Identification of novel androgen receptor target genes in prostate cancer
- Unnati Jariwala†1,
- Jennifer Prescott†2,
- Li Jia3,
- Artem Barski1,
- Steve Pregizer1,
- Jon P Cogan1,
- Armin Arasheben1,
- Wayne D Tilley5,
- Howard I Scher6,
- William L Gerald6,
- Grant Buchanan2, 3, 5,
- Gerhard A Coetzee†2, 3 and
- Baruch Frenkel†1, 4Email author
© Jariwala et al; licensee BioMed Central Ltd. 2007
Received: 13 March 2007
Accepted: 06 June 2007
Published: 06 June 2007
The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression.
Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1) – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation.
AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.
Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer-related mortality in men . Prostate development and carcinogenesis are highly androgen dependent [2, 3]. By regulating cell proliferation, differentiation and apoptosis the androgen receptor (AR) plays a pivotal role in PCa progression, as well as in normal prostate development [2–4]. AR-mediated PCa growth is initially hormone-dependent, and men failing surgical and radiation therapy are therefore subjected to androgen ablation therapy . Androgen ablation in these cases almost always leads to tumor regression, but this is inevitably followed by recurrence of PCa due to the development of castrate-resistant and often metastatic disease.
Although most recurrent PCa tumors are castrate-resistant, AR expression and function are maintained in advanced disease [6, 7] and the growth of ablation-resistant PCa cells remains AR dependent as exemplified by the following three lines of evidence. Disruption of the AR by a specific antibody or ribozyme inhibited proliferation in ablation-resistant PCa cells in the absence of androgens . Increased AR expression was necessary and sufficient to convert androgen-sensitive PCa to an ablation-resistant state . Finally, specific expression in mouse prostate epithelial cells of an AR transgene containing a gain-of-function mutation (with increased basal activity and response to coregulators), resulted in PCa development in 100% of the animals  proving that aberrant AR signaling was sufficient to cause PCa and that under certain conditions the AR acts as an oncogene.
As AR is a transcription factor, its oncogenic functions are likely mediated through specific target genes. Prostate specific antigen (PSA), the best studied AR target gene, is thought to contribute to PCa progression through its protease activity  and its ability to induce epithelial-mesenchymal transition and cell migration . Other AR target genes implicated in PCa progression are FGF8 , Cdk1 and Cdk2 , as well as PMEPA1  and TMPRSS2 . Interestingly, the AR response mechanism of TMPRSS2 drives oncogenic Ets family members in many castrate resistant tumors due to TMPRSS2:Ets chromosomal translocations [17, 18]. However, additional, yet unidentified target genes most likely contribute to the tumorigenic activity of the AR in PCa. The present study was undertaken to identify such genes based on their physical interaction with the AR. C4-2B human PCa cells, a model for castrate-resistant disease, were subjected to a procedure called Chromatin Immunoprecipitation (ChIP) Display (CD)  and 19 novel regions occupied by the AR were discovered. The expression patterns of genes within the AR-occupied loci, along with functions attributed to these genes, render some of them potential PCa therapeutic targets.
ChIP Display of AR targets in C4-2B cells: an example
AR targets identified in this study
Ava II – Ava II3
Position of CD Hit Relative to gene
30,152,547 – 30,152,728
Nearest gene is 626-kb away
178,433,288 – 178,433,456
241,348,804 – 241,348,990
intron 23/exon 24
53,169,093 – 53,169,401
6,644,411 – 6,644,619
72,483,118 – 72,483,317
72,922,165 – 72,922,474
143,094,298 – 143,094,518
Nearest gene is 197kb away 
24,584,349 – 24,584,579
126,072,189 – 126,072,473
1,017,234 – 1,017,529
62,532,814 – 62,532,977
134,102,859 – 134,103,167
Nearest gene is 315-kb away
86,510,091 – 86,510,360
Nearest gene is 959-kb away
3,446,846 – 3,447,080
exon 1/intron 1
TRPV3 [60, 61]
77,480,155 – 77,480,527
PYCR1 [36, 50]
SIRT7 [71, 72]
22,655,127 – 22,655,462
exon 4/intron 4
38,101,611 – 38,101,989
48,707,684 – 48,707,956
The example shown in Figure 1A entails the amplification and PAGE of two independent AR-ChIPs and two mock ChIPs using one of the 36 primer combinations – the 'AC' and the 'TG' primer (see Methods and Additional file 1). The arrowheads in Figure 1A point to bands more prominently amplified in the AR ChIPs as compared to the IgG ChIPs. These bands, representing a putative AR binding region, were excised, reamplified and further characterized by secondary restriction digests and agarose gel electrophoresis (Figure 1B). The major Hae III digestion product from each of the two ChIPs was sequenced and mapped to human chromosome 17q25.3, 1.5-kb upstream of the MAFG gene and 2.5-kb 3' of the PYCR1 gene (Figure 1C). The Ava II fragment displayed in the original PAGE (Figure 1A), depicted in Figure 1C as "hit", does not contain repetitive sequences and is located between two canonical Androgen Receptor Elements (AREs) (Figure 1C). A 2.4-kb CpG island is present adjacent to the hit (Figure 1C).
