Oncogene (2011) 30, 619–630 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ONCOGENOMICS Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells K Takayama1,2,3, S Tsutsumi4, S Katayama5, T Okayama6, K Horie-Inoue3, K Ikeda3, T Urano1,2,3, C Kawazu5, A Hasegawa5, K Ikeo6, T Gojyobori6, Y Ouchi2, Y Hayashizaki5, H Aburatani4 and S Inoue1,2,3 1Department of Anti-Aging Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; 2Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; 3Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Hidaka-shi, Saitama, Japan; 4Genome Science Division, Research Center for Advanced Science and technology, University of Tokyo, Meguro-ku, Japan; 5Riken Omics Science Center, RIKEN Yokohama Institute, Kanagawa, Japan and 6National Institute of Genetics, Mishima, Shizuoka, Japan The androgen receptor (AR) is a critical transcriptional clusters in our study were previously confirmed as factor that contributes to the development and the androgen target genes in PCa. The integrated high- progression of prostate cancer (PCa) by regulating the throughput genome analyses of CAGE and ChIP-chip transcription of various target genes. Genome-wide provide useful information for elucidating the AR- screening of androgen target genes provides useful mediated transcriptional network that contributes to the information to understand a global view of AR-mediated development and progression of PCa. gene network in PCa. In this study, we performed 50-cap Oncogene (2011) 30, 619–630; doi:10.1038/onc.2010.436; analysis of gene expression (CAGE) to determine andro- published online 4 October 2010 gen-regulated transcription start sites (TSSs) and chro- matin immunoprecipitation (ChIP) on array (ChIP-chip) Keywords: androgen receptor; prostate cancer; CAGE; analysis to identify AR binding sites (ARBSs) and histone Chip-chip H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (Po0.01), and ChIP-chip analysis identified 2872 andro- gen-dependent ARBSs (Po1e-5) and 25 945 AcH3 sites Introduction (Po1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were Androgen action is essential for the development, determined as androgen target genes. Besides prototypic proliferation and subsequent progression of prostate androgen-regulated TSSs in annotated gene promoter cancer (PCa). Androgen binds to its cognate receptor, regions, there are many androgen-dependent TSSs that androgen receptor (AR), a member of the nuclear are widely distributed throughout the genome, including receptor superfamilies that functions as a ligand- those in antisense (AS) direction of RefSeq genes. Several dependent transcriptional factor (Shang et al., 2002; pairs of sense/antisense promoters were newly identified Wang et al., 2005; Dehm and Tindall, 2006). It has been within single RefSeq gene regions. The integration of shown that AR and its downstream signals are deeply CAGE and ChIP-chip analyses successfully identified a involved in the pathophysiology of hormone-dependent cluster of androgen-inducible miRNAs, as exemplified by and hormone-independent PCas (Suzuki et al., 2003; the miR-125b-2 cluster on chromosome 21. Notably, the Chen et al., 2004; Debes and Tindall, 2004). Therefore, number of androgen-upregulated genes was larger in elucidation of the entire AR signaling pathways will LNCaP cells treated with R1881 for 24 h than for 6 h, and reveal the precise mechanisms underlying the develop- ONCOGENOMICS the percentage of androgen-upregulated genes accompa- ment and the progression of PCa and will identify novel nied with adjacent ARBSs was also much higher in cells molecular targets for cancer therapy. Recent advances in treated with R1881 for 24 h than 6 h. On the basis of the high-throughput gene analysis technology have enabled Oncomine database, the majority of androgen-upregu- to identify a number of target genes that are associated lated genes containing adjacent ARBSs and CAGE tag with diseases at various statistical thresholds. Our group and others have successfully determined bonafide AR binding sites (ARBSs) in the human genome by Correspondence: Professor S Inoue, Department of Anti-Aging chromatin immunoprecipitation (ChIP) analysis com- Medicine, Graduate School of Medicine, University of Tokyo, bined with genome tiling arrays (ChIP-chip) (Massie Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: [email protected] et al., 2007; Takayama et al., 2007, 2009; Wang et al., Received 22 April 2010; revised 4 August 2010; accepted 12 August 2010; 2007, 2009). ChIP-chip analysis is also useful to published online 4 October 2010 determine epigenetic alterations in the genome. We CAGE and ChIP-chip in prostate cancer cells K Takayama et al 620 have identified that amyloid b-precursor protein is promoter region of a representative androgen