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 expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen 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 (AR) is a critical transcriptional clusters in our study were previously confirmed as factor that contributes to the development and the androgen target 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 (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 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 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 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 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). 1 929 416; at 6 h, 682 111; and at 24 h, 586 083). Regions containing multiple tags were referred to as tag clusters (TCs); these clusters were diversely distributed through- Genome-wide identification of ARBSs in prostate cancer out the genome. By using Fisher’s exact test, we cells identified TCs whose distributions were significantly ChIP-chip analysis revealed 6366 (Po1e-4), or 2872 regulated by androgen. The total number of androgen- (Po1e-5) specific ARBSs in the genome of LNCaP cells. dependent TCs obtained was 13 110 (Po0.01); 1572 TCs To compare our ChIP-chip data with previous studies, were obtained using a stringent threshold (1e-6.11 with we found that 77 and 74% of our AR binding (Po1e-5) Bonferroni correction). Among the androgen-regulated were involved in the ChIP-chip data (Wang et al., 2009) TCs, 78.9% were upregulated and the remaining was and the ChIP-Seq data (Yu et al., 2010), respectively. downregulated at 6 or 24 h after stimulation. In the We also identified 25 945 AcH3 sites (Po1e-4) as

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 621 epigenetic markers that were associated with activated AcH3 sites and the CAGE TCs, 41% of the CAGE TCs promoters (Bernstein et al., 2005). Among 2872 ARBSs were overlapped with AcH3 sites and 49% were (Po1e-5), 420 ARBSs (15%) were overlapped with overlapped with AcH3 sites within a distance o3 kb. AcH3 sites. In addition, approximately 37.6% of AcH3 On the other hand, most of the ARBSs were found to be sites were mapped to the proximal regions of the TSSs of widely distributed across the human genome, and only RefSeq genes (o3 kb from the TSSs), including many 4.8% of the ARBSs were mapped to promoter regions at sites located in the downstream proximal regions of o1 kb from the TSSs (Figure 1c). TSSs (Figure 1c), as consistent with a previous report in We randomly selected 25 ARBSs in the vicinity of regard to genome-wide analysis of active promoters known androgen targets such as ACSL3, and intergenic (Kim et al., 2005). In terms of the overlap between the regions on 15, 20 and X to perform conventional ChIP analysis for AR binding in LNCaP cells (Figure 2a). When compared with four genomic Chr 6 (p21.32-p21.2) regions as negative controls, most of the investigated regions were confirmed to be androgen-dependent ARBSs. To further investigate the effects of the histone 0-6 h acetylation status on the AR-dependent transcriptional 0-24 h activity, we performed ChIP analysis for AR, SRC1, TC

CAGE AcH3 and RNA Pol II recruitment for the group of 6-24 h ARBSs that overlapped with AcH3 sites (AcH3 overlap AcH3 ( þ )) and the group of ARBSs distinct from the AcH3 ChIP AR sites (AcH3 overlap (À)) (Figure 2b). Significant RefSeq androgen-dependent recruitment of both SRC1 and RNA Pol II was detected (41.5-fold) in the AcH3 overlap ( þ ) group, but not in the AcH3 overlap (À) 0-6 h group. 0-24 h TC CAGE 6-24 h ChIP-chip and CAGE analyses reveal AR-dependent AcH3 regulation of androgen target genes Novel ARBSs and AcH3 sites adjacent to CAGE TCs ChIP AR were identified in the androgen-regulated genes. For FKBP5 instance, we found novel ARBSs in the intronic regions 35700000 35750000 35800000 of a-methyl-CoA racemase, which is a prognostic marker for PCa (Luo et al., 2002) (Figure 3a). Interest- >10 kb Intergenic ingly, AR binding was also observed in an intronic down region of insulin-like growth factor-1 receptor, whose expression has been previously reported to be dependent AS on the nongenomic action of androgen (Pandini et al.,

1-10 kb down <1 kb down 10-100 kb Figure 1 Genome-wide cap analysis of gene expression (CAGE) up and chromatin immunoprecipitation on DNA chips (ChIP-chip) of the androgen-regulated gene network in hormone-naı¨ve prostate <0.1 kb 1-10 kb up cancer LNCaP cells. (a) Mapping of androgen-regulated promoters down and androgen receptor binding sites (ARBSs) or acetylated histone <0.1 kb up <1 kb up H3 (AcH3) sites in the vicinity of FKBP5 on chromosome 6. Top panel, cytogenetic location of the representative androgen-regu- lated target gene FKBP5 on chromosome 6. Middle panel, UCSC genome browser view of androgen-dependent CAGE tag clusters (TCs) and AR/AcH3 sites determined by ChIP-chip at the 6p21 region. Androgen-upregulated CAGE TCs were mapped for three different time points after R1881 treatment (Tx) (0–6 h Tx, 6-h Tx versus 0-h; 0–24 h Tx, 24-h Tx versus 0-h; 6–24 h Tx, 24-h Tx versus 6-h). Bottom panel, browser view of CAGE TCs and AR/AcH3 sites in the vicinity of FKBP5 gene and its promoter region. (b) Distribution of androgen-regulated CAGE TCs (n ¼ 13,110, % of Binding Sites Po0.01) relative to the transcription start sites (TSSs) of the RefSeq genes. TCs located 4100 kb upstream of the TSSs of the RefSeq genes are classified as intergenic TCs. AS represents the TCs in the antisense regions of the RefSeq genes. (c) Distances of the AR/AcH3 sites from the TSSs of RefSeq genes. The y axis Distance from TSS (bp) represents the percentages of the total sites that were identified as ARBSs or AcH3 sites.

