Selected AR Binding Sites Play Transcriptional Roles

We and others have previously found that RNA Pol II associates with the AR binding regions of the PSA enhancer and promoter, which is critical for PSA transcription (Louie et al., 2003; Shang et al., 2002; Wang et al., 2005). To address whether Pol II is recruited to the AR binding sites on chromosomes 21 and 22, we performed Pol II ChIP on several AR binding sites. Interestingly, Pol II was significantly recruited to 50% of binding sites tested in an androgen-dependent manner (≥3-fold) (Figure 2D), supporting the assertion that a significant number of binding sites play transcriptional regulatory roles. We further performed a comparative genomic analysis of 90 AR binding sites between genomes of seven vertebrate species (chimp, dog, mouse, rat, chicken, fugu, and zebrafish). The results showed a strong conservation at the center of the AR binding sites (Figure S1), consistent with the notion that noncoding sequences that have survived selection play important roles in gene regulation (Hardison, 2000).

Most AR Binding Sites Are Far from the Promoters of Androgen-Regulated Genes

To correlate AR binding with androgen-dependent gene expression, we examined the gene expression profile in LNCaP cells in response to DHT using an expression microarray. LNCaP cells were deprived of hormone for 72 hr and then treated with vehicle or DHT for 4 and 16 hr. Total RNA was extracted and then hybridized to Affymetrix U133 plus 2.0 expression array. We found 62 transcripts on chromosomes 21 and 22 that were differentially expressed in response to androgen (Table S2). Consistent with previous studies (Nelson et al., 2002; Velasco et al., 2004), we find that the expression of only a small number of transcripts on chromosomes 21 and 22 is very highly changed by androgens. In order to avoid a bias toward this small number of highly differentially expressed genes, we have included all transcripts that were differentially expressed by at least 1.1-fold. Real-time RT-PCR validation of five selected genes and PSA as a positive control confirmed the expression array results (Figure S2). Interestingly, on chromosomes 21 and 22, most AR binding sites are far from transcription start sites of androgen-regulated genes (>10 kb) (Table S3). In total, only 34 out of 90 binding sites (38%) were located within 500 kb of transcriptional start sites of genes regulated by androgens under these conditions in LNCaP cells.

Noncanonical AR-Responsive Elements Mediate AR-Dependent Transcription

We next performed a DNA-binding motif search on the 90 AR binding regions. Surprisingly, we found that, although 78% of the binding regions contain the AR binding half-site motif TGTTCT, only 10% of the binding regions have a canonical class I NR (AGAACAnnnTGTTCT) binding motif when we allow up to two positions to vary from the palindromic consensus with 3 nt spacing (for detail, see “Sequence Analysis” in the Supplemental Experimental Procedures) (Verrijdt et al., 2003) (Figure 2E and Table S4). Of the 90 binding sites, 68% have one or more noncanonical AREs including isolated ARE half-sites, head-to-head AREs (AGAACA [0–8 n] TGTTCT, n ≠ 3), tail-to-tail AREs (TGTTCT [0–8 n] AGAACA), and ARE direct repeats (AGAACA [0–8 n] AGAACA) (Table S4 and “Sequence Analysis” in the Supplemental Experimental Procedures).

To further address whether AR binding regions containing noncanonical AREs can function as enhancers, we subcloned 10 AR binding sites and the recently characterized enhancer of the androgen-regulated gene FKBP5 (Magee et al., 2006) as a positive control, upstream of an SV40 promoter in the pGL3 luciferase vector. The pGL3 luciferase vector with the minimal SV40 promoter alone served as a negative control. These constructs were transfected into androgen-depleted LNCaP cells, and luciferase activity was measured after 24 hr treatment with vehicle alone or in the presence of saturating concentrations of DHT. As shown in Figure 2F, all AR binding regions tested demonstrate enhancer activity (≥2-fold) with varying degrees of androgen dependence comparable to the activity of the FKBP5 enhancer. To confirm that the enhancer characteristics of the AR binding sites require AREs, we deleted the canonical ARE within the B21 and the noncanonical ARE within the B39 enhancer constructs and transfected these mutated vectors into LNCaP cells. As expected, deletion of the single typical ARE within B21 completely abolished its enhancer activity (Figure 2F). More importantly, deletion of the single noncanonical (head-to-head, 8 nt spacing) ARE from B39 also completely abolished the enhancer activity, thus demonstrating that the noncanonical AREs within AR binding regions are required for their enhancer function (Figure 2F).

