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Biomedicum Helsinki, Institute of Biomedicine (Z.K., O.A.J., J.J.P.), and Department of Clinical Chemistry (O.A.J.), University of Helsinki and University of Helsinki Central Hospital, FI-00014 Helsinki; and Department of Medical Biochemistry (J.J.P.), University of Kuopio, FI-70211 Kuopio, Finland
Address all correspondence and requests for reprints to: Jorma J. Palvimo, Ph.D., Department of Medical Biochemistry, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland. E-mail: jorma.palvimo{at}helsinki.fi or jorma.palvimo{at}uku.fi.
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There are two distinct classes of activities that regulate the accessibility of promoters to transcription and DNA replication machinery, both of which contribute to the generation of a dynamic chromatin structure (10). The first class includes ATP-dependent chromatin remodeling complexes, such as SWI/SNF, ISWI, NURD, WINAC, that are thought to reorganize chromatin structure through DNA sliding or conformational changes in the nucleosomes to expose DNA (11, 12, 13). The second class includes enzymes that catalyze posttranslational modifications in histones (14). Dynamic changes in multiple posttranslational modifications of the N-terminal tails of core histones, i.e. the histone code, can control chromatin packaging and create binding sites for chromatin-associated proteins (15, 16). In their unmodified state, the N-terminal tails of core histones are positively charged and interact with DNA and core histone regions on the same or neighboring nucleosomes. The best characterized histone modification is acetylation that is a dynamic process regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Acetylation has been linked to transcriptionally active genes, with the rate of gene transcription correlating positively with the degree of histone H3 and H4 acetylation (14, 17). The importance of covalent histone modifications in nuclear receptor signaling is shown by the fact that many holo-receptor-interacting proteins, such as the steroid receptor p160 coactivator family members steroid receptor coactivator-1, glucocorticoid receptor-interacting protein 1 (GRIP1)/transcription intermediary factor 2/NCoA-2 and activator of thyroid and retinoic acid receptor/amplified in breast cancer 1/retinoic acid receptor coactivator 3/p300/CBP (cAMP response element binding protein-binding protein)-interacting protein/thyroid receptor coactivator molecule 1, the cointegrator CBP/p300 and the coactivator PCAF, possess or recruit HAT activity (6, 7, 8, 9). Likewise, nuclear receptor corepressor proteins NCoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoic and thyroid hormone receptors) that interact with antagonist-occupied receptors form complexes with HDAC (18).
Unlike histone acetylation, increased histone lysine methylation that may occur as a mono-, di-, or tri-modification has been linked to both transcription activation and repression (19, 20, 21). Methylation, especially trimethylation of H3 at K4 (H3-K4), is generally associated with transcriptional activation in yeast, whereas methylation of H3 at K9 (H3-K9) tracks with repression (21, 22, 23). In addition to lysine residues, histones may be methylated at arginines. Interestingly, two arginine-specific histone methyltransferases, protein arginine methyltransferase 1 (PRMT1) and coactivator-associated arginine methyltransferase 1 (CARM1), function as nuclear receptor coactivators (24, 25). PRMT1 catalyzes methylation of H4 at R3, whereas CARM1 methylates H3 primarily at R17 (24, 26, 27). Methylation of H4-R3 by PRMT1 may facilitate subsequent acetylation by p300, providing a possible molecular explanation for the coactivator activity of PRMT1 (24, 28). Lysine and arginine methylations appear to cooperate with other histone modifications in the regulation of gene transcription (29, 30). In contrast to acetylation, methylation does not alter the charge of a histone. The influence of histone methylation on chromatin structure has remained elusive, and very little is known about the role of lysine methylation in connection with nuclear receptor signaling. The methyl groups may block protein binding or create new docking sites for binding of a protein in a fashion similar to that of acetylation, which generates binding sites for bromodomain proteins.
Chromatin immunoprecipitation (ChIP) is a powerful technique to follow covalent modifications of histones within gene regulatory regions in vivo (31). In this work, we have examined the kinetics of various histone H3 tail modifications associated with the promoter and enhancer of prostate-specific antigen (PSA) and kallikrein 2 (KLK2) genes during androgen induction in human prostate cancer cells. We have also compared AR transcription complex assembly onto these regulatory regions in cells exposed to pure or partial antiandrogens by utilizing quantitative ChIP assays.