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 622

Vehicle 120 R1881

90

60

Fold Enrichment 30

0 #1#2#3#4 ST7 NFIB ChrX LAF4 TPT1 NC EPN2 CHN2 SMA5 Chr20 LAMA ANKH SARG Chr15 CUTL1 CUTL2 SNX25 ACSL3 FOLH1 MKLN1 NRXN3 TRPM3 TMEM2 IMMP2L SLC7A1 PPP3CA

FKBP5 Fold change TACC2 AcH3 3 TRIM36 Overlap ACACA (+) 0 PPFIBP2 VDAC1 -3 UGT2B10 AcH3 SOX4 Overlap EFNA5 (-) R1881 TP53INP1 -Tx: - + -+-+-+-+ ChIP: IgG AR SRC1 AcH3 Pol II Figure 2 ChIP–qPCR validation of ARBSs determined by ChIP-chip. (a) Conventional ChIP analysis of 25 randomly selected ARBSs. AR binding was determined in LNCaP cells treated with R1881 or vehicle for 24 h by ChIP. NC represents negative control locus that did not show any specific AR binding (Horie-Inoue et al., 2006). ARBSs are denoted as the gene symbols of the RefSeq genes to which they were adjacent to as the respective chromosome numbers if they were located in the intergenic regions. (b) Effect of AcH3 sites overlap with ARBS on the binding of androgen-regulated transcriptional factors. Representative ARBSs with or without AcH3 sites overlap were examined for their occupancy of AR, SRC1, AcH3 and RNA Pol II in the promoter regions of the indicated genes. IgG was used as a control for nonspecific binding.

2009); thus, we presume that the genomic activity of AR abundant in the proximal regions of the TSSs of RefSeq will also contribute to the transcriptional modification genes (Figure 4b). We identified 39 RefSeq genes with of insulin-like growth factor-1 receptor (Figure 3b). bidirectional S/AS pairs of CAGE TCs in the human Identification of ARBSs in other androgen-regulated genes, genome. Among these S/AS pairs, 15 of 39 S/AS pairs such as NK3 1 (Prescott et al., 1998) (Figure 3c) exhibited reciprocal alteration of tag numbers in and jagged 1 (Santagata et al., 2004) (Figure 3d), also response to androgen, whereas other pairs exhibited provide further evidence that direct actions of AR is concordant alteration. With regard to the androgen- required for their transcriptional regulation. dependent reciprocal patterns of S/AS pairs, one pattern consisted of an upregulated sense TC present in the TSS region of a RefSeq gene and a downregulated intronic S/AS transcriptional regulation by androgen showed antisense TC; this pattern was observed for the diverse alteration patterns androgen-inducible gene DNM1L (Figure 4c). The other Genome-wide promoter exchange such as bidirectional pattern consisted of a downregulated sense TC present or sense/antisense (S/AS) transcriptional regulation is in the TSS region of a RefSeq gene and an upregulated known to occur in the human genome. Antisense intronic sense TC, as exemplified by the androgen- transcripts associate with neighboring genes in complex repressed gene SLC7A1 (Figure 4d). The contribution of loci to form chains of linked transcriptional units an antisense promoter to the expression of a gene needs (Katayama et al., 2005). We investigated the number to be studied; nevertheless, CAGE analysis is a useful of antisense-directed TCs that were associated with technology to clarify the complexity of androgen- ARBSs: of the 1572 TCs, 173 (11% with Bonferroni regulated transcription in PCa cells. correction) were situated on the antisense strand of RefSeq genes and 34 of these 173 TCs (19%) were associated with ARBSs within a 100-kb region ChIP-chip analysis reveals miRNA regulation by AR (Po1e-5). Previous studies on expression profiling in PCa have Antisense TCs were mostly abundant in the 30-regions revealed that B20-nt short RNAs or miRNAs are of RefSeq genes (Figure 4a). However, sense TCs were closely associated with the progression of the disease.