To validate AR occupancy at the region described above, we performed conventional ChIP assays with locus-specific primers (see Additional file 1). Four independent experiments with C4-2B cells showed that the PYCR1/MAFG locus was enriched in AR ChIPs as compared to paired IgG control ChIPs (Table 1, and see a representative result in Figure 1D).
ChIP Display discloses 19 novel AR binding sites in PCa cells
The CD procedure, exemplified above for the 'AC' and 'TG' primer pair, was performed using all 36 possible primer combinations , resulting in the identification of 19 novel AR-occupied regions in C4-2B cells (Table 1). AR occupancy at the novel AR binding regions was confirmed in independent conventional ChIP assays of C4-2B cells (Table 1). Whereas only four of the 19 AR binding regions were up to 10-kb 5' of the nearest gene (indicated by bolded and italicized text in Table 1), many of the binding regions were either within the body of annotated genes (8 of the 19 regions) or up to 4-kb 3' of the nearest gene (3 of the 19 regions), indicating that AR-bound regions are not preferentially found within so-called 5'-flanking gene regulatory sequences. Four of the 19 CD hits were mapped to regions more than 197-kb away from any annotated gene (Table 1).
An important enigma in prostate cancer research is the molecular nature of the transition from androgen-dependent to castrate-resistant disease. In this context, the C4-2B cell line serves as a model of the latter, whereas its parent cell line, LNCaP, serves as a model of the former . We speculated that many of the AR-occupied regions in C4-2B cells could become targets for this transcription factor only during the transition from androgen dependence to castrate-resistance and would therefore not be occupied by the AR in LNCaP cells. However, results of ChIP analysis in LNCaP cells were inconsistent with this notion, as 16 of 17 regions that we tested, which were occupied by the AR in C4-2B cells, were also occupied in LNCaP cells at least in one conventional ChIP assay (Table 1). Be that as it may, several of the AR-occupied regions are located near genes that have been linked to prostate or other cancers (see Discussion below and references in Table 1).
AR occupied regions are associated with DHT-stimulated and DHT-repressed genes in C4-2B cells
AR-dependent, DHT-independent regulation of OAT and MRFAP1
As controls for the AR siRNA experiments we also measured expression of AR itself and the DHT-stimulated genes PSA , PRKCD, PYCR1 and MRFAP1 (Figure 2C, F, J). As expected, the AR knockdown (Figure 3B) was associated with loss of DHT-stimulation (Figure 3C, D, and 3E). Interestingly, however, one of these controls, MRFAP1, displayed an unexpected phenotype. In addition to the DHT-stimulation, it was reproducibly stimulated in cells treated with AR siRNA (Figure 3F). Taken together, our data suggest that unliganded AR supports basal OAT expression (Figure 3A) without further stimulation by DHT (Figure 2L), while basal MRFAP1 expression is suppressed by unliganded AR (Figure 3F), yet stimulated by DHT (Figure 2J).