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RESULTS |
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We first compared loading of AR and recruitment of Pol II between the promoter and the enhancer regions of the two genes. As shown in Figs. 1
and 2, the dynamics of holo-AR loading and that of Pol II recruitment onto the PSA promoter and enhancer were essentially indistinguishable from those occurring on the analogous regions of the KLK2 gene. In agreement with our previous results (36), initial loading of holo-AR onto the promoters occurred in a cyclical fashion. After the first wave of promoter occupancy peaking at approximately 1 h, AR binding reached its maximum at approximately 3 h after androgen exposure, and the occupancy remained at this level throughout the time course (Fig. 1, A and C). By contrast, the receptor’s residence time was more transient on the enhancer; after the first peak of holo-AR loading at approximately 30 min, the second, stronger wave peaked at approximately 2 h, after which the receptor levels declined rapidly (Fig. 1, B and D). Quantitatively, the maximal AR occupancy on the enhancer was approximately 20-fold higher than that on the promoter, and enhancer AR level exceeded that on the promoter throughout the time course. The dynamics of Pol II recruitment onto the promoters generally mirrored that of holo-AR for both the PSA and the KLK2 gene (c.f. Figs. 1 and 2). Some Pol II was recruited onto the enhancer, but in quantitative terms, its amount on the enhancer represented only 5–10% of that on the promoter. Likewise, the basal Pol II level (that before androgen exposure) associated with the enhancer was one twentieth of that on the promoter (Fig. 2). The concentration of T used (100 nM) was saturating, in that it elicited maximal AR loading and Pol II recruitment onto the PSA promoter and enhancer at 2 h as well as maximal PSA mRNA accumulation in LNCaP cells at 24 h. 5
-Dihydrotestosterone (DHT) brought about indistinguishable responses at 100 nM, but lower concentrations of DHT (1 and 10 nM) were 1.5- to 2-fold more active than the corresponding T concentrations (data not shown). The quantitative differences in Pol II recruitment and AR loading between the PSA promoter and enhancer are not a feature peculiar to LNCaP cells because similar results were seen in PC-3 cells in which transcription of the endogenous PSA gene was activated by ectopically expressed AR (data not shown).
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, A and B). In agreement with the pure antagonistic nature of CDX, this antiandrogen elicited no or marginal recruitment of Pol II, GRIP1, and p300 onto the promoter or enhancer of the PSA gene (Figs. 3, B and C, and 4). Instead, AR-CDX complex recruited corepressors NCoR and SMRT onto the promoter (Fig. 5). Although some NCoR was detected on the enhancer, the relative amount of NCoR tethered by the AR-CDX complex onto the PSA promoter exceeded that on the enhancer by approximately 40-fold.
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). Likewise, RU486 exhibited clear mixed agonist/antagonist activity in LNCaP cells, in that, besides stimulating loading of AR and recruitment of Pol II and p300, the AR-RU486 complex recruited NCoR onto the PSA promoter (Figs. 3–5). The relative amounts of coactivators and corepressors recruited by the RU486-bound AR differed, however, from those recruited by the AR-CPA complexes. In all cases, the levels of coregulators on the promoter exceeded those on the enhancer.
Acetylation and Phosphorylation of H3 Tails on the PSA Promoter and Enhancer
The tail of H3 is the longest of the core histone tails, and it plays a fundamental role in the compaction of chromatin (38). Modification of the H3 tail by acetylation and phosphorylation weakens its chromatin packaging ability. Moreover, the H3 tail can concomitantly be acetylated at K9 and phosphorylated at S10 (phosphoacetylated) (39, 40). In addition to H3 acetylation at K9 and K14 (H3-K9/K14), we monitored by ChIP assays the level of H3-S10 phosphorylation and H3-S10/K9 phosphoacetylation on the PSA promoter and enhancer. The levels of acetylated, phosphorylated, and phosphoacetylated H3 on the promoter and enhancer increased rapidly in LNCaP cells after inclusion of T in culture medium (Fig. 6). However, there were clear differences in the dynamics of different H3 modifications, especially between the promoter and the enhancer. In general, these modifications peaked and declined more rapidly on the enhancer than the promoter, with H3-K9/K14 acetylation turning over faster than the other modifications. The extent of H3-S10/K9 phosphoacetylation and H3-S10 phosphorylation, but not that of H3-K9/K14 acetylation, saturated to different levels on the promoter and the enhancer. Treatment of LNCaP cells with CDX resulted in a slight decrease in H3-K9/K14 acetylation and H3-S10 phosphorylation on the PSA promoter compared with vehicle-treated control cells (Fig. 6, A and E, and data not shown). In contrast to androgen-induced changes in H3 modifications associated with regulatory regions of the PSA gene, no alterations were detected in the total amounts of these modifications, as judged by immunoblotting with the modification-specific antibodies of histones extracted from LNCaP cell nuclei (data not shown).