Characterization of the TMPRSS2-Regulatory Region

As mentioned above, most prostate cancers have recently been shown to harbor either TMPRSS2:ERG or TMPRSS2: ETV1 fusions (Tomlins et al., 2005). However, how the TMPRSS2 gene or the TMPRSS2-ETS fusions are regulated by AR is unknown. Using quantitative ChIP, we confirmed AR binding to site B39, which is ~13.5 kb upstream of the TMPRSS2 mRNA start site both in LNCaP cells that do not harbor a rearranged TMPRSS2 gene (Figures 2A) and in VCaP cells that contain the TMPRSS2:ERG fusion (Figure 3A). In order to test whether this AR binding site functions as an AR-dependent enhancer and communicates with the TMPRSS2 promoter, we performed a ChIP combined with chromosome conformation capture (5C) assay in order to detect looped chromatin structures in vivo (Wang et al., 2005). Hormone-depleted cells were stimulated with vehicle or androgen. The fixed chromatin was digested with EcoRI followed by anti-AR ChIP and ligation. After reversing the crosslinks, one primer in the TMPRSS2 enhancer and one primer in the promoter region were used for PCR amplification of equal amounts of eluted DNA. As shown in Figure 3B, the specific PCR product is formed only in the presence of ligase and is significantly enhanced by androgen stimulation, while control PCR for the level of input chromatin was equivalent under all conditions (Figure 3B). The 383 bp PCR amplified band was sequenced to confirm that it indeed represents the product of the correct ligation of the TMPRSS2 enhancer and promoter. This result demonstrates that the TMPRSS2 enhancer is in close proximity to the promoter in vivo. As negative controls, the interaction of the PSA enhancer with the TMPRSS2 promoter and the TMPRSS2 enhancer with the PSA promoter was tested by 5C. In neither case was a specific PCR product formed (data not shown), supporting the specificity of the 5C product formed between the TMPRSS2 enhancer and promoter. In addition, a control region 9 kb upstream of the TMPRSS2 transcription start site that contains two putative AR-responsive element (ARE) motifs that were not found to bind AR did not contact the TMPRSS2 promoter by 5C (Figure S3). Interestingly, an AR binding site 462 kb downstream of TMPRSS2 (B38) also failed to form a loop with the TMPRSS2 promoter (Figure 3B). This result serves as another negative control and is consistent with the conclusion that regions upstream of TMPRSS2 that are retained in the TMPRSS2-ETS fusions rather than those downstream harbor the important functional elements. These data demonstrate that the –13.5 kb AR binding site specifically interacts with the TMPRSS2 promoter region, supporting the formation of a chromosomal loop between this upstream enhancer and the proximal promoter. The identification of this upstream TMPRSS2 enhancer also provides a molecular explanation of how expression TMPRSS2-ETS factor fusions are driven in an androgen-dependent manner. Interestingly, a newly identified prostate cancer oncogene resulting from the fusion of TMPRSS2 and the ETS transcription factor ETV4 does not include any of the TMPRSS2 coding exons, as the breakpoint is ~8 kb upstream of the mRNA start site though ~5.5 kb downstream of this newly identified TMPRSS2 enhancer (Tomlins et al., 2006), suggesting that this enhancer acts to drive ETV4 expression in this setting.

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Figure 3

Characterization of TMPRSS2-Regulatory Regions

(A) ChIP analyses of AR occupancy on the PSA and B39 in VCaP cells. VCaP cells (a prostate cancer cell line that expresses TMPRSS2:ERG fusion gene) were treated with 100 nM DHT for 1 hr. ChIP assays were performed with an anti-AR antibody.