Differential regulation of genes near AR-occupied regions in LNCaP versus C4-2B cells
Although most of the regions occupied by the AR in C4-2B cells (a model of castrate-resistant PCa) were also occupied in LNCaP cells (a model of androgen-dependent PCa) (Table 1), we suspected that the functional consequences of AR occupancy at these loci might differ between the two cell lines. We therefore complemented the DHT time course studies in C4-2B cells (Figure 2, solid lines) with parallel expression analysis of the same genes in LNCaP cells under a similar experimental protocol (Figure 2, dashed lines). Both similarities and differences between the two cell lines were observed. The three genes most strongly stimulated by DHT in C4-2B cells – DDT, CRELD2 and PRKCD – were also stimulated in LNCaP cells (Figure 2A, B, C), although the stimulation of DDT and PRKCD was more modest in LNCaP cells. Genes that were more moderately stimulated by DHT in C4-2B cells were slightly (PYCR1, Figure 2F) or not at all stimulated in LNCaP cells (GSTT2, AP2A2, ACBD6, and SIRT7; Figure 2, panels D, G, H, I). Repressed genes displayed a mirror image. The four genes most strongly repressed in C4-2B cells – MUC6, CHRM1, KIAA1217, and WBSCR28 – were also repressed in LNCaP cells, but repression generally occurred faster in the C4-2B cells (Figure 2ZC, ZD, ZE, ZF). Subtle responses to DHT were less consistent between C4-2B and LNCaP cells, although three genes, CARKL, MAN2B2, and LHX4, displayed remarkably similar expression patterns (Figure 2, panels M, N, O).
Clinical relevance of novel AR target genes
AR occupancy is not biased towards 5' promoter-proximal regions
The classical view of gene regulation places 5'-flanking sequences at the center stage. Consistent with this view, functional AREs have been mapped within 0.5-kb upstream of the AR-responsive genes probasin, KLK2 and KLK3 (PSA) [26–28]. While four of the 19 AR-occupied regions disclosed in our study were located within 10-kb upstream of annotated transcription start sites, many more were found within gene bodies (8/19) or within the 4-kb sequences downstream from the 3' ends of annotated genes. Our findings are consistent with several recent genome-wide location analyses of other transcription factors. For example, only 4% of estrogen receptor (ER) binding sites were mapped to 1-kb promoter-proximal regions by ChIP-chip analysis . Similarly, genome-wide location analysis indicates that p53 has no preference for binding to 5' promoter-proximal regions . Thus, accumulating evidence suggest that promoter-proximal regions constitute only a small fraction of mammalian gene regulatory sequences.
Many of the AR-occupied regions identified in the present study, which cannot be designated classical 5' promoter regions, were still close to annotated genes that they could potentially regulate. There is no consensus as to how far a transcription factor-binding region should be in order to be considered a putative cis-acting regulatory domain for a given gene. Values 1-kb, and up to 100-kb from the transcription start site have been used by various investigators [29, 30], but experimental evidence in support of any value is scarce. Systematic analyses of transcription factor binding regions across the genome have become feasible only recently. Such studies, including the present one, illustrate the need for mutagenesis of transcription factors binding regions that are distant from annotated genes in order to identify functionally relevant regions. In particular, it would be interesting to decipher the role of binding regions located hundreds of kbs away from the nearest annotated gene. Such regions may still regulate distant annotated genes on the same  or even other chromosomes , or they may regulate nearby unannotated transcripts .
AR location analysis discloses ligand-independent, AR-dependent gene regulation
Comprehensive gene expression analysis is frequently employed for the discovery of target genes for transcription factors, including the AR [24, 34–36]. Such expression analyses cannot differentiate between direct and indirect targets and they do not provide information on the location of regulatory elements. Another, frequently under-appreciated limitation of expression studies is that they only disclose target genes that respond to the transcription factor of interest under the specific experimental conditions utilized by the investigator. In contrast, ChIP Display and other approaches for location analysis (see below) rely on physical interaction, not gene expression. These approaches allow the discovery of target genes that expression studies would potentially miss. For example, in the present study, we discovered OAT as an AR-regulated gene, although it did not respond to DHT. AR-target genes could also be missed in expression-based studies because of the limited sensitivity and specificity of microarray hybridization as compared to RT-qPCR. Experimental approaches for location analysis are constantly improving and include many that are far more comprehensive than ChIP Display, for example ChIP-chip [37–39], SABE , STAGE , ChIP-PET , GMAT , SACO  and DamID . However, the present study demonstrates that important information can be obtained with ChIP Display, a relatively inexpensive sampling method that can be performed in any molecular biology laboratory. Of course, each of the expression and the location approaches for target identification should ideally be complemented appropriately. In the present study, we showed that many (but not all) of the genes near AR-occupied regions are DHT-responsive. For OAT, which was disclosed here by ChIP Display and could not have been disclosed by comprehensive analysis of gene expression in response to androgen treatment, we used siRNA knockdown to demonstrate the ligand-independent regulation by the AR. Furthermore, of the genes near AR-occupied regions that responded to neither DHT nor AR siRNA in our study, some may be AR-regulated under specific, possibly transient physiological or pathological conditions not modeled by the experimental systems we employed. This is particularly important in the context of castrate-resistant PCa, where AR activation can occur through various signaling pathways, including Her2, AKT, and MAPK .