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 623 miR-21 is surrounded by a 1-kb upstream AcH3 site and Scale 10 kb a 28-kb downstream ARBS (Supplementary Figure 3a). 34050000 chr5: We validated that miR-21 expression was upregulated 0-24 h by R1881 in LNCaP cells (Supplementary Figure 3b). 0-6 h

CAGE Furthermore, we focused on the miRNA-clustered 6-24 h region of miR-125b-2 on chromosome 21; miRNAs acH3 let-7c, miR-99a and miR-125b-2 were clustered in the

ChIP AR intronic regions of C21orf34 gene (Figures 5a and b). AMACR These results will also provide evidence that androgen AMACR genomic actions regulate miRNAs, as miR-125b has been shown to be an androgen-inducible miRNA that Scale 100 Kb can be associated with PCa proliferation (Shi et al., Chr15: 97000000 2007), and let-7c is reported as an upregulated miRNA 0-24 h in metastatic PCa (Leite et al., 2009). miR-99a is located at 131 kb from the nearest ARBS, although the miRNA 0-6 h

CAGE has not been well-characterized in PCa. Quantitative 6-24 h RT–PCR (qRT–PCR) revealed that miR-125b-2, let-7c acH3 and miR-99a exhibited androgen-dependent upregula- AR ChIP tion 48 h after R1881 treatment (Figure 5c), thereby FAM169B indicating that all of the three miRNAs on the IGF1R miR-125b-2 locus are putative AR-regulated targets in PCa cells. Scale 50 kb Chr8: 23550000 23600000 Identification of AR regulated genes by ChIP-chip 0-24 h analysis 0-6 h We explored androgen-regulated genes based on the CAGE 6-24 h data of ChIP-chip and CAGE analyses. Within a region acH3 of 100 kb from the ARBSs, 44, 66 and 134 androgen- upregulated CAGE TCs were identified by the compar- ChIP AR ison of time points at 6 versus 0 h, 24 versus 6 h and 24 NHX3-1 versus 0 h, respectively (Figure 6a). mRNA microarray analysis revealed that 254 and 1477 genes were Scale 20 kb upregulated (4twofold) after 6 and 24 h of androgen chr20: 10600000 treatment, respectively (Figure 6b). Among the genes 0-24 h: upregulated by androgen, the rate of ARBS positivity 0-6 h (at o100 kb) was 21.6% at 24 h and 7.2% at 6 h

CAGE (Figure 6b). AR binding bias was not significantly 6-24 h shown in upregulated genes at 6 h only versus the acH3 genomic background (P ¼ 0.23). In contrast, ARBS

ChIP AR positivity was highly enriched in the upregulated genes JAG1 at 24 h versus the genomic background (P ¼ 1.4e-19). JAG1 These results show that the AR binding will contribute Figure 3 Contribution of AR-dependent promoters and AR to the androgen-dependent transcription by 24 h. More- occupancy to the transcriptional regulation of representative over, representative qRT–PCR data showed that known androgen-regulated genes. UCSC genome browser views of and putative androgen target genes exhibited time- androgen-dependent CAGE TCs and AR/AcH3 sites determined dependent upregulation and that the androgen-depen- by ChIP-chip in the vicinity of AMACR (a), IGF1R (b), NKX3.1 (c) and JAG1 (d). dent upregulation was continuously observed up to at least for 48 h after treatment (Figure 6c). Taken together, we identified 258 novel upregulated genes (4twofold change at 24-h treatment), which may be On the basis of ChIP-chip data, we searched miRNAs in putative direct targets of AR. the vicinity of ARBSs; we identified four miRNAs in three miRNA clusters located at a distance of o100 kb from the ARBSs. We also identified four additional Androgen target genes are associated with tumorigenesis miRNAs that were relatively adjacent to the ARBSs or To elucidate the role of putative androgen target genes AcH3 sites, which were recently shown to be relevant in in the tumorigenesis of PCa, we reviewed the expression the progression of PCa (Shi et al., 2007; Leite et al., profiles of 508 genes that were differentially regulated 2009; Ribas et al., 2009; Sun et al., 2009) (Table 1). in our experiments (258 androgen-upregulated genes Among them, miR-21 is a characterized AR-regulated and 250 androgen-downregulated genes) in normal and miRNA that promotes hormone-dependent and -inde- cancer (PCa) regions of prostate tissues. cDNA micro- pendent PCa growth (Ribas et al., 2009). In our analysis, array data for 62 primary tumors and 41 normal tissue

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 624 0 = TSS 1 = End of RefSeq Gene 40 30 25 30 20 20 15 10 10 Number of RefSeq Genes Number of 5 0 RefSeq Genes 0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 S TC Location AS TC Location

