTS
 
We thank Guntram Suske, Institute of Molecular Biology and Tumor Research Marburg, for providing rabbit polyclonal anti-Sp1 and anti-Sp3 antibodies (30 ) and other reagents, an isotope laboratory, critical reading of the manuscript, and constant encouragement; Andreas Meinhardt, Institut für Anatomie und Zellbiologie Giessen, for encouragement and help during revision; Suada Fröhlich and Tamara Henke, Institut für Anatomie und Zellbiologie Giessen, for expert technical assistance; Emily Slater, IMT Marburg, for providing the tk-Luc construct; Hannes Westphal, IMT Marburg, for providing NF1-DBD; Jan Trapman and Hetty van der Korput for pRIT2TAR and Miguel Beato, Center for Genomic Regulation Barcelona, for teaching science (J.K.).


    FOOTNOTES
 
This work was supported by a grant from the Graduiertenkolleg 533 "Cell-Cell-Interaction in Reproduction" (to F.X.).

Current address for F.X.: Department of Molecular Biology and Oncology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9148.

First Published Online July 14, 2005

Abbreviations: AF, Activation function; AR, androgen receptor; ARE, androgen response element; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DHS, DNase I-hypersensitive site; DHT, dihydrotestosterone; FP, footprint; GR, glucocorticoid receptor; HSV-tk, Herpes simplex virus-thymidine kinase; NF1, nuclear factor 1; NF-Y, nuclear factor Y; PSA, prostate-specific antigen; RLU, relative light unit; SCGB, secretoglobin; SDS, sodium dodecyl sulfate.

Received for publication October 13, 2004. Accepted for publication July 8, 2005.


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