Differential basal gene expression in C4-2B versus LNCaP cells
The AR plays critical roles during all stages of PCa progression [5, 9, 45]. It is not clear, however, whether AR regulates different sets of genes before and after ablation therapy. In our study, AR occupancy at most of the regions disclosed by CD was similar in LNCaP and C4-2B cells, models of early and late stage PCa, respectively. Our data is therefore consistent with the idea that the AR continues to regulate the same genes before and after ablation therapy, but that the nature of this regulation alters during disease progression. For many genes near AR-occupied regions, ligand-bound AR had the same qualitative effects in the two cell lines, except they were stronger in the C4-2B as compared to the LNCaP model (e.g., DDT, PRKCD, GSTT2, PYCR1; Figure 2). Some other genes near AR-occupied regions were found to express at higher basal levels in C4-2B as compared to LNCaP cells (e.g., OAT, GSTT2, TRPV3; Figure 4). The higher basal expression of these genes could be a direct result of ligand-independent activation by the AR due to, for example, cofactor expression [46, 47] and/or chromatin reorganization . Accumulation of mutations during the derivation of C4-2B from LNCaP cells could also contribute to differential basal and DHT-responsive expression, although the two cell lines are mostly isogenic as indicated by our microsatellite analysis (see Additional file 2).
Evidence from PCa biopsies for in vivo AR-mediated regulation of genes disclosed by ChIP Display
In the present study, we included analysis of the ChIP Display-disclosed genes for their expression in PCa biopsies. This analysis provided evidence that AR regulates in vivo several of the ChIP Display-disclosed genes. This notion, however, remains tentative because the clinical material used for the microarray expression analysis is no longer available for confirmation by RT-PCR. To increase our confidence in the microarray data, we only considered results from probesets that are considered highly reliable (Affymetrix' grade A annotation). Results from the PCa biopsies were consistent with those from the in vitro gene expression analysis for the positively regulated genes PYCR1, DDT, PRKCD, and CRELD2. However, ALG12 and TRPV1, which appear to be stimulated by the AR in vivo, were not responsive to either DHT or AR siRNA in vitro. The in vivo and in vitro analyses were less well correlated for negatively regulated genes. Only one of six probesets interrogating KIAA1217 expression indicated repression in vivo, although this gene was strongly inhibited in vitro. CHRM1 was also strongly repressed by androgens in vitro, yet it was found to be stimulated in vivo. OAT, which appears to be downregulated by the AR in vivo, was stimulated in vitro in a ligand independent manner. It remains to be seen whether these inconsistencies result from differential requirements for AR-mediated gene stimulation/repression in vitro and in vivo, the presence of multiple splicing isoforms, or simply erroneous microarray expression scores. Be that as it may, further investigation of genes highlighted by our study, and especially genes for which results from the PCa biopsies are apparently inconsistent with the in vitro results, will have to start with validation of the regulation of such genes in PCa in vivo.
Novel AR target genes: potential mechanisms contributing to PCa progression
PSA (KLK3) remains the most well studied AR target gene in the PCa literature to date. Since its approval in 1986, serum PSA is routinely used to aid the early diagnosis and prognosis of PCa in men. However, AR-driven PSA expression alone does not fully explain the role of AR in PCa development and progression. Although additional AR target genes have been recently discovered, e.g., FKBP5  and TMPRSS2 , most remain elusive. Some of the AR target genes discovered in the present study, and more to be discovered in the future, may open new research avenues and help develop novel therapeutic approaches to manage PCa.
Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate (P5C) to proline. Stimulation of PYCR1 by the AR could contribute to PCa progression because P5C is pro-apoptotic  and proline is anti-apoptotic . Indeed, a role for PYCR1 in PCa was suggested by a 4-fold increased expression in human prostate tumors compared to adjacent normal tissue . In the present study we demonstrate AR occupancy at the PYCR1 locus in living PCa cells, the functionality of which is suggested by DHT-mediated stimulation of gene expression. Consistent with these in vitro data, we also demonstrate decreased PYCR1 expression in PCa biopsies from men undergoing androgen ablation therapy as compared to untreated controls. The highest PYCR1 expression in our PCa samples was found in biopsies from metastatic tumors, possibly as a result of atypical AR activation. Of the AR targets discovered in the present study, PYCR1 is a strong candidate for mediating the oncogenic action of AR signaling in PCa.
Interestingly, another AR target gene discovered in this study also participates in proline metabolism. Ornithine aminotransferase (OAT) converts ornithine to glutamate γ-semialdehyde, which spontaneously cyclizes to form pyrroline-5-carboxylate (P5C), a proline precursor and the substrate for PYCR1. The functional evidence for AR-mediated regulation of OAT is weaker than that for PYCR1. While OAT mRNA was not significantly altered in response to DHT, it was repressed after siRNA-mediated knockdown of the AR, and also displayed a 3.3-fold higher basal expression in C4-2B as compared to LNCaP cells. Interestingly, OAT's expression pattern in our tumor samples does not suggest AR-mediated stimulation, but rather repression, possibly reflecting interactions of AR signaling with input from other cell types or components of the extracellular matrix, which only occur in vivo.
In this study, we mapped AR occupancy to a region 0.8-kb upstream of the gene encoding Protein Kinase C delta (PRKCD), which has received much attention in the PCa literature. PRKCD mRNA levels were higher in DHT-treated as compared to untreated PCa cell cultures and were reduced in PCa biopsies from patients undergoing androgen ablation therapy as compared to those from untreated patients. The observed high PRKCD mRNA levels in PCa metastases, which may reflect ligand-independent AR activation, possibly play a role in late stage disease because PRKCD is implicated in growth, migration and invasion of cancer cells, including PCa [51, 52]. PRKCD has also been implicated in the control of cell survival, although most studies suggest it is in fact pro-apoptotic [53–55]. Future studies will have to address how PRKCD's pro-apoptotic activity is overcome in advanced PCa cells.
CRELD2 and DDT
The gene expression data form both the cell culture models and the clinical tumor samples suggest androgen-mediated stimulation of CRELD2 and DDT. Although a role for CRELD2 in carcinogenesis remains to be investigated, this Cysteine-rich with EGF-like Domains 2 (CRELD2) protein has been shown to interact with neuronal acetylcholine receptors . Likewise, no role in tumor progression has been assigned yet to DDT, a protein with homology to macrophage migration inhibitory factor (MIF) .
Among the few genes near AR-occupied regions that were repressed by DHT was MUC6. Mucin 6 is a secreted glycoprotein that forms a protective gel layer around the producing cell . Other mucins are aberrantly expressed in cancer  and MUC2 was ascribed a tumor suppression function . Conceivably, AR-mediated MUC6 repression can contribute to PCa progression. Notably, however, MUC6 mRNA was neither increased in the androgen-ablated compared to the untreated tumors, nor was it absent in the metastatic samples. Although our in vivo data does not support a role for MUC6 in PCa progression, MUC6 could still play a transient role during a short period of time not captured by our clinical samples.
TRPV3 and GSTT2
Like the androgen-repressed MUC6, evidence for roles for the androgen-stimulated TRPV3 and GSTT2 genes in PCa progression is suggested only from our in vitro data. These two genes are not only androgen stimulated but are also expressed in C4-2B cells more strongly than in LNCaP cells. TRPV3 is a member of the transient receptor potential (TRP) family of thermosensory ion channel genes. Another member of this family, TRPV6, potentiates calcium-dependent cell proliferation , and its expression has been linked to human PCa progression . Glutathione S-transferase theta 2 (GSTT2) belongs to a family of detoxification enzymes, overexpression of which is thought to provide cells with protection against oxidative stress and various drugs .