Scale 20 kb 200 chr12: 32750000 DNM1L S 0-24 h 150 0-6 h S AS

CAGE 6-24 h 100 acH3 AR 50 ChIP DNM1L Number of TCs AS DNM1L 0 DNM1L R1881-Tx: 0 6 24 (h)

Scale 20 kb 100 SLC7A1 chr13: 29050000 80 0-24 h 60 0-6 h S S 40 CAGE 6-24 h AS acH3 20 AS Number of TCs

ChIP AR 0 SLC7A1 R1881-Tx: 0 6 24 (h)

Figure 4 Identification of androgen-regulated CAGE TCs present in the antisense regions. (a) Distribution of antisense TCs relative to the proportional positions of RefSeq genes. Positions 0 and 1 on the x axis represent the TSSs and ends of RefSeq Genes, respectively. (b) Distribution of sense (S) TCs relative to the proportional positions of RefSeq genes. (c, d) Reciprocal regulation of S/ AS promoter pairs of RefSeq genes in response to androgen. The sense promoter at the TSS of DNM1L was upregulated, whereas the antisense promoter at an intron of DNM1L was downregulated (c). The sense promoter at the TSS of SLC7A1 was downregulated, whereas the antisense promoter at an intron of SLC7A1 was upregulated (d).

Table 1 miRNAs adjacent to ARBSs miRNA chr Location Region ARBS (kb) AcH3 (kb)

miR-222 X 45362675–45362784 Intergenic 9.6 103.3 miR-221 X 45361839–45361948 Intergenic 10.5 104.2 miR-21 17 55273409–55273480 30-downstream (TMEM49) 28.3 1.0 miR-125b-2 21 16884428–16884516 Intron (C21orf34) 80.2 1.1 miR-218-1 4 20206167–20206276 Intron (SLIT2) 111.3 172.2 miR-218-2 5 168127729–168127838 Intron (SLIT3) 116.9 151.7 let-7c 21 16834019–16834102 Intron (C21orf34) 130.6 84.7 miR-100 11 121528147–121528226 Intron (LOC399959) 307.2 42.3

Abbreviations: AcH3, histone H3 acetylated; ARBSs, androgen receptor binding sites; chr, chromosome; miRNA, microRNA.

samples (Lapointe et al., 2004) were downloaded from depicted as normal 4PCa (blue square). The normal the Stanford University website (http://microarray- oPCa cluster containing androgen-upregulated genes pubs.stanford.edu/prostateCA); genes upregulated and (group I) included genes such as ACACA and a-methyl- downregulated by androgen were hierarchically clus- CoA racemase (related to metabolism) and tered using the pairwise average-linkage method the normal 4PCa cluster containing androgen-upregu- depending on the gene expression features of normal lated genes (group II) included genes such as EGFR and against tumor predominancy (Figure 6d). The cluster of EFNA5 (related to signal transduction). The normal genes with higher expression in PCa tissues than in oPCa cluster containing androgen-downregulated normal prostate tissues is depicted as normal oPCa (red genes (group III) included genes such as NFIX and square), and the other cluster of genes with higher SOX4 (related to transcriptional regulation), and the expression in normal prostate than in PCa samples is normal 4PCa cluster containing androgen-downregu-

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 625 Scale 200 kb regulation is closely associated with the development chr21: 16500000 17000000 of PCa. 0-24 h Finally, we extracted novel androgen-inducible genes 0-6 h that are regulated by direct AR actions. We selected 569

CAGE 6-24 h RefSeq genes that exhibited androgen-inducible CAGE acH3 TCs 24 h after treatment at o0.1 kb from their TSSs.

ChIP ChIP AR Among them, 51% were induced by 41.5-fold at 24 h C21orf34 after R1881 treatment, indicating that the majority of C21orf34 CAGE TCs function as bonafide androgen-regulated hsa-mir-99a promoters. We analyzed novel AR target genes with hsa-let-7c CAGE TCs in the promoter regions or within the genes. hsa-mir-125b-2 Of the AR target genes, 31% (82 genes) included androgen-regulated CAGE TCs (at least 20 t.p.m.) Scale 20 kb (Figure 6e). In the Oncomine database (Wilson and chr21: 16850000 Gigue` re, 2007), we identified 112 genes that were 0-24 h upregulated by androgen (Po1e-4) in PCa tissues 0-6 h

CAGE (Figure 6f). Among these androgen-upregulated genes, 6-24 h 48 of 82 genes (58%) are associated with adjacent acH3 CAGE TCs, whereas 64 of 176 (36%) did not exhibit ChIP AR C21orf34 CAGE TCs (Po0.001). C21orf34 This study revealed a global approach for mapping of hsa-mir-99a AR binding and AR-dependent promoters, which hsa-let-7c provides the systemic database regarding the AR- hsa-mir-125b-2 regulated gene network that essentially contributes to the development and progression of PCa. Notably, this 8 study successfully confirmed that known androgen- *** R1881-Tx regulated or PCa-specific genes are androgen-inducible, 0 h 6 *** as exemplified by ACACA (Lapointe et al., 2004), 24 h ABCC4 (Ho et al., 2008), TRIM36 (Balint et al., 2004) 48 h and amyloid b-precursor protein (Takayama et al., 4 *** 2009)(Table 2). On the basis of global mapping analysis, we identified novel androgen-upregulated genes, such ** 2 as FGFRL1, CAMKK2 and C16orf60, although their