(B) Spatial communication between the TMPRSS2 enhancer and promoter. 5C was performed using fixed or EcoRI- or BtgI-digested chromatin from vehicle- or DHT-treated LNCaP cells. Primers (Table S1) flanking the –13.5 kb or 462 kb AR binding region and –700 bp promoter region were used to PCR amplify DNA after ligation. Control PCR was performed using chromatin before restriction enzyme digestion.

(C) Schematic representation of the TMPRSS2 14 kb upstream regulatory region. Potential five ARE clusters and fourteen 1 kb fragments are shown.

(D) ChIP analyses of AR recruitment to five potential ARE regions. ChIP assays were performed with anti-AR antibodies in LNCaP cells treated with vehicle or 100 nM DHT for 4 or 16 hr.

(E) Systematic mapping of enhancer elements within the TMPRSS2 14 kb upstream regulatory region. Fourteen 1 kb TMPRSS2 upstream sequences were subcloned into the pGL3-promoter vector and transfected into LNCaP cells. Cells were treated with vehicle or 100 nM DHT for 24 hr. The data are presented as the mean ± SE of two to three replicates, (A), (D), and (E).

Although we found that the –13.5 kb B39 binding site in the TMPRSS2 gene is a functional androgen-regulated enhancer (Figure 2F) and is in close proximity to the TMPRSS2 promoter in vivo (Figure 3B), we investigated the possibility that other functionally important AR binding sites located between –13.5 kb and the transcription start site may have been missed by the ChIP-on-chip assay. We therefore performed an AR motif search within the 14 kb region upstream of the TMPRSS2 gene and found five clusters of potential AREs (Figure 3C). Despite the presence of AREs, quantitative AR ChIP analyses found only significant AR binding to ARE region V corresponding exactly to the region identified by ChIP-on-chip (–13.5 kb) (Figure 3D). In addition, to further address whether other regions within the 14 kb upstream sequence might play transcriptional roles, we systematically subcloned fourteen 1 kb regions (Figure 3C) upstream of an SV40 promoter in the pGL3 luciferase vector and transfected these into androgen-depleted LNCaP cells. As shown in Figure 3E, construct A (–14 to –13 kb) corresponding to the B39 element had the most significant enhancer activity. In addition, construct N (–1 kb to +24 bp) that contains the TMPRSS2 promoter also demonstrated weak enhancer activity in this reporter assay. Significantly, quantitative AR ChIP showed that AR was only weakly recruited to the endogenous TMPRSS2 promoter region. Therefore the B39 binding site is the primary site of AR binding and functional enhancer activity within the 14 kb upstream of the TMPRSS2 gene. Of course, we cannot rule out the possibility that other cis-regulatory regions outside of the 14 kb region (Figure S4) may also play a role in TMPRSS2 expression.

Identification of AR-Collaborating Transcription Factor Complexes

We hypothesized that other transcription factors might play important collaborative roles in AR function. To search for these AR partners, we mapped all of the mammalian transcription factor motifs in the TRANSFAC database (Matys et al., 2003) to the 90 AR binding regions to identify motifs significantly enriched compared to the chromosomes 21 and 22 genomic background. While there was only a moderate enrichment (1.55-fold, p value 3.00E-3) for the AR half-site, the co-occurrence of an AR half-site motif with each of three DNA motifs, Forkhead, GATA, and Oct, was significantly enriched compared with the genomic background (AR half-site+ Forkhead, 5.33-fold, p value 1.63E-08; AR half-site + GATA, 3.19-fold, p value 1.16E-04; AR half-site + Oct, 2.03-fold, p value 3.09E-05) (Figure 4A). These associations strongly suggested a functional interaction between AR and sequence-specific DNA-binding transcription factors that recognize each of these motifs.

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Figure 4

Collaborative Transcription Factors Are Recruited to AR Binding Regions

(A) Schematic representation of association of AR half-site and transcription factor motifs within AR binding regions. Each pairwise association is designated by a circle with different color. The area of the circle is proportional to the frequency of the association.