AR occupancy at PCa-linked loci
Two of the AR-occupied regions disclosed by ChIP Display were near loci previously linked to PCa: (i) the hereditary prostate cancer 1 (HPC1) locus, which has been mapped to 1q24-25 ; and (ii) the 8q24 locus, recently linked to PCa through admixture mapping in African American men . Although fine mapping of specific genetic elements has not been achieved yet for either of these loci, their contribution to PCa progression in a mechanistic sense may be related to the observed AR occupancy.
We have identified 19 novel AR-occupied regions in PCa cells, many of which are associated with genes that are regulated by the AR in either a ligand-dependent or ligand-independent manner. Furthermore, some of the newly identified AR target genes are differentially regulated in cell models for, and/or biopsies from, different stages of PCa progression. These genes provide opportunities for future research to better understand the role of the AR in PCa and eventually improve patient care, especially in the context of castrate-resistant disease.
Cell culture and materials
Human C4-2B cells, a model for castrate-resistant PCa, were obtained from ViroMed Laboratories Inc. (Minnetonka, MN) and their parental, androgen-dependent LNCaP cells, were obtained from ATCC. (Manassas, VA). The close relationship between the two cell lines was confirmed by microsatellite analysis at ten loci (see Additional file 2). Both C4-2B and LNCaP cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; Invitrogen). Dihydrotestosterone (DHT; Sigma Chemical Co., St. Louis, MI) was administered in phenol red-free RPMI-1640 supplemented with 5% charcoal-stripped FBS (CSS; Gemini, West Sacremento, CA). An N-terminal AR antibody (N20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
ChIP was carried out essentially as described previously . C4-2B and LNCaP cells were cultured for 3 days in phenol red-free RPMI-1640 supplemented with 5% CSS, then treated for 4-hr with 10 nM DHT, followed by cross-linking with 1% formaldehyde for 10 minutes. After sonication, chromatin was immunoprecipitated overnight at 4°C with either anti-AR antibodies or isotype-matched IgG. AR occupancy was assessed by PCR with locus-specific primers (see Additional file 1) using material from several independent ChIPs. Serial dilutions of genomic DNA were amplified to ensure that PCR was performed within a dynamic range.
ChIP Display (CD)
We have recently described the CD procedure in detail . Briefly, DNA from AR ChIP and IgG control ChIP was dephosphorylated using shrimp alkaline phosphatase (NEB, Ipswich, MA) and digested with Ava II (NEB). The Ava II fragments were subjected to ligation-mediated PCR using each of 36 combinations of eight primers. Each primer had A or T at the +3 position of the Ava II site, and A,T,G, or C at the so-called +6 position, immediately internal to the Ava II site . In the present paper, primers are named by the nucleotides occupying these two positions. For example, the PCR primer 'AC' is the one with A at the +3 and C at the +6 position. Each PCR reaction in the present study was performed in duplicate, with a 1°C difference in the annealing temperature (see Fig. 1A). The amplified material form 2–3 independent AR ChIPs and 2–3 controls was resolved by polyacrylamide gel electrophoresis (PAGE), and bands enriched in the AR ChIPs were excised and reamplified. They were then subjected to secondary digestion with Hae III, Hinf I and Msp I (NEB), and sub-fragments were isolated by agarose gel electrophoresis and sequenced. The sequences were mapped to the human genome using the SSAHA program on ENSEMBL .
C4-2B cells (1.5 × 105 cells/well in 6 well plates) were cultured for two days in phenol red-free RPMI-1640 supplemented with 5% CSS. The cells were then transfected using OligofectAMINE (Invitrogen) with 100 nM of either AR-specific or non-specific siRNA (see Additional file 1) as previously described . After two days, cells were treated for 16 hours with 10 nM DHT or ethanol vehicle prior to analysis of gene expression.
Cells were grown in six-well plates and RNA was extracted using Biorad's total RNA mini kit according to manufacturer's protocols (Biorad, Hercules, CA). RNA quality was assessed spectrophotometrically and by agarose gel electrophoresis. High quality RNA (200–1000 ng) was reverse-transcribed with random hexamers using the Taqman reverse transcription reagents kit (Applied Biosystems, Foster City, CA). cDNAs of interest were amplified using gene specific-primers (see Additional file 1) and the iQSYBR Green supermix (Biorad). Amplification was performed in triplicate in a 96-well format and monitored in real time using the Opticon 2 DNA Engine (Biorad). Negative controls without RNA, without RT and without cDNA were always included to rule out contamination. Expression levels were determined using standard curves for each gene and corrected for 18S ribosomal RNA levels.