Relative miRNA Level Relative miRNA functions remain to be studied in PCa (Table 2). 0 Notably, the application of pathway analysis using miR-125b let-7C miR-99a Database for Annotation, Visualization and Integrated ** P < 0.01 *** P < 0.005 Discovery Bioinformatics Resources 6.7 (Dennis et al., Figure 5 Androgen-dependent transcriptional regulation of 2003; Huang et al., 2009) further shows that metabolic miR-125b-2 microRNA locus on chromosome 21. (a) UCSC genome pathways, such as diabetes pathways, metabolism of browser view of the miR-125b-2 locus, including hsa-mir-99a, hsa-let-7c and lipoproteins, or amino sugar and nucleotide and hsa-mir-125b-2. (b) Androgen-dependent expression of miRNAs sugar metabolism, were enriched in our CAGE data in the miR-125b-2 locus. qRT–PCR was performed using TaqMan probes; the data represent relative miRNA levels normalized to the (Supplementary Table 2). level of small nuclear RNA U6.

Discussion lated genes (group IV) included genes such as TGFBR3 and FOXO1A (related to signal transduction and To understand the mechanism underlying AR regulation transcriptional regulation). Among the androgen-upre- of the progression of PCa, we identified its transcriptional gulated genes, the proportion of group I genes was network in the genome of human PCa LNCaP cells. In the significantly higher than that of group II genes. The previous studies, we identified direct AR target genes by proportion of group IV was also substantially higher using the ChIP-cloning technique (Takayama et al., 2008) than that of group III genes among the androgen- or tiling arrays for the Encyclopedia Of DNA Elements downregulated genes. The rates of the genes with the regions of the human genome (Takayama et al., 2007) expressions altered by androgen were significantly or chromosomes 21 and 22 (Takayama et al., 2009). higher in the clinical microarray data overlapped with A number of genes adjacent to ARBSs were found LNCaP data compared with the randomly selected to be upregulated or downregulated by androgen. In genes overlapped with the clinical data (P ¼ 3.5e-9 for this study, we used the high-throughput CAGE method upregulated genes and P ¼ 0.001 for downregulated for mapping androgen-regulated promoters in the genes). This clustering analysis of microarray data will human genome. To validate the present CAGE and indicate that the androgen-dependent transcriptional ChIP-chip results, we mapped the data on the genome

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 626 browser adjacent to prototypic AR-regulated genes. We performed both CAGE and microarray analyses Androgen treatment substantially increased the number for comparing the two transcriptome technologies. of CAGE tags found in the promoter regions of AR- Microarray analysis is useful for understanding global regulated genes such as PSA/KLK3, TMPRSS2 and expression profiles of protein-coding genes, whereas FKBP5. CAGE is useful for detecting alterations in expressions

24 h > 0 h

R1881-Tx ARBS 0 6 24 h positivity 79 55 11 7.2% 24 h > 6 h Upregulated genes Upregulated CAGE TCs at 6 h only (2-fold) ARBS 24 h > 0 h 6 h > 0 h 0624 positivity 21.6% 126 8 (P < 1e-8) 36

KLK4 15 ACSL3 KCNN2 12 LIFR PGM3 9 HOMER2 MCCC2 6 PSA TWIST1 mRNA Level

Fold Change of GADD45G 3 WIPI1 0 Upregulated genes at 24 h (>2-fold) 0 12 24 48 (h) R1881-Tx

Normal PCa Normal PCa

II IV

I

III

Upregulated genes Downregulated genes (>2-fold) (>2-fold) Normal < PCa Normal > PCa

300 + CAGE TCs + ARBSs 250 P < 0.001 200 150 335 82 176

+ ARBSs 100 Number of 50 Upregulated Genes 0 Androgen-upregulated Genes Total + TCs - TCs (>1.5-fold) Confirmed in PCa