target an androgen-inducible gene that could be a novel gene FKBP5 (Velasco et al., 2004), we detected a time- prognostic and therapeutic target of PCa by screening dependent increase in tag numbers by androgen genes in the vicinity of both ARBSs and acetylated stimulation (21.2, 80.6 and 474.3 tags accumulated per histone H3 (AcH3) sites (Takayama et al., 2009). million tags (t.p.m.) at 0, 6 and 24 h after R1881 In terms of transcriptome, cDNA microarray has been treatment, respectively) (Figure 1a). Androgen-depen- extensively used for years as a convenient and cost- dent increase in CAGE TCs was also shown in the effective technique for analyzing gene expression signa- promoter regions of PSA/KLK3 and TMPRSS2 (Sup- tures. cDNA microarray, however, is not designed to plementary Figure 1). The 50-termini of RefSeq genes identify novel genes without probes, thus it has limita- are often different from the TSSs obtained by CAGE tions to analyze the expression of noncoding RNAs, analysis (Katayama et al., 2007). For example, our study including short RNAs. As some microRNAs (miRNAs) shows that TRIM36 includes an intronic CAGE TC and novel RNA transcripts have been reported to be within the second AcH3 site, which is situated in the relevant to PCa (Louro et al., 2007; Shi et al., 2007), downstream of the intronic ARBS (Supplementary an alternative high-throughput transcriptome technol- Figure 2). Only 35% of CAGE TCs were located at a ogy will be also required to analyze noncoding RNA distance of o1 kb from the TSSs of known RefSeq expression as well as coding RNA profiles. genes (Figure 1b). In total, 13% of the TCs were In this study, we investigated AR actions across the identified at intergenic regions that were located entire human genome to elucidate the androgen- 4100 kb upstream of TSSs and did not overlap with mediated transcriptional network by performing ChIP- any RefSeq gene. In all, 7% of the TCs were genome- chip and cap analysis gene expression (CAGE). We widely distributed at the antisense regions of RefSeq could determine novel AR-mediated genomic actions, genes, and 33% were transcribed from the RefSeq in particular, those dependent on antisense (AS) or regions, most of which were from novel TSSs. Global intergenic transcription start sites (TSSs) that have not transcriptome analysis using the CAGE technique and been previously identified by any other methods. Thus, large-scale cDNA sequencing in the FANTOM3 project our integrated approach identify novel androgen target has recently shown that a large proportion of the genes and provides potential diagnostic and therapeutic mammalian genome can transcribe genes from both molecular targets of PCa, which can be applied to the sense (S) and antisense strands, and that many of the clinical management of the advanced disease. unknown transcripts are noncoding RNAs (Carninci et al., 2005). Taken together, these findings suggest that androgen-regulated transcripts are widely distributed Results across annotated genes and noncoding RNAs. We also compared the androgen responsiveness of our Genome-wide analysis of androgen-activated promoters CAGE TCs with RefSeq genes. Among protein-coding in prostate cancer cells genes with an increasing number of CAGE TCs (420 CAGE involves the preparation and sequencing of t.p.m. by 24 h) at their promoter regions (o0.1 kb from concatemers of 20-nucleotide (nt) DNA tags that were TSS), 51% of genes were also significantly upregulated derived from the 50-end of capped mRNA, and mapping (41.5-fold) by 24-h treatment with R1881 (1 nM)in the CAGE tags to the genome (Shiraki et al., 2003). This LNCaP cells based on the microarray data set method is cost-effective and enables high-throughput GSE14028 (Yu et al., 2010, P ¼ 2.0e-24). In contrast, genome-wide analyses of TSSs and promoter usage. For 40% of protein-coding genes with a decreasing number preparing CAGE samples, we stimulated LNCaP cells of TCs were significantly downregulated (o0.8-fold) by with R1881 (10 nM), which were cultured in a hormone- R1881 in LNCaP cells (Yu et al., 2010, P ¼ 2.7e-5). On depleted medium for 72 h. We extracted total RNA from the basis of another microarray data GSE7868 (Wang cells stimulated for 6 and 24 h, and synthesized first- et al., 2007), 44% of the RefSeq genes with an increasing strand cDNA by reverse transcription. After cutting number of CAGE TCs were upregulated in LNCaP cells and amplifying the 20-nt CAGE tags derived from treated with dihydrotestosterone (100 nM) for 16 h the 50-ends of mRNAs, we sequenced the tag-ligated (P ¼ 4.2e-28) and 45% of the RefSeq genes with a concatemers. We then mapped the sequenced CAGE decreasing number of TCs were downregulated by tags to the human genome (total tags at 0 h were dihydrotestosterone (P ¼ 1.7e-36).