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, increased H3-R17 di-methylation and recruitment of CARM1 occurred concomitantly on the PSA promoter and in conjunction with holo-AR loading. In quantitative terms, CARM1 was recruited onto the PSA enhancer to levels similar to those on the promoter; however, only a marginal change in H3-R17 di-methylation occurred on the enhancer, suggesting that H3-R17 may not be the primary target for CARM1’s methyltransferase activity on the enhancer.
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, A and B). Comparable modification was observed on the analogous regulatory regions of the KLK2 gene (Fig. 8, E and F). Similar to the pattern of acetylated H3 induced by T, the level of H3-K4 tri-methylation associated with the promoter increased until 4 h and persisted at this level for another 4 h, whereas on the enhancer, the modification peaked transiently at 2 h after T exposure. Interestingly, the basal level of H3-K4 tri-methylation linked to PSA and KLK2 promoters was much higher than that of the enhancers. The appearance of H3-K4 di-methylation on the PSA regulatory regions after T exposure suggests that it precedes tri-methylation (Fig. 8 and data not shown). The induction of H3-K4 tri-methylation was specific for androgen, as the antiandrogen CDX failed to elicit this modification, and if anything, it decreased the level of H3-K4 tri-methylation below that in vehicle-treated cells (30-, 60-, and 120-min time points; Fig. 8A). Androgen treatment failed to alter the level of di- or the tri-methylated H3-K9 on the PSA promoter (Fig. 9), indicating that holo-AR action is linked to H3-K4 methylation, but not to a more wide-spread H3 N-terminal lysine methylation. Androgen-induced alterations in H3-K4 tri-methylation on the regulatory regions of the PSA gene were not limited to LNCaP cells, as ectopically expressed holo-AR increased the extent of tri-methylated H3-K4 on the same gene also in PC-3 cells (data not shown). No T-induced changes in the total level of di- or tri-methylated H3-K4 of LNCaP cells was observed by immunoblotting with the modification-specific antibodies (data not shown).
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DISCUSSION |
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20 times) holo-AR was loaded onto the enhancers, indicating that PSA AREIII and the corresponding region of the KLK2 gene, both harboring multiple AR-binding sites (34, 35), are the primary platforms for AR loading. This is in agreement with previous in vitro results showing that the AREs on both the promoter and the enhancer are needed for maximal activity of PSA enhancer/promoter reporter constructs (34). Although some Pol II was recruited onto the enhancer, the principal Pol II-containing complex was assembled on the promoters, implying that the distal ARE-bound receptors cooperate with those on the promoter in transcription complex formation. This may occur through DNA loop formation and/or via mutual contacts with coactivators. A similar quantitative difference in AR loading onto promoter and enhancer regions has been reported for DHT-treated LNCaP cells (41). Androgen-induced recruitment of Pol II onto the PSA enhancer was also observed by Shang et al. (42) who did not, however, address quantitative differences between the two AR target regions.
Antiandrogen Action
We and others (36, 42) have recently demonstrated that AR complexed with the nonsteroidal antiandrogen bicalutamide is also loaded onto the PSA promoter in vivo, but the pure antagonist-bound AR is not compatible with formation of a productive AR transcription complex, as evidenced by the lack of recruitment of coactivators and Pol II. Our present quantitative data show that, although loading of the PSA promoter by T-occupied AR was comparable to that by the AR-CDX complex, the antagonist-bound receptor failed to get loaded onto the PSA enhancer. A possible explanation for this interesting difference is that the off-rate of the AR-CDX complex from DNA is considerably faster than that of the AR-T complex and that the AR-CDX complex on the PSA promoter is stabilized via the influence of cis-acting DNA elements or by interacting proteins that are present on the promoter but not on the enhancer. Live cell imaging analyses have shown that the CDX-bound AR indeed exhibits faster mobility than the agonist-bound receptor (43). The notion that heterologous protein-protein interactions are involved in anchoring the CDX-occupied receptor to the promoter is supported by the finding that significantly more NCoR was recruited onto the promoter than enhancer in the presence of CDX. A similar difference in the recruitment of NCoR and SMRT between the PSA promoter and the enhancer has been observed by others (42). From a quantitative point of view, our results suggest, however, that in comparison to NCoR, SMRT plays a minor role in the formation of CDX-dependent repressor complexes in LNCaP cells.
