following treatment with 1 nM and 10 nM DHT. DHT (at both 1 nM and 10 nM concentrations) did not have an effect on expression of the alternative mRNA isoform derived from RIMS1, however expression changes were seen with 1 nM R1881. For other transcripts, an overall trend was observed towards relative increased expression of androgen-regulated mRNA isoforms in keeping with the data with 10 nM R1881. These findings suggest that the functional cellular response to androgens mediated by alternative mRNA isoform expression may be dependent on the local (physiological) concentration of endogenous ligand. To determine whether the identified androgen-dependent mRNA isoforms are cancer-specific and cell line-specific, we examined the expression of alternative mRNAs in other tumourigenic androgen-dependent (CWR22Rv1) and androgen- independent (LNCaP-AI, LNCaP-cdxR) PCa cell lines, as well as a non-tumourigenic androgen-independent prostate epithelial cell line (BPH-1) (Figure S3). Since we were examining the steady-state levels of mRNA expression (rather than dynamic changes in response to androgens), we focussed on the alternative mRNAs (derived from NDUFV3, ZNF121, PDE4D, and WEE1) that could be specifically detected with our RT-PCR assays in steady-state conditions (culture medium containing androgens). Androgen- responsive exons were not exclusive to cancer cells (expressed in BPH-1 cells), nor to PCa cell lines that exhibit an androgenic growth response (LNCaP, and CWR22Rv1). In addition, alternative mRNA isoforms were present in LNCaP cells cultured in medium devoid of steroids (LNCaP-AI) and with anti- androgens (LNCaP-cdxR) (Figure S3A). To demonstrate that AR is required to mediate androgen-regulated expression of alternative mRNAs, we examined expression of alternative exons in PCa cell lines that do not exhibit an androgenic growth response (DU145, PC-3, PC-3M) (Figure S3B). Although alterna- tive exons were expressed in these cell lines, treatment with androgens did not alter their expression levels. Finally, we studied expression of alternative mRNA isoforms (derived from NDUFV3, ZNF121, PDE4D, and WEE1) in prostate biopsies from patients with PCa (Figure S3C), and found that although alternative exons were expressed in clinical PCa, there was no association with disease progression (p.0.05). Taken together, the above data suggest that in addition to androgens, expression of alternative mRNAs may be regulated by other trans- acting factors as well as cis-regulatory elements in a cell type- and tissue-specific manner. Expression of novel mRNA isoforms is both directly and indirectly regulated by androgens Recent studies have used chromatin immunoprecipitation (ChIP) and direct next generation sequencing (ChIP-Seq) for unbiased mapping of AR genomic binding sites in LNCaP cells in response to androgens [3,34]. To determine whether the association of AR within candidate genes identified in our study may drive the expression of alternative mRNA isoforms, we compared the previously published AR binding profiles [3,34] to the predicted locations of alternative events (Figures 4A and S4). The genomic loci of AR binding sites were uploaded to UCSC genome browser [24], and interrogated for positively-validating genes of interest from our present study (Figure 2). In keeping with our hypothesis, AR binding (Figure 4A, LNCaP AR ChIP-seq peaks, and Figure S4) was observed close to alternative promoters predicted to yield variant androgen-regulated mRNA isoforms with alternative 59 exons generated from alternative transcription- al initiation sites (PDE4D, TACC2, TSC2, RIMS1, and WEE1). However, we did not observe AR binding within genes encoding transcripts identified to possess androgen-regulated cassette exons Androgen-Dependent Exons (NDUFV3 and ZNF121) suggesting that these splicing events may be due to the indirect effect of androgens. To test this hypothesis, we co-treated LNCaP cells with or without 10 nM R1881 (as above) in conjunction with the translational inhibitor cycloheximide or vehicle (dimethylsulfoxide; DMSO) (Figure 4B). Using multiplex RT-PCR, we monitored exon skipping and exon inclusion within transcripts derived from NDUFV3 and ZNF121, respectively. Cycloheximide completely inhibited androgen-dependent NDUFV3 exon 3 skipping and ZNF121 exon 5 inclusion. These data indicate that the effect of androgens on NDUFV3 and ZNF121 splicing is indirect and requires intermediate protein synthesis. Ligand-activated AR is rapidly recruited to an alternative internal promoter within the TSC2 gene To determine whether AR can directly regulate alternative mRNA expression from alternative transcription start sites, we explored the association of AR with the TSC2 gene. To test our hypothesis that the alternative TSC2 mRNA isoform is directly androgen-regulated, we firstly examined expression of TSC2 mRNAs from LNCaP cells co-treated with or without 10 nM R1881 (as described above) in conjuction with cycloheximide or vehicle (DMSO) (Figure 5A). Cycloheximide did not have any significant effect (P.0.05) on expression of androgen-induced mRNA isoform expression indicating that the ligand-dependent effect of AR on the TSC2 gene is direct and does not require intermediate protein synthesis. To confirm this hypothesis, we examined the dynamic recruitment of ligand-activated AR to alternative internal promoters (Figure 4A) of TSC2 by ChIP at early timepoints following androgen stimulation. LNCaP cells were cultured in steroid-deplete medium to quiesce AR activity, and subsequently treated with 10 nM R1881 as the only available steroid and AR ligand. Chromatin was harvested at 60-minute intervals following androgen treatment, and subjected to immunoprecipitation using anti-AR antisera. Recovered genomic DNA was analysed by qPCR to examine recruitment of AR protein to the DP1 and 2 (Figure 4B, left and right panels, respectively) as well as a region of the TSC2 gene that was not predicted to bind AR. Maximal recruitment of AR to both DP1 and 2 was observed after 120 minutes following androgen stimulation, but not to the non- AR-binding region within GREB1. Overall these data show that AR is dynamically recruited to DP1 and 2 within TSC2, in response to ligand activation. Due to close proximity of the promoters (separated by 524 nucleotides), it was difficult to cleanly resolve the association of AR-promoter association. Since AR-dependent gene expression is a dynamic process [17], we measured the expression of the shorter AR-regulated TSC2 isoform by RT-PCR at timepoints up to 24 hours following androgen stimulation (Figure 4C). We observed an increasing temporal trend in expression of the alternative TSC2 isoform, which reached statistical significance at 16 hours following androgen stimulation (p,0.05). Taken together, these data suggest that recruitment of ligand- activated AR to internal androgen-regulated promoters controls expression of an alternative TSC2 mRNA isoform. Although we observed a lag in alternative isoform expression ratios following AR recruitment to the DP1 and 2 promoters, this is likely explained by the stability of the constitutive mRNA isoform and the time taken to establishment of steady-state levels. Discussion In the present study, we have undertaken gene expression analysis at exon level resolution to identify androgen-regulated PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29088