Gene expression analysis in clinical PCa specimens
Expression of genes disclosed by ChIP Display was analyzed in prostate cancer samples using microarray data collected as part of our previous studies . Briefly, clinical samples were from 40 primary prostate cancers obtained during radical prostatectomy and 7 AR-positive metastatic prostate cancer lesions. Twenty-three of the primary tumors were from patients receiving no therapy before surgery and the remaining 17 were from patients after 3 months of goserelin plus flutamide androgen-ablation therapy. All tissues were obtained during routine clinical management at the Memorial Sloan-Kettering Cancer Center, New York, NY, under protocols approved by the Institutional Review Board. RNA was extracted from manually-microdissected tissue consisting of 60–80% prostate cancer cell nuclei, and analyzed as previously described  using the Affymetrix U95 A-E array set. The results are displayed as a heat-map generated using 'Heatmap Builder' (Stanford University), with data routed to 50 equal gates for each probeset (row) using a linear grey scale gradient from white (lowest value) to black (highest value). Data for each gene was generated using only those high-fidelity probesets with a grade A annotation as defined by Affymetrix.
We employed the unpaired t-test using GraphPad Instat version 3.0 for PC to compare mRNA levels for each gene between DHT-treated and vehicle-treated cells at each time point, and between the basal levels in LNCaP versus C4-2B cells. Unless otherwise stated, differences referred to in the text were assigned a p value of less than 0.05. The individual p values assigned to each of the comparisons are provided in additional files 3 and 4.
polyacrylamide gel electrophoresis
prostate specific antigen 1
charchoal stripped serum
fetal bovine serum
androgen responsive element
real time quantitative polymerase chain reaction
serial analysis of binding elements
sequence tag analysis of genomic enrichment
chromatin immunoprecipitation paired-end ditag sequencing
genome mapping technique
serial analysis of chromatin occupancy
tethered Dam methyltransferase identification
cysteine-rich with EGF-like Domains 2
protein kinase C delta
glutathione S-transferase theta 2
transient receptor potential (TRP) subfamily V member 3
pyrroline-5-carboxylate reductase 1
adaptor-related protein complex 2 alpha 2 subunit
Acyl-Coenzyme A binding domain containing 6
silent mating type information regulation 2 (sirtuin 7)
mof4 family associated protein 1
v-maf musculoaponeurotic fibrosarcoma oncogene homolog G
ornithine amino transferase
carbohydrate kinase like
mannosidase 2 alpha B2
LIM homeobox 4
alpha 1,6 mannosyltransferase
centrosomal protein 350 kDa
bromodomain adjacent to zinc finger domain 1B
kinesin family member 1A
phospholysine phosphohistidine inorganic pyrophosphate phosphatase
transient receptor potential (TRP) subfamily V member 1
williams beuren syndrome chromosome region 27
solute carrier family 22 (organic anion transporter)
frizzled homolog 9
mitogen-activated protein kinase kinase kinase 7 interacting protein 1
cholinergic receptor, muscarinic 1
williams beuren syndrome chromosome region 28.
This work was supported by the following grants: W81XWH-05-1-0025 from the Department of Defense (to BF), CA109147 (to GAC) and DK071122 (to BF) from the National Institutes of Health, 299048 & 453662 from the National Health and Medical Research Council of Australia (to WDT, ID#453662) P50 CA92629 and MSKCC SPORE in prostate cancer from the National Institutes of Health, a grant from PepsiCo Foundation (to HIS), grants from the Prostate Cancer Foundation (to HIS and GAC). GB is a recipient of a National Health and Medical Research Council of Australia CJ Martin Biomedical Fellowship. SP was partially supported by NIH training grant T 32 GM067587. BF holds the J. Harold and Edna L. LaBriola Chair in Genetic Orthopaedic Research at the University of Southern California. The experiments were conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 (RR10600-01, CA62528-01, RR14514-01) from the NIH/NCRR.
Dr. David Van Den Berg (Norris Comprehensive Cancer Centre, Genomics Core Facility, University of Southern California, Los Angeles, CA, USA) provided technical support for genotype analysis.
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