We also compared two steroidal partial agonists/antagonists in the formation of AR transcription complex. Cyproterone acetate, a progestin, possesses both agonistic and antagonistic activities in vivo and in reporter gene assays (44, 45, 46). In LNCaP cells, which express a mutant form of AR (T877A) with altered ligand-binding characteristics, CPA elicits the same level of reporter gene activity as full androgen agonists (47). RU486 has antiandrogenic properties in vitro and in vivo (47, 48, 49), but at high concentrations, it induces PSA expression in LNCaP cells and activates wild-type AR in reporter gene assays (46, 49). In this study, both CPA and RU486 were used at concentrations that exhibited significant agonistic activity. In keeping with the activation of PSA transcription, loading of the PSA promoter and enhancer by CPA- and RU486-bound ARs resulted in recruitment of Pol II and p300, even though the compounds differed somewhat in their ability to promote AR loading and coactivator recruitment. In contrast to T-occupied AR, CPA-, and RU486-bound receptors recruited concomitantly the corepressor NCoR. Therefore, our results with endogenous proteins do not agree with data from studies with overexpressed proteins, which have suggested that NCoR actively inhibits the function of pure agonist-bound AR (50, 51). If anything, T exposure of LNCaP cells decreased the level of NCoR (and SMRT) on the PSA promoter below that in vehicle-treated cells. We favor a model in which distinct AR conformations are induced by agonists, partial agonists/antagonists and pure antagonists that, in turn, result in exposure of different binding surfaces leading to the formation of pure coactivator assemblies, mixed coactivator/corepressor complexes or corepressor assemblies. It is also likely that the ratio of corepressor and coactivator concentrations within a given cell ultimately dictates the responses to mixed androgen agonists/antagonists in a target gene-specific fashion. A surprising recent finding was that increased amount of AR may also shift the balance from receptor-corepressor interactions to receptor-coactivator interactions even in the presence of a pure antiandrogen, in that CDX was demonstrated to possess agonistic activity in LNCaP cells with amplified AR levels. This antagonist-to-agonist conversion in CDX function was linked to altered recruitment of coactivators and corepressors onto the PSA and KLK2 promoters (52).
Histone Modifications
Several lines of evidence support the importance of histone acetylation in transcriptional regulation by nuclear receptors (7, 8, 53). Our results show that occupancy of both the PSA promoter and enhancer by holo-AR is accompanied by accumulation of K9 and K14 acetylated H3, as could have been expected from the recruitment of HAT activity to these regions. By contrast, CDX exposure decreased somewhat the basal level of acetylation on the promoter, implicating recruitment of HDAC by corepressors. We also examined whether H3 phosphorylation is involved in AR-dependent transcription by using an antibody specific for phosphorylated S10 which does not recognize the epitope when K14 of H3 is acetylated (39, 40, 54). Similar to di-acetylation of H3, increased phosphorylation of H3-S10 on the PSA promoter and enhancer was associated with transcriptional activation by holo-AR, whereas in the presence of CDX, H3-S10 phosphorylation was unaltered or decreased below the level in vehicle-treated cells. By using a dual H3-S10/K9 phosphoacetylation-specific antibody, we also demonstrated that increased phosphorylation and acetylation can occur on the same H3 tails. Phosphorylation of H3-S10 is coupled to acetylation at H3-K14 in response to epidermal growth factor stimulation (55), whereas acetylation and phosphorylation of H3 tails are independently and dynamically regulated on the c-jun promoter during immediate-early gene induction (40). Acetylation, phosphorylation, and phosphoacetylation patterns on the PSA regulatory regions observed in this study agree with the notion that acetylation does not require prior phosphorylation. Interestingly, phosphorylation of H3-S10 has recently been functionally linked to the activity of retinoic acid receptor ß on its own promoter (56) and to glucocorticoid-induced, but not progesterone-induced, activation of stably integrated mouse mammary tumor virus promoter (57). Moreover, activation of the collagenase gene is connected to a transient increase in H3-S10 phosphorylation on the promoter (58). Mitogen- and stress-activated protein kinase 1 and 2 are strong candidates for H3-S10 kinases (59). However, in the case of the collagenase promoter, increased phosphorylation of S10 was reported to parallel with the occupancy of the promoter by another kinase, RSK2 (59). Whether one of these kinases is responsible for increased H3 phosphorylation in response to AR action and whether the putative kinase is recruited to the PSA promoter/enhancer via interaction with CBP or p300 remain to be elucidated (58, 60).