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 627 of individual promoters for both noncoding and gene network, as SOX4, one of the developmental protein-coding genes. The similarity between the two transcription factors overexpressed in PCa (Liu et al., data sets is the expression alterations of protein-coding 2006), was found to contain a functional ARBS within genes with CAGE tags in their promoters or within the the gene. The calcium-dependent protein kinase genes. This tendency is well observed among the genes CAMKK2 (Table 2) seems to be an interesting novel with CAGE tags in their promoters with a high TC. In target of AR identified in this study. CAMKK2 can contrast, the correlation between CAGE and microarray affect cell growth by upregulating AMPK phosphoryla- is lower for genes with multiple promoter regions as tion (Yu et al., 2009), and it is also known to be shown by the CAGE study in myeloid leukemia THP-1 upregulated in PCa. cells (FANTOM Consortium, 2009). In this study, we determined that transcriptional Our combined analyses can clarify the direct role of regulation was also mediated by antisense promoters in AR in the transcriptional regulation of androgen target LNCaP cells. The antisense-directed TSSs were - genes, including genes that were not previously con- tively abundant in the 30-regions of RefSeq genes. This sidered to be regulated by androgen. Notably, we finding is consistent with those of previous studies that identified an intronic ARBS in the insulin-like growth reported that antisense transcripts are often transcribed factor-1 receptor gene, which indicates direct AR- from the 30-untranslated regions of genes. The relevance mediated transcriptional regulation of this known of antisense promoter-mediated transcriptional regula- androgen nongenomic target of androgen. The identifi- tion in carcinogenesis has been reported in HCT116 cation of a novel ARBS in the JAG1 gene, a canonical colorectal cancer cells, as the binding of b-Catenin to its ligand for Notch1, also suggests that AR through this target at the E2F4 30-untranslated region induces binding site has a critical role in the progression and an E2F4 antisense transcript, which downregulates metastasis of PCa. In this study, we also confirmed the E2F4 protein levels and suppresses the binding of involvement of transcription factors in the AR-mediated E2F4 to its specific target genes (Yochum et al.,

Table 2 Known and novel androgen-upregulated genes Chr Start End Gene TC location CAGE tag CAGE tag Fold change Fold change Function symbol (distance from count count 6 versus 0 h 24 versus 0 h TSS) at 0 h (t.p.m.) at 24 h (t.p.m.)

Known androgen targets 21 26464837 26465023 APP 47 656.7 909.4 1.4 2.8 Cell proliferation 13 94751591 94751758 ABCC4 À4 39.9 158.7 2.9 11.1 Transport 17 32790079 32790210 ACACA 20 82.9 148.4 2.6 3.6 Metabolism 18 53967444 53967515 NEDD4L 104686 3.1 32.4 1.8 2.9 E3 ligase (HECT) 2 168812181 168812305 STK39 117337 20.2 63.1 1.5 3.4 Kinase 2 223551207 223551314 ACSL3 22 7.8 145 1.5 4.4 Metabolism 8 134378640 134378679 NDRG1 3 0.5 27.3 0.8 4.6 Differentiation

Novel androgen targets 5 114533435 114533504 TRIM36 10638 8.3 42.7 2.1 2.6 E3 ligase (RING) 15 53369273 53369337 RAB27A À10 8.8 68.2 2.3 6 Ras GTPase 1 36359091 36359198 THRAP3 À12 124.4 175.7 1.8 2.5 Transcription 16 79597576 79597706 C16orf60 1 19.7 264.5 1 2.9 Unknown 12 120197208 120197268 CAMKK2 1599 17.1 102.4 3.1 7.4 Ca-dependent kinase 4 995386 995471 FGFRL1 À29 35.8 63.1 1 2.1 Cell adhesion

Abbreviations: Ca, calcium; CAGE, cap analysis of gene expression; Chr, chromosome; TC, tag count; TSS, transcription start site.

Figure 6 Identification of androgen-regulated genes exhibiting AR binding. (a) The number of CAGE TCs adjacent to ARBSs (o100 kb) that were significantly upregulated by androgen in LNCaP cells (Po1e-6.11). Top panel: Venn diagrams for the number of androgen-upregulated TCs at 24 h of treatment versus 0 h or 24 h versus 6 h of treatment. Bottom panel: Venn diagrams for the number of androgen-upregulated TCs at 24 h of treatment versus 0 h or 6 h of treatment versus 0 h. (b) Androgen-upregulated genes (4twofold) determined by mRNA microarray analysis. Note that the rate of ARBS positivity (o100 kb of the TSS) was 21.6% in the group of androgen-upregulated genes at 24 h of treatment, whereas this rate was only 7.2% in the group of androgen-upregulated genes at 6 h of treatment. (c) qRT–PCR analysis of androgen-upregulated genes in LNCaP cells. Data show fold change of mRNA levels in R1881 (10 nM)-treated cells versus vehicle-treated controls. (d) Expression of AR-regulated genes in normal prostate or cancer specimens. Microarray data for 508 genes selected by our experiments (258 androgen-upregulated and 250 androgen-downregulated genes) were retrieved from the Stanford University website. Note that the rate of genes predominant in cancer was higher in androgen- upregulated genes, whereas the rate of genes predominant in normal prostate was rather higher in androgen-downregulated genes. (e) Venn diagrams for the number of AR-upregulated genes containing androgen-activated CAGE TCs or ARBSs. CAGE TCs (420 t.p.m.) were located within RefSeq genes or their promoter regions in the sense direction. (f) The rate of androgen-upregulated genes confirmed in prostate cancers by the Oncomine database (Po1e-4) is higher in the presence of CAGE TCs compared with the absence of CAGE TCs.