(B) The recruitment of collaborative factors to AR binding regions. LNCaP cells were cultured in hormone-depleted medium for 3 days and then treated with vehicle or DHT for 1 and 16 hr. ChIP analyses were then performed with indicated antibodies or control IgG. DNA precipitates were measured by real-time PCR using primers spanning the PSA enhancer and nine AR binding regions on chromosomes 21 and 22. The results are shown as the average of two to three replicates ± SE. The occurrence of ARE, Forkhead, GATA, and Oct motifs in each region is also shown.

Given the three significantly enriched DNA motifs, we were faced with the question of which of the large number of transcription factors that can potentially bind the motifs were in fact playing a functional role. To generate hypotheses on which factors might be responsible, we searched gene expression profiles from several human prostate cancer studies available on the Oncomine database (Rhodes et al., 2004) and found the three transcription factors FoxA1, GATA2, and Oct1 to be of potential interest on the basis of their high expression in prostate cancers. FoxA1 and GATA2 have been shown to be required for androgen-induced PSA gene transcription in reporter gene assays (Gao et al., 2003; Perez-Stable et al., 2000). Moreover, FoxA1 and Oct1 are known to interact with AR in vivo (Gao et al., 2003; Gonzalez and Robins, 2001). To examine whether the three collaborating transcription factors are actually recruited to AR binding regions on chromosomes 21 and 22, we performed quantitative ChIP assays using antibodies against each of the three factors or IgG (control) using primers spanning ten of the validated AR binding regions and the well-characterized PSA enhancer as a control. As shown in Figure 4B, the three collaborating factors are recruited (≥2-fold) to 80% of the AR binding sites tested. Significant binding of each of the three factors to the AR binding regions was observed in the absence of androgen while androgen did increase specific factor binding at specific sites. These results demonstrate that AR and various combinations of each of the three potential collaborating transcription factors are recruited to specific regions of the genome. Given the complex transcriptional response to androgen, it is not surprising that there is a wide variation in the degree of factor occupancy and androgen dependence seen at these sites.

To explore the physical association of AR and the three transcription factors, we first performed coimmunoprecipitations of the endogenous proteins in vivo. Each of the three transcription factors was immunoprecipitated from DHT-treated or untreated LNCaP cells and subsequently probed by western blot with anti-AR antibodies. As shown in Figure 5A, AR interacts with GATA2 and Oct1 in a hormone-dependent manner, but its interaction with FoxA1 is not affected by androgen. To address whether AR forms a single complex with all three transcription factors, we immunoprecipitated GATA2 from LNCaP cells followed by western blotting with anti-Oct1 antibodies. The results showed that GATA2 does not interact with Oct1 (data not shown), suggesting that AR may form independent complexes with each of the collaborating factors.

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Figure 5

AR Interacts with Collaborative Transcription Factors In Vivo

(A) AR coimmunopreciptates with its collaborating factors in vivo. LNCaP cells were grown in the presence or absence of DHT for 24 hr. Whole-cell lysates were immunoprecipitated (IP) with indicated antibodies or control IgG. Western blot (WB) was performed with indicated antibodies.

(B) AR-collaborating factor complexes form on chromatin. LNCaP cells were treated with or without DHT for 16 hr. ChIP assays were performed with anti-AR antibodies. The immunoprecipitated chromatin was eluted, reimmunoprecipitated with the indicated antibodies or control IgG, and amplified by PCR using primers flanking the PSA and B38 enhancer regions.

(C and D) Colocalized cis-active elements are required for AR-dependent transcription. A wild-type PSA reporter construct and PSA reporter construct with deleted GATA or Oct motifs within the PSA enhancer or promoter regions (C) or B39 reporter wild-type construct and B39 reporters with GATA or Oct motif deletions (D) were transfected into LNCaP cells treated with vehicle or 100 nM DHT for 24 hr. Luciferase activities were determined, and results are presented as the mean ± SE of the triplicate experiments.