Histone core tails are also subject to methylation. Thus far, the function of CARM1 as an AR coactivator has been examined only under transient transfection conditions (23), and PRMT1 has been shown to function as a coactivator for AR in a Xenopus oocyte transcription system (24). Our results with endogenous proteins provide evidence for the involvement of H3-R17 methylation and recruitment of CARM1 onto AR targets also in vivo. Previous ChIP assays have shown that recruitment of CARM1 and methylation of H3-R17 occur concomitantly during estrogen receptor-dependent activation of the PS2 gene (27, 28), and that the level of CARM1 and CARM1-methylated form of H3 increase on stably integrated mouse mammary tumor virus promoters upon dexamethasone induction (61).
An interesting feature of H3 lysine methylation is that modifications occurring at nearby residues lead to opposite regulatory consequences (23). Methylation at K4 of H3 is generally associated with transcriptionally active genes, whereas methylation at K9 is linked to gene silencing and generation of chromodomain-binding sites for the heterochromatin protein 1 (22, 62, 63). Yeast studies have indicated that tri-methylated H3-K4 is present solely on active genes and that the occurrence of di-methylated K4 correlates with a poised state of chromatin, in which genes are either active or potentially active (22). Recent work on chicken genes has shown that di- and tri-methylation of H3-K4 is linked to active transcription also in metazoa (64). Our present results demonstrate that activation of androgen-responsive regions of the PSA and KLK2 genes is accompanied by increased di- and tri-methylation of H3-K4, but without corresponding changes in the methylation of H3-K9. The kinetics suggest that di-methylation precedes tri-methylation. The modifications are agonist specific because they were not brought about by the pure antiandrogen CDX. Our data linking di- and tri-methylation of H3-K4 to androgen-induced transcription are also in line with a recent report that activation of the collagenase gene is connected to a transient accumulation of H3-K4 di- and tri-methylation on the promoter (58). Moreover, Chakrabarti et al. (65) have demonstrated that the proximal insulin promoter is hyper-di-methylated at H3-K4 in mouse ß-cells and that this modification occurs concomitantly with recruitment of the histone methyltransferase SET7/9 onto the promoter. Potential mechanisms by which H3-K4 methylation controls transcription include blockage of the access of histone acetylase NuRD repression complex to H3 N-terminal tails (66) and facilitation of the association of Isw1p remodeling enzyme with chromatin (67). In contrast to these results connecting the di- and tri-methylation of H3-K4 to transcriptional activation, Kim et al. (68) have reported that activation of the PSA gene was accompanied by rapid decreases in di- and tri-methylation of H3-K4 on the promoter and enhancer. The reason for this discrepancy is currently unclear.
Although lysine histone methylation has been known for several decades, most of the known histone methyltransferases have been identified only very recently, and they all contain a conserved SET methyltransferase domain (21, 23). SET methyltransferases demonstrate a high degree of site specificity, as exemplified by the specificity of SET7/9 and SET1 toward H3-K4 and SUV39H1 toward H3-K9 (69, 70, 71). Interestingly, one of the AR-interacting proteins, ARA267 (NSD1), is a SET domain protein that enhances AR-dependent transcription in a fashion that is additive to the histone acetyltransferase PCAF (72). NSD1 appears to be specific for H3-K36, and also this modification correlates with transcriptional activation (73).
Conclusions
Our results indicate that activation of the PSA and KLK2 genes by AR in vivo is linked to a cascade of distinct covalent histone modifications. In addition to previously known histone acetyltransferase and arginine methyltransferase activities, agonist-bound AR recruits directly or indirectly H3 lysine 4 methyltransferase(s) and H3 serine 10 protein kinase(s). Moreover, the pure antiandrogen bicalutamide and partial agonists/antagonists cyproterone acetate and mifepristone exhibit clear differences in their ability to induce AR-dependent loading of histone-acetylating and histone-deacetylating activities. And finally, there are distinct quantitative differences between the promoter and enhancer regions of the PSA and KLK2 genes with regard to AR loading, Pol II recruitment and histone modifications.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture
LNCaP human prostate carcinoma cells from American Type Culture Collection (Manassas, VA) were maintained in RPMI 1640 medium with 10% fetal calf serum (FCS), 2 mM glutamine, penicillin (25 U/ml), and streptomycin (25 µg/ml) in a 5% CO2 atmosphere at 37 C. At approximately 50% confluency, the medium was changed to RPMI 1640 containing 10% charcoal-stripped FCS for 4 d to reach approximately 90% confluency. Medium was changed, and the cells were cultured for another 24 h before the exposure to T or antiandrogen for various periods before harvesting for ChIP assays. PC-3 cells from American Type Culture Collection were maintained in Nutrient Mixture F-12 (HAM) containing penicillin (25 U/ml), streptomycin (25 U/ml), and 7% FCS and supplemented with L-glutamine (250 mg/liter).