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 628 2007). We also found novel pairs of S/AS promoters in Materials and methods individual RefSeq genes. Both reciprocal and concor- dant alterations in promoter transcription are known to Cell culture and reagents occur in PCa. Taken together, the abundant expression of Androgen-sensitive human PCa LNCaP cells (ATCC, Manassas, androgen-regulated antisense promoters in LNCaP cells VA, USA) were maintained in RPMI medium supplemented will indicate the physiological significance of antisense with 10% fetal bovine serum, 50 U/ml penicillin and 50 mg/ml streptomycin. Before androgen treatment, cells were cultured mechanisms in the AR-mediated gene network in PCa. in phenol red-free medium containing 5% charcoal-stripped This integrated study also identified short RNAs or fetal bovine serum for 48–72 h. Antibodies for AR, AcH3 and miRNAs that are regulated by AR. We observed that SRC1 and RNA Pol II were used for ChIP assay as described CAGE TCs and ARBSs accumulate in the vicinity of the previously (Takayama et al., 2007). miR-125b-2 cluster on chromosome 21. All miRNAs in the miR-125b-2 locus were upregulated by androgen. CAGE This result is consistent with a recent study in which let- CAGE libraries derived from total RNAs of LNCaP cells before 7c was found to be overexpressed in PCa tissues with (0 h) or after 6- or 24-h treatment with R1881 (10 nM) were high Gleason score (Leite et al., 2009). The global generated as previously described (Maeda et al., 2008). Briefly, mapping of AR-mediated gene regulation will further first-strand cDNAs, which were transcribed to the 50-end reveal novel short RNAs involved in the pathophysiol- of capped RNAs, were attached to CAGE ‘bar code’ tags ogy of PCa. Whether the adjacent ARBSs directly (AATAG for 0 h, ATTAT for 6 h and ATTGG for 24 h), and regulate the miRNA expression remains to be studied by digested into 20-nt tags by MmeI (New England Biolabs, Ipswich, MA, USA). The CAGE tags were concatenated, ligated to performing such as chromosome conformation capture sequencer-specific DNA adaptors and analyzed by 454 FLX experiments. Sequencing (Roche Diagnostics, Penzberg, Germany). The Hierarchical cluster analysis of prostate tissue positions of the CAGE tags on the human genome (NCBI samples from a published microarray database and a version 35) were determined by using Vmatch alignment tool comparative study of the Oncomine database revealed (by Professor Stefan Kurtz, University of Hamburg, Germany), that the androgen-dependent overexpression of AR and 1 929 416, 682 111 and 586 083 CAGE tags were determined target genes and the increased activity of target gene from 0-, 6- and 24-h libraries, respectively. CAGE tags are grouped promoters that is mediated by androgen are well-related intoTCsinwhichthemembertagsmaptothesamestrandofa to the signature of PCa. In contrast, a large cluster of chromosome and overlap by at least 1 bp. Fisher’s exact test was androgen-downregulated genes is associated with a performed to compare tag distributions between the 0-, 6-, and 24-h samples; 2 Â 2 contingency matrix constructed tag numbers in feature of normal prostate tissues. a TC against tag number in the other TCs by a sample pair. It is also notable that partner genes involved in PCa- We determined whether there were significant differences related chimeric transcripts were validated as direct AR (Po0.01) among tag distributions for each TC at 6 and 24 h targets in our study. We have previously shown that after androgen treatment (total number of TCs ¼ 13 110). Also FOXP1 is a that is also regulated by using the Bonferroni correction (Po0.01/n; where n is total ARBSs (Takayama et al., 2008). Like the well-known TCs, 381 554), we identified 1572 androgen-regulated TCs. For TMPRSS2–ETV1 transcript in PCa, the upregulation of calculating genomic locations of TCs relative to the closest FOXP1–ETV1 transcript was reported in PCa cells Refseq genes or Refseq on the antisense strand, we defined the (Hermans et al., 2008). Other androgen-regulated genes location of TC that has maximal frequency at the TSS regions. such as ACSL3 (Attard et al., 2008) and NDRG1 All data will be released from the website of the Genome Network Platform at National Institute of Genetics, Japan (Pflueger et al., 2009) were also identified as putative AR (http://genomenetwork.nig.ac.jp/download/dataset_e.html). targets genes in this study (Table 2). In PCa, ACSL3 is fused to ETV1, whereas NDRG1 is fused to ERG. Microarray Further characterization will discover new partner genes Total RNAs were extracted from LNCaP cells treated with R1881 for fusions from our database. (10 nM) for 6 and 24 h, using ISOGEN reagent (Nippon Gene, In summary, our integrated study is a powerful tool Tokyo, Japan). Affymetrix (Santa clara, CA, USA) U133Plus2.0 for elucidating the diversity of the AR-regulated expression microarrays were used. Data analysis was performed transcriptional network, which alters the expression of using the Affymetrix Microarray Suite software. For comparing various types of target genes throughout the genome, arrays, normalization was performed using data from all probe sets. especially those present in the intergenic and antisense regions of RefSeq genes. The strand-specific transcrip- ChIP-chip tome data combined with AR binding information will ChIP-chip analysis was performed in biological triplicates by provide a basic pipeline for the screening of novel AR using antibodies for AR and AcH3 as previously described target genes and expand the global information avail- (Takayama et al., 2007). Briefly, in vitro transcription was able on the genome-wide data of direct AR-mediated performed twice for amplification of ChIP materials. Ampli- transcriptional regulation; it will also offer new insights fied DNA was fragmented, labeled with biotin and hybridized into unknown nongenomic regulations of genes and to Human Tiling 1.0R microarrays (7 Chip Set) (Affymetrix). Data were analyzed as described previously. Using a stringent identify cancer-regulated biomarkers of unknown P-value cutoff 1e-5, 2800 AR ChIP-enriched regions were mechanisms. ‘Oncomining’ of the genome-wide land- identified as significant ARBSs. Using a stringent P-value scape database will identify new classes of molecular cutoff 1e-4, 25 954 AcH3 ChIP-enriched regions were identified targets that can be exploited for improving the diagnosis as significant AcH3 sites. For comparison study of our data and treatment of PCa. with previous AR binding data, we retrieved the ChIP-chip