To demonstrate that AR and the three transcription factors form complexes on chromatin, we performed serial ChIP experiments (re-ChIP). The crosslinked chromatin was first immunoprecipitated with anti-AR antibodies and reimmunoprecipitated with either control IgG or antibodies against each of the three transcription factors. The PSA enhancer and B38 regions were enriched after re-ChIP in a ligand-dependent manner (Figure 5B), demonstrating that AR can act together on chromatin with FoxA1, GATA2, or Oct1.

While the above ChIP, coimmunoprecipitation, and re-ChIP assays demonstrated the protein-protein interaction between AR and potential collaborating transcription factors, it is not clear if these interactions are based on functional interactions between colocalized cis-active motifs with the AR binding regions. To address this issue, we generated GATA and Oct motif deletion mutants in the PSA enhancer within the full-length 6 kb PSA-regulatory region upstream of a luciferase gene. As a control, we also generated an Oct motif deletion mutant in the PSA promoter region. We transiently transfected these mutated constructs along with the wild-type PSA construct into hormone-depleted LNCaP cells, and luciferase was measured after 24 hr DHT stimulation. Consistent with a previous report (Perez-Stable et al., 2000), deletion of the GATA motif within the PSA enhancer region dramatically decreased androgen-stimulated transcriptional activities (Figure 5C). Interestingly, deletion of the Oct motif within the PSA enhancer, but not the promoter region, also significantly attenuated androgen-induced transcriptional activity (Figure 5C). These data suggest a functional interaction among colocalized cis-active elements within the PSA enhancer region. Significantly, both the Oct and GATA motifs were also required for the activity of the TMPRSS2 enhancer (Figure 5D).

ChIP and Re-ChIP

ChIP and re-ChIP were performed as previously described (Shang et al., 2000; Wang et al., 2005). Details are available in the Supplemental Experimental Procedures.

Cell Proliferation Assay

Cell proliferation was measured using a WST-1 kit (Roche, Indianapolis, IN).

Expression Array Experiments

Hormone-depleted LNCaP cells were grown in the absence or presence of 100 nM DHT for 4 and 16 hr. Total RNA was isolated using an RNeasy kit (QIAGEN, Valencia, CA). Total RNA from three biological replicates was hybridized at the Dana-Farber Cancer Institute Microarray Core Facility to human U133 plus 2.0 expression array. The data were analyzed by using dChip microarray software (Li and Wong, 2001).

ChIP Combined Chromosome Conformation Capture Assay

5C was performed as previously described (Wang et al., 2005). The primers used are listed in Table S1. The PCR amplified band was sequence verified.

Construction of Plasmids

AR binding sites were PCR amplified from LNCaP genomic DNA. Fourteen 1 kb TMPRSS2 upstream sequences were PCR amplified from BAC RP11-814F13. These PCR products were subcloned into pGL3-promoter vector (Promega, Madison, WI). ARE, GATA, and Oct motif deletion mutants were sequence verified. The PCR primers are listed in Table S1.

Reporter Gene Assays

Hormone-depleted LNCaP cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Details are available in the Supplemental Experimental Procedures.

Coimmunoprecipitation and Western Blotting

Coimmunoprecipitation and western blotting were performed as previously described (Wang et al., 2002) with a few modifications. Details are available in the Supplemental Experimental Procedures.

Small Interfering RNA

Small interfering RNA (siRNA) duplexes were transfected into LNCaP cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after siRNA transfection, cells were treated with DHT or vehicle and then harvested. The siRNA sequences are listed in Table S1.

Real-Time RT-PCR

Real-time RT-PCR was performed as previously described (Wang et al., 2005). Primers for RT-PCR are listed in Table S1.

This work was supported by the Claudia Adams Barr Program in Innovative Basic Cancer Research (M.B.), Prostate Cancer SPORE Grants P50 CA90381 (M.B.) and P50 CA69568 (K.J.P.), Department of Defense award W81XWH-05-01-0023 (Q.W.), and European Union contract LSHM-CT-2005-018652 (O.A.J.). K.J.P. is an American Cancer Society Clinical Research Professor. The authors also thank Drs. Jérôme Eeckhoute and Kirsten Fertuck for helpful discussions.