ChIP
ChIP assays were performed as previously described (36). In brief, after cross-linking with 1% formaldehyde, glycine was added to a final concentration of 125 mM for 10 min at 22 C, and the cells were rinsed twice with cold PBS, harvested into lysis buffer, and nutated for 10 min at 4 C. Lysates were centrifuged, resuspended in wash buffer, and nutated for 10 min at 4 C. The resulting nuclei were pelleted by centrifugation and resuspended in RIPA buffer. Chromatin was sonicated to an average DNA length of 500-1000 bp using Fibra Cell 375W sonicator (Misonix, Farmingdale, NY) with a microtip (6 x 10 sec at maximum power). Sonicated samples were centrifuged, precleared by incubation with normal rabbit serum and protein G beads, and subjected to immunoprecipitation with specific antibodies in the presence of 100 µg/ml of sonicated salmon sperm DNA with rotation overnight at 4 C. Immunocomplexes were collected by adsorption onto protein G beads, and the beads were washed sequentially with TSE I, TSE II, and buffer III. Precipitates were washed three times with TE buffer [10 mM Tris-HCl (pH 8.0), and 1 mM EDTA], and antibody-bound chromatin fragments were eluted from the beads with 1% sodium dodecyl sulfate in 0.1 M NaHCO3. Cross-links were reverted by heating at 65 C overnight. DNA was recovered using QIAquick PCR purification system (QIAGEN, Valencia, CA). Input samples (half of the amount used for immunoprecipitation with specific antibody) were treated in the same way except that no immunoprecipitation was performed. Each ChIP assay was performed on at least three independent occasions.
Real-Time PCR
DNA samples from ChIP preparations were quantified by real-time PCR using LightCycler system and LightCycler FastStart DNA Master Hybridization Probes reagent mix (Roche Diagnostics, Indianapolis, IN) with dual labeled probes (TIB Molbiol, Berlin, Germany). The probes are labeled with a reporter dye at 5'-end and a quencher at the 3'-end. The primers and the probes were: PSA enhancer, forward primer, 5'-GCCTGGATCTGAGAGAGATATCATC-3'; reverse primer, 5'-ACACCTTTTTTTTTCTGGATTGTTG-3'; probe, 5'-6-FAM-TGCAAGGATGCCTGCTTTACAAACATCC-BHQ-1–3'; PSA promoter, forward primer, 5'-CCTAGATGAAGTCTCCATGAGCTACA-3'; reverse primer, 5'-G-GGAGGGAGAGCTAGCATTG-3'; probe, 5'-6-FAM-CAATTACTAGATCACCCTGGATGCACCAGG-DQ-1–3'; KLK2 enhancer, forward primer, 5'-GTTGAAAGCAGACCTACTCTGGA-3'; reverse primer, 5'-CTGGACCATCTTTTCAAGCAT-3'; probe, 5'-6-FAM-CCTTGCAAGATGGTATCGCCTTCAGA-DQ-3'; KLK2 promoter, forward primer, 5'-GGGAATGCCTCCAGACTGAT-3'; reverse primer, 5'-CTTGCCCTGTTGGCACCTA-3'; probe, 5'-6-FAM-AAGTGCTGGCTCTCCCTCCCCTTCC-DQ-3'. Triplicate PCRs for each sample were carried out. The results are given as percentages of inputs and represent the mean ± SE of at least three independent experiments. Control ChIP assays with nonspecific antisera were performed in each ChIP experiment series at 0 min and 120 min after the exposure of cells to T or antagonists. PSA promoter or enhancer sequences in these control ChIP assays were in all cases below the detection limit.
Real-Time RT-PCR
Total cellular RNA was isolated using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. A two-step RT-PCR method was employed. Avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and oligo-(deoxythymidine) were used to synthesize single-stranded cDNA. Target genes were analyzed by real-time PCR using the LightCycler system and LightCycler-FastStart DNA Master SYBR Green I dye (Roche Diagnostics). The primers were: PSA forward, 5'-GGCAGGTGCTTGTAGCCTCTC-3'; PSA reverse, 5'-CACCCGAGCAGGTGCTTTTGC-3'; S9 forward, 5'-GATGAGAAGGACCCAC-GGCGTCTGTTCG-3'; S9 reverse, 5'-GAGACAATCCAGCAGCCCAGGAGGGACA-3'. Triplicate PCRs were performed. S9 mRNA abundance was analyzed in each sample. The results are presented as PSA/S9 mRNA ratios. The specificities of RT-PCR products were monitored by melting curve analysis and also checked by agarose gel electrophoresis.
Acid Extraction of Histones and Immunoblotting
Acid extraction of proteins from LNCaP cells was performed after the extraction protocol from Upstate Biotechnology (www.upstatebiotech.com). In brief, the cells were scraped from the plates and the cell pellet was suspended in 15 vol PBS and centrifuged at 200 x g for 10 min. The resulting pellet was suspended in 10 vol lysis buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol and 1.5 mM phenylmethylsulfonyl fluoride] and HCl was added to a final concentration of 0.2 M and the mixture was incubated on ice for 30 min. The supernatant recovered by centrifuging at 11,000 x g for 10 min at 4 C was dialyzed twice for 2 h against 0.1 M acetic acid and three times (1 h, 3 h, and overnight) against H2O. The proteins were separated on 15% SDS-PAGE and immunoblotted using the modification-specific antibodies (described above) with the ECL Western blotting detection reagents from Amersham Biosciences (Arlington Heights, IL) and the Kodak Image Station 440 CF system (Kodak, Rochester, NY).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AR, Androgen receptor; ARE, androgen response element; CARM1, coactivator-associated arginine methyltransferase 1; CBP, cAMP response element binding protein-binding protein; CDX, bicalutamide; ChIP, chromatin immunoprecipitation; CPA, cyproterone acetate; DHT, dihydrotestoserone; FCS, fetal calf serum; GRIP1, glucocorticoid receptor-interacting protein 1; HAT, histone acetyltransferase; HDAC, histone deacetylase; KLK2, kallikrein 2; NCoR, nuclear receptor corepressor; nt, nucleotide; PCAF, p300/CBP-associated factor; Pol II, RNA polymerase II; PSA, prostate-specific antigen (KLK3); SET, Su(var)3–9, enhancer-of-Zeste, Trihorax; SMRT, silencing mediator for retinoic and thyroid hormone receptors; T, testosterone.
Received for publication June 16, 2004. Accepted for publication August 4, 2004.
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REFERENCES |
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in prostate cancer cells. J Biol Chem 276:40417–40423NURSA Molecule Pages Link:
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 M. C. Hodgson, H. C. Shen, A. N. Hollenberg, and S. P. Balk Structural basis for nuclear receptor corepressor recruitment by antagonist-liganded androgen receptor Mol. Cancer Ther., October 1, 2008; 7(10): 3187 - 3194. [Abstract] [Full Text] [PDF]
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 A. M. Kivela, E. Kansanen, H.-K. Jyrkkanen, T. Nurmi, S. Yla-Herttuala, and A.-L. Levonen Enterolactone Induces Heme Oxygenase-1 Expression through Nuclear Factor-E2-Related Factor 2 Activation in Endothelial Cells J. Nutr., July 1, 2008; 138(7): 1263 - 1268. [Abstract] [Full Text] [PDF]
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A. Yemelyanov, J. Czwornog, L. Gera, S. Joshi, R. T. Chatterton Jr., and I. Budunova Novel Steroid Receptor Phyto-Modulator Compound A Inhibits Growth and Survival of Prostate Cancer Cells Cancer Res., June 15, 2008; 68(12): 4763 - 4773. [Abstract] [Full Text] [PDF]
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 U. Moehren, M. Papaioannou, C. A. Reeb, A. Grasselli, S. Nanni, M. Asim, D. Roell, I. Prade, A. Farsetti, and A. Baniahmad Wild-type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development FASEB J, April 1, 2008; 22(4): 1258 - 1267. [Abstract] [Full Text] [PDF]
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 H. V. Heemers and D. J. Tindall Androgen Receptor (AR) Coregulators: A Diversity of Functions Converging on and Regulating the AR Transcriptional Complex Endocr. Rev., December 1, 2007; 28(7): 778 - 808. [Abstract] [Full Text] [PDF]
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M. C. Hodgson, I. Astapova, A. N. Hollenberg, and S. P. Balk Activity of Androgen Receptor Antagonist Bicalutamide in Prostate Cancer Cells Is Independent of NCoR and SMRT Corepressors Cancer Res., September 1, 2007; 67(17): 8388 - 8395. [Abstract] [Full Text] [PDF]
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 Y. Dai, D. Ngo, L. W. Forman, D. C. Qin, J. Jacob, and D. V. Faller Sirtuin 1 Is Required for Antagonist-Induced Transcriptional Repression of Androgen-Responsive Genes by the Androgen Receptor Mol. Endocrinol., August 1, 2007; 21(8): 1807 - 1821. [Abstract] [Full Text] [PDF]
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U. Moehren, M. Papaioannou, C. A. Reeb, W. Hong, and A. Baniahmad Alien Interacts with the Human Androgen Receptor and Inhibits Prostate Cancer Cell Growth Mol. Endocrinol., May 1, 2007; 21(5): 1039 - 1048. [Abstract] [Full Text] [PDF]
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 A. Domanskyi, F.-P. Zhang, M. Nurmio, J. J. Palvimo, J. Toppari, and O. A. Janne Expression and localization of androgen receptor-interacting protein-4 in the testis Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E513 - E522. [Abstract] [Full Text] [PDF]
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 C. J Burd, L. M Morey, and K. E Knudsen Androgen receptor corepressors and prostate cancer Endocr. Relat. Cancer, December 1, 2006; 13(4): 979 - 994. [Abstract] [Full Text] [PDF]
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S. Chen, Y. Xu, X. Yuan, G. J. Bubley, and S. P. Balk Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1 PNAS, October 24, 2006; 103(43): 15969 - 15974. [Abstract] [Full Text] [PDF]
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M. A. Choudhry, A. Ball, and I. J. McEwan The Role of the General Transcription Factor IIF in Androgen Receptor-Dependent Transcription Mol. Endocrinol., September 1, 2006; 20(9): 2052 - 2061. [Abstract] [Full Text] [PDF]
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R. Nielsen, L. Grontved, H. G. Stunnenberg, and S. Mandrup Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery Mol. Cell. Biol., August 1, 2006; 26(15): 5698 - 5714. [Abstract] [Full Text] [PDF]
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S. Carascossa, J. Gobinet, V. Georget, A. Lucas, E. Badia, A. Castet, R. White, J.-C. Nicolas, V. Cavailles, and S. Jalaguier Receptor-Interacting Protein 140 Is a Repressor of the Androgen Receptor Activity Mol. Endocrinol., July 1, 2006; 20(7): 1506 - 1518. [Abstract] [Full Text] [PDF]
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B. B. Cheung, J. Bell, A. Raif, A. Bohlken, J. Yan, B. Roediger, A. Poljak, S. Smith, M. Lee, W. D. Thomas, et al. The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal J. Biol. Chem., June 30, 2006; 281(26): 18246 - 18256. [Abstract] [Full Text] [PDF]
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K. M.A. Dreijerink, K. W. Mulder, G. S. Winkler, J. W.M. Hoppener, C. J.M. Lips, and H.Th. M. Timmers Menin Links Estrogen Receptor Activation to Histone H3K4 Trimethylation. Cancer Res., May 1, 2006; 66(9): 4929 - 4935. [Abstract] [Full Text] [PDF]
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H.-G. Yoon and J. Wong The Corepressors Silencing Mediator of Retinoid and Thyroid Hormone Receptor and Nuclear Receptor Corepressor Are Involved in Agonist- and Antagonist-Regulated Transcription by Androgen Receptor Mol. Endocrinol., May 1, 2006; 20(5): 1048 - 1060. [Abstract] [Full Text] [PDF]
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 K. J. Pienta and D. Bradley Mechanisms underlying the development of androgen-independent prostate cancer. Clin. Cancer Res., March 15, 2006; 12(6): 1665 - 1671. [Full Text] [PDF]
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 J. A. Magee, L.-w. Chang, G. D. Stormo, and J. Milbrandt Direct, Androgen Receptor-Mediated Regulation of the FKBP5 Gene via a Distal Enhancer Element Endocrinology, January 1, 2006; 147(1): 590 - 598. [Abstract] [Full Text] [PDF]
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 H. I. Scher and C. L. Sawyers Biology of Progressive, Castration-Resistant Prostate Cancer: Directed Therapies Targeting the Androgen-Receptor Signaling Axis J. Clin. Oncol., November 10, 2005; 23(32): 8253 - 8261. [Abstract] [Full Text] [PDF]
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R. Ilagan, L. J. Zhang, J. Pottratz, K. Le, S. Salas, M. Iyer, L. Wu, S. S. Gambhir, and M. Carey Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model Mol. Cancer Ther., November 1, 2005; 4(11): 1662 - 1669. [Abstract] [Full Text] [PDF]
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 P. Farla, R. Hersmus, J. Trapman, and A. B. Houtsmuller Antiandrogens prevent stable DNA-binding of the androgen receptor J. Cell Sci., September 15, 2005; 118(18): 4187 - 4198. [Abstract] [Full Text] [PDF]
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