Tip60 and Histone Deacetylase 1 Regulate Androgen Receptor Activity through Changes to the Acetylation Status of the Receptorстатья из журнала
Аннотация: The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, is thought to play an important role in the development of prostate cancer. The AR is a hormone-dependent transcription factor that activates expression of numerous androgen-responsive genes. Histone acetyltransferase-containing proteins have been shown to increase activity of several transcription factors, including nuclear hormone receptors, by eliciting histone acetylation, which facilitates promoter access to the transcriptional machinery. Conversely, histone deacetylases (HDACs) have been identified which reduce levels of histone acetylation and are associated with transcriptional repression by various transcription factors. We have previously shown that Tip60 (Tat-interactive protein, 60 kDa) is a bona fideco-activator protein for the AR. Here we show that Tip60 directly acetylates the AR, which we demonstrate is a requisite for Tip60-mediated transcription. To define a mechanism for repression of AR function, we demonstrate that AR activity is specifically down-regulated by the histone deacetylase activity of HDAC1. Furthermore, using both mammalian two-hybrid and immunoprecipitation experiments, we show that AR and HDAC1 interact, suggestive of a direct role for down-regulation of AR activity by HDAC1. In chromatin immunoprecipitation assays, we provide evidence that AR, Tip60, and HDAC1 form a trimeric complex upon the endogenous AR-responsive PSA promoter, suggesting that acetylation and deacetylation of the AR is an important mechanism for regulating transcriptional activity. The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, is thought to play an important role in the development of prostate cancer. The AR is a hormone-dependent transcription factor that activates expression of numerous androgen-responsive genes. Histone acetyltransferase-containing proteins have been shown to increase activity of several transcription factors, including nuclear hormone receptors, by eliciting histone acetylation, which facilitates promoter access to the transcriptional machinery. Conversely, histone deacetylases (HDACs) have been identified which reduce levels of histone acetylation and are associated with transcriptional repression by various transcription factors. We have previously shown that Tip60 (Tat-interactive protein, 60 kDa) is a bona fideco-activator protein for the AR. Here we show that Tip60 directly acetylates the AR, which we demonstrate is a requisite for Tip60-mediated transcription. To define a mechanism for repression of AR function, we demonstrate that AR activity is specifically down-regulated by the histone deacetylase activity of HDAC1. Furthermore, using both mammalian two-hybrid and immunoprecipitation experiments, we show that AR and HDAC1 interact, suggestive of a direct role for down-regulation of AR activity by HDAC1. In chromatin immunoprecipitation assays, we provide evidence that AR, Tip60, and HDAC1 form a trimeric complex upon the endogenous AR-responsive PSA promoter, suggesting that acetylation and deacetylation of the AR is an important mechanism for regulating transcriptional activity. androgen receptor nuclear hormone receptor activation function-1 p300/CBP-associating factor histone acetyltransferase CREB-binding protein histone deacetylases factor acetyltransferase DNA-binding domain Tat-interactive protein, 60 kDa fetal calf serum trichostatin A β-galactosidase Prostate cell growth, development, and homeostasis are critically dependent upon the androgen receptor (AR),1 an androgen-responsive transcription factor that activates expression of target genes in response to hormonal signals derived from the testis. The AR is a member of the nuclear hormone receptor (NHR) family and, in common with other family members, is a modular protein composed of numerous independently functioning domains (1Jenster G. Vanderkorput H. Vanvroonhoven C. Vanderkwast T.H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (421) Google Scholar, 2Jenster G. Vanderkorput J. Trapman J. Brinkmann A.O. J. Steroid Biochem. Mol. Biol. 1992; 41: 671-675Crossref PubMed Scopus (64) Google Scholar, 3Brinkmann A.O. Jenster G. Kuiper G. Ris C. Vanlaar J.H. Vanderkorput J. Degenhart H.J. Trifiro M.A. Pinsky L. Romalo G. Schweikert H.U. Veldscholte J. Mulder E. Trapman J. J. Steroid Biochem. Mol. Biol. 1992; 41: 361-368Crossref PubMed Scopus (32) Google Scholar). Upon binding to androgens within the cytoplasm, the AR translocates to the nucleus (4Ozanne D.M. Brady M.E. Cook S. Gaughan L. Neal D.E. Robson C.N. Mol. Endocrinol. 2000; 14: 1618-1626Crossref PubMed Google Scholar) where it recognizes and binds specific promoter elements and activates transcription of target genes through the concerted action of two transcriptional activation domains, namely activation function-1 (AF-1) and -2 (AF-2) (5Brinkmann A.O. Blok L.J. de Ruiter P.E. Doesburg P. Steketee K. Berrevoets C.A. Trapman J. J. Steroid Biochem. Mol. Biol. 1999; 69: 307-313Crossref PubMed Scopus (257) Google Scholar). The AF-2 domain of NHRs plays a fundamental role in receptor-mediated transcriptional activation. Upon ligand-binding, the C-terminal AF-2 undergoes a shift in conformation generating a platform suitable for protein-protein interaction with co-activator molecules (6Weatherman R.V. Fletterick R.J. Scanlan T.S. Annu. Rev. Biochem. 1999; 68: 559-581Crossref PubMed Scopus (292) Google Scholar, 7Bourguet W. Germain P. Gronemeyer H. Trends Pharmacol. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). To date, numerous co-activator molecules have been identified that function to enhance the transcriptional potential of NHRs (8Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-457Crossref PubMed Scopus (1412) Google Scholar). The majority of co-activators identified share the capacity to elicit histone acetyltransferase (HAT) activity, a catalytic process heavily implicated in target gene activation via chromatin remodeling (9Berger S.L. Curr. Opin. Cell Biol. 1999; 11: 336-341Crossref PubMed Scopus (141) Google Scholar, 10Utley R.T. Ikeda K. Grant P.A. Cote J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (447) Google Scholar). Of the identified HAT-containing co-activators, several have emerged to play significant roles in NHR activity, including the p160 (11Hong H. Kohli K. Garabedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (497) Google Scholar, 12Leo C. Chen J.D. Gene (Amst.). 2000; 245: 1-11Crossref PubMed Scopus (440) Google Scholar, 13Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1070) Google Scholar) and CBP (CREB-binding protein)/p300 families (14Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Crossref PubMed Scopus (255) Google Scholar, 15Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar) and PCAF (p300/CBP-associating factor) (16Blanco J.C.G. Minucci S., Lu, J. Yang X.J. Walker K.K. Chen H. Evans R.M. Nakatani Y. Ozato K. Genes Dev. 1998; 12: 1638-1651Crossref PubMed Scopus (340) Google Scholar). Whereas histone acetylation is important for initiating and maintaining transcriptionally active genes, the recruitment of factors involved in deacetylating target promoters is deemed to be a requisite for gene silencing. Numerous co-repressor molecules have been identified, such as Sin3 (17Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1086) Google Scholar) and SMRT (18Chen J.D. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7567-7571Crossref PubMed Scopus (222) Google Scholar), which play an active role in transcriptional repression by numerous transcription factors such as unliganded class II NHRs (19Nagy L. Kao H.Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar). These repressors are found in complex with histone deactylases (HDACs); enzymes that actively reduce the level of histone acetylation. To date, three groups of histone deacetylases have been identified. The class I family is composed of 4 members, HDAC1–3 and HDAC8, and are homologues of the yeast protein RPD3 (20Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar). Six class II HDACs have been characterized, HDAC4–7, -9, and -10, which bear significant homology to the HDA protein of yeast (20Gray S.G. Ekstrom T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar). Class III HDACs are homologous to the yeast Sir2 protein, but as yet, are not well characterized. The finding that several transcription factors, such as p53 (21Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, 22Juan L.J. Shia W.J. Chen M.H. Yang W.M. Seto E. Lin Y.S. Wu C.W. J. Biol. Chem. 2000; 275: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) and MyoD (23Mal A. Sturniolo M. Schiltz R.L. Ghosh M.K. Harter M.L. EMBO J. 2001; 20: 1739-1753Crossref PubMed Scopus (208) Google Scholar, 24Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y.J. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar), are targets for direct acetylation and deacetylation suggests that factor acetyltransferase (FAT) and HDAC proteins, respectively, play an active role in regulating transcription factor function, in which the status of acetylation at both the histone and transcription factor level heavily influences gene expression profiles. Recently, the AR has been found to be a substrate for p300- and PCAF-mediated FAT activity (25Fu M.F. Wang C.G. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Acetylation of three lysine residues in the short lysine-rich motif KLKK, flanking the DNA-binding domain (DBD), increases transcriptional activity of the AR, implicating this post-translational modification as a mechanism for regulating AR activity. Tip60 (Tat-interactive protein, 60 kDa) was first identified in complex with the Tat protein of human immunodeficiency virus-1 (26Kamine J. Elangovan B. Subramanian T. Coleman D. Chinnadurai G. Virology. 1996; 216: 357-366Crossref PubMed Scopus (242) Google Scholar) and was later demonstrated to directly acetylate histones H2A, H3, and H4 via a C-terminal HAT domain (27Yamamoto T. Horikoshi M. J. Biol. Chem. 1997; 272: 30595-30598Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). We previously identified Tip60 as an AR-interacting protein and showed Tip60 as a bona fideco-activator for the AR (28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Moreover, we have recently demonstrated that Tip60 is a class I NHR-specific co-activator implicating an important role for Tip60 in steroid hormone receptor function (29Gaughan L. Brady M.E. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2001; 276: 46841-46848Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To further define the role of Tip60 in AR-mediated gene expression, we provide evidence that Tip60 directly acetylates the AR in vivo, which is a requisite for Tip60-mediated AR co-activation. We next investigated the potential for HDACs to influence AR transcriptional activity. Here we demonstrate that the AR is specifically down-regulated by the histone deacetylase activity of HDAC1, the effect of which can be reversed by the HAT activity of Tip60. In mammalian two-hybrid and immunoprecipitation experiments, we show that HDAC1 interacts directly with the AR. Using chromatin immunoprecipitation assays, we demonstrate that Tip60 and HDAC1 associate with the endogenous AR-responsive PSA promoter in LNCaP cells, implicating an important physiological role for acetylation and deacetylation in AR regulation. Together, the data suggests that the acetylation status of the AR is a dominant factor in regulating transcriptional activity, and is the first evidence that HDAC1 can down-regulate a member of the class I nuclear hormone receptor family. The following plasmids have been described previously: pPSALuc, UASTKLuc, pCMV-β-gal, pcDNA3-AR (28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), pM-AR-DS (29Gaughan L. Brady M.E. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2001; 276: 46841-46848Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), pBJ5-FLAG-HDAC1 and pBJ5-FLAG-HDAC1H141A (gifts from Stuart Schreiber, Harvard Medical School) (30Hassig C.A. Fleischer T.C. Billin A.N. Schreiber S.L. Ayer D.E. Cell. 1997; 89: 341-347Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 31Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (332) Google Scholar), pME18S-FLAG-HDAC2 (gift from Robert Eisenman, Fred Hutchinson Cancer Research Centre and Research Institute) (32Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar), pCMV-FLAG-HDAC3 (gift from Cheng-Wen Wu, Institute of Biomedical Sciences, Academia Sinica) (22Juan L.J. Shia W.J. Chen M.H. Yang W.M. Seto E. Lin Y.S. Wu C.W. J. Biol. Chem. 2000; 275: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), pcDNA-His-HDAC5 and pcDNA-His-HDAC6 (gifts from Saadi Khochbin, Institut Albert Bonniot) (33Verdel A. Khochbin S. J. Biol. Chem. 1999; 274: 2440-2445Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 34Lemercier C. Verdel A. Galloo B. Curtet S. Brocard M.P. Khochbin S. J. Biol. Chem. 2000; 275: 15594-15599Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), pcDNA3-AR630 and pcDNA3-AR632/633 (gifts from Richard Pestell, the Albert Einstein Cancer Centre, Albert Einstein College of Medicine) (25Fu M.F. Wang C.G. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). The full-length Tip60 construct was generated by PCR, incorporating TipF (ATGGACTACAAAGACGACGATGACAAAGCGGAGGTGGGGGAGATAATCGAG (anneals to the start codon of Tip60 and incorporates a FLAG tag)) and TipR (TCAACCACTTCCCCCTCTTGCTCCA (anneals to the stop codon of Tip60)), using POZ-Tip60 (gift from Yoshihiro Nakatani, Dana-Farber Cancer Research Institute) (35Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar) as template and Bio-Taq DNA polymerase enzyme (Bioline). The product was cloned into the TA-vector (Invitrogen) and then subcloned into pCMV vector via the EcoRI site. The Tip60 HAT-defective mutant, Tip60Q377E/G380E, was generated by PCR using POZ-Tip60Q377E/G380E (gift from Tsuyoshi Ikura) (35Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar) as template and Bio-Taq DNA polymerase enzyme, incorporating TipF and TipR. The product was cloned into the TA-vector and then subcloned into the pCMV vector via the EcoRI site. To generate pVP16AD-HDAC1, PCR was performed with HDAC1F, GAATTCATGGCGCAGACGCAGGGCACCCGG (anneals to the start codon of HDAC1), and HDACR, GGATCCTCAGGCCAACTTGACCTCCTCCTTGAC (anneals to the stop codon of HDAC1), incorporating pBJ5-FLAG-HDAC1 as template and Bio-Taq DNA polymerase. The product was cloned into the TA-vector system as before and then subcloned into pVP16AD (CLONTECH), via theBamHI and EcoRI sites. All constructs were fully sequenced to confirm integrity. Tip60-specific antibody was generated by injecting rabbits with a Tip60 peptide (amino acids 283–297) and then the antibody was affinity purified. Cell culture and DNA transfection were performed as described previously (28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). COS-7 cells were maintained in RPMI 1640 media containing 10% fetal calf serum (FCS) (Invitrogen), 1% penicillin, and 1% streptomycin. 1 × 104 COS-7 cells were routinely plated per well in 24-microtiter plates (Corning). After 24 h, the cells were transfected using Superfect (Qiagen) according to the manufacturer's recommendations. After 2 h, cells were washed and incubated either in FCS-containing media prior to treatment with 100 nmtrichostatin A (TSA), or in RPMI 1640 media containing 10% FCS that had been stripped of steroids by treatment with dextran-coated charcoal prior to experimentation with 10 nm R1881 (synthetic androgen analogue). After 48 h, cells were harvested and assayed for luciferase activity according to the manufacturer's guidelines (Promega). Luciferase activity was corrected for the corresponding β-galactosidase activity to give relative activity as described previously (28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In general, TSA treatment lasted 12 h prior to cell harvesting, whereas treatment with R1881 lasted the duration of transfection. The prostate cancer cell line LNCaP was cultured as above. For transfections, a superfect:DNA ratio of 3:1 for COS-7 cells was increased to 5:1 for LNCaP cells and the incubation period for transfection mixtures was increased from 2 h for COS-7 cells to 3 h for LNCaP cells. In general, co-transfection experiments using both COS-7 and LNCaP cells incorporated 50 ng of each expression vector and 200 ng of each reporter construct. Fold increases were determined for 50 ng of expression vector by comparing the activity with empty pCMV-driven vector. Each experiment was performed in triplicate and repeated a minimum of three times. COS-7 cell lysates were boiled in SDS sample buffer (100 mm dithiothreitol, 125 mmTris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.005% bromphenol blue) for 10 min and equivalent amounts of protein were resolved on 12% polyacrylamide gels. Proteins were subsequently transferred to HybondTM membrane (Amersham Biosciences) and detected by specific antibodies (see Figs. 1, 3, and 6) using the ECL system (Amersham Biosciences) according to the manufacturer's recommendