Androgen-Dependent Exons Figure 4. Expression of novel mRNA isoforms is both directly and indirectly regulated by androgens. (A) UCSC Genome Browser showing chromosomal locations and annotated transcript details for each novel androgen-regulated alternative mRNA isoform. Custom tracks (black bars below ‘‘LNCaP AR ChIP-seq peaks’’) for each gene show locations of AR binding site(s) within the LNCaP genome as determined by ChIP-Seq [4]. (B) Fluorescent quantification of capillary agarose gel electrophoretic analysis of RT-PCR products amplified from mRNA from LNCaP cells pretreated for 1 hour with vehicle (DMSO) or 1 g/ml cycloheximide (CHX) prior to addition of 10 nM R1881 for 24 hours. The relative normalised expression ratio is the contribution of the PCR product representing the alternative (androgen-regulated) transcript/constitutive transcript. Data from four independent replicates were used to obtain the means 6 SE. doi:10.1371/journal.pone.0029088.g004 production of both novel genes and endogenous variant mRNA isoforms. Gene expression pathway analysis revealed clusters of novel androgen-regulated genes associated with, and including the tyrosine kinase LYN, which is required for PCa cell proliferation [23]. Our experimental data indicates that LYN expression is normally repressed by androgens both at the mRNA and the protein level. We also identified androgen-dependent up-regula- tion of the LYN-associated kinase mTOR. mTOR kinase activity has been previously found to be up-regulated in PCa cells in response to androgens [35], and the mTOR pathway is important in clinical PCa, particularly in tumours lacking expression of PTEN (phosphatase and tensin homolog). Since AR gene targets can remain active in CRPCa, the mTOR complex may be a possible therapeutic target for CRPCa [35,36]. Exon resolution analyses further identified a number of mRNA isoform switches that occur in response to androgen treatment, including selection of alternative 59 exons and inclusion of cassette exons. The oestrogen receptor (ER) has been recently shown to regulate expression of many mRNA isoforms from alternative transcriptional initiation sites as are result of alternative promoter selection [13]. ER was found to associate with alternative promoters regions, and ER-regulated alternative mRNA isoforms influenced breast cancer cell behaviour. Similarly, based upon published data [3], we predicted the association of AR to alternative promoter regions within candidate genes, and con- firmed AR binding to an alternative promoter region (comprising DP1 and 2) within the TSC2 gene. This association directly regulated the dynamic accumulation of a shorter TSC2 mRNA predicted to encode a 508 aa C-terminus peptide up to 24 hours after androgen addition (GenBank protein accession: BAG52831.1). This peptide lacks an interaction domain needed to form a heterodimer with TSC1 (hamartin), which is required to regulate mTOR kinase function. Although primarily characterised as an upstream regulator of mTOR, TSC2 also has been associated with mitotic structures and cell cycle control, and in particular the C-terminus of the full-length protein has been localised to the nucleus and associated with the transcriptional activity of FOXO transcription factors [37]. We are currently exploring our hypothesis that this novel AR-regulated TSC2 mRNA isoform might play an important role in mTOR regulation in PCa cells. In principle, ExonArrays should provide an extremely accurate measure of gene expression [38] since data are gathered along the full length of the transcript. Compared with other published datasets on androgen-regulated gene expression [16,17,18,19, 20,21,22], the results of our analysis have the most extensive overlap with other microarray analyses. Although ,50% of gene expression changes in our dataset were novel, these findings may be in part due to experimental differences between previously published studies rather than platform-specific differences (Table S2). Indeed, a more detailed analysis suggested that many of the novel hits are in fact functionally related to known androgen- regulated genes (see Table S2, column 5). Of the 1279 putative androgen-regulated alternative events identified, only ,25% mapped to known alternative splicing events or alternative first/ last exons. Furthermore, the low positive validation rate (,23%) highlights the technical challenges of using ExonArrays to identify alterations in alternative mRNA isoform expression on a background of large changes in gene expression [15]. Taken together, our analyses suggest that the bulk of androgen-regulated effects on the transcriptome are at the level of overall gene expression rather than alterations in the expression of variant mRNAs. In summary, we have found that androgens not only have quantitative effects on gene expression but also have some qualitative effects on transcript composition. While different molecular mechanisms may underlie androgen-regulated expres- sion of alternative mRNAs (including alternative splicing, transcriptional initiation or RNA stability) in a cell line- and tissue-specific manner, our data suggest that androgens directly regulate expression of alternative 59 exons (as a result of promoter selection), and indirectly regulate alternative splicing. The androgen-dependent variant transcripts identified in this study are predicted to yield protein isoforms that may mediate the cellular response to androgens, although the functional roles of these mRNA isoforms in clinical PCa is as yet unknown, and warrants further investigation. Materials and Methods Antibodies The following antibodies were used: anti-LYN mouse mono- clonal antibody (65038-1-1 g, Proteintech), anti-AR rabbit anti- sera (C19, Santa Cruz Biotechnology), anti-actin rabbit antisera (A-2066, Sigma), and normal rabbit IgG (sc-2027, Santa Cruz Biotechnology). Cell line samples for array analysis and RT-PCR validation All cells were grown at 37uC in 5% CO2. LNCaP cells (CRL- 1740, ATCC) were maintained in RPMI-1640 with L-Glutamine (PAA Laboratories, R15-802) supplemented with 10% Fetal Bovine Serum (FBS) (PAA Laboratories, A15-101). For details of other cell lines see Materials and Methods S1. Where indicated, medium was supplemented with 10% dextran charcoal stripped FBS (PAA Laboratories, A15-119) to produce a steroid-deplete medium. In experiments performed in quadruplicate, LNCaP cells were cultured for 72 hours, following which 10 nM synthetic androgen analogue methyltrienolone (R1881) (Perkin–Elmer, NLP005005MG) was added (treatment; n = 4) or absent (control; n = 4) for 24 hours. Cells were then harvested and total RNA extracted using TRIzol (Invitrogen, 15596-026) according to manufacturer’s instructions as previously described [9]. To ensure array findings were not due to batch effects and to confirm the reproducibility of validation methodologies, the above experiment was performed independently in triplicate and all samples analysed by qPCR and RT-PCR in an identical manner. Where indicated, LNCaP cells were pre-treated for 1 hour with vehicle (dimethyl- sulfoxide; DMSO) (Sigma, C1988) or 1 mg/ml cycloheximide (Sigma, D2438) prior to addition of 10 nM R1881 for 24 hours as previously described [39]. PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29088