Oncogene CAGE and ChIP-chip in prostate cancer cells K Takayama et al 629 (Wang et al., 2009) and ChIP-Seq (Yu et al., 2010) data and website (http://microarray-pubs.stanford.edu/prostateCA) mapped all the ARBS data to the human genome hg17 using and both androgen-upregulated and downregulated genes liftOver tool in the UCSC Genome Bioinformatics (http:// were clustered in a manner dependent on the gene expression genome.ucsc.edu/). features of normal or tumor predominancy, using a hierarch- iral clustering by Cluster3.0 software (developed by Eisen ChIP and quantitative PCR (qPCR) et al., 1998, extended by de Hoon et al., 2004), based on the ChIP was performed as previously described (Takayama et al., pairwise average-linkage method. 2007). Fold enrichments relative to immunoglobulin G or the input control were quantified by real-time PCR using SYBR green Pathway analysis PCR master mix (Applied Biosystems, Foster City, CA, USA) and Pathway analysis was performed by Database for Annotation, the ABI Prism 7000 system (Applied Biosystems) based on SYBR Visualization and Integrated Discovery Bioinformatics green E´fluorescence. Relative differences in the amounts of PCR Resources 6.7 (Dennis et al., 2003; Huang et al., 2009), using products among the treatment groups were evaluated by the REACTOME (http://www.reactome.org/) and Kyoto Ency- comparative cycle threshold (Ct) method, using glyceraldehyde-3- clopedia of Genes and Genomes (http://www.genome.jp/kegg/) phosphate dehydrogenase as an internal control. Primer sequences databases as annotation sources. for ARBSs are listed in the Supplementary Table 1.

Quantitative reverse transcription–PCR (qRT–PCR) Conflict of interest First-strand cDNA was synthesized using the Primescript RT reagent kit (TAKARA, Kyoto, Japan). Primer sequences are The authors declare no conflict of interest. listed in the Supplementary Table 1. qRT–PCR for miRNA was performed in triplicates by TaqMan miRNA RT–PCR system (Ambion, Austin, TX, USA), using RUN6B (Applied Acknowledgements Biosystems) as an internal control. This study was supported by the Genome Network Project, Hierarchical cluster analysis for androgen-regulated genes Cell Innovation Program and Support Project of Strategic in prostate samples Research Center in Private Universities from the MEXT, Unsupervised hierarchical clustering was performed for 508 grants from the Japan Society for the Promotion of Science, genes selected by our experiments (258 androgen-upregulated grants-in-aid from the MHLW and the Program for Promo- and 250 androgen-downregulated genes). cDNA microarray tion of Fundamental Studies in Health Sciences of the NIBIO. data for 62 primary tumors and 41 normal samples (Lapointe We are grateful to T Murata for helpful discussion and Hiromi et al., 2004) were retrieved from the Stanford University Sano and Kazumi Yamaguchi for their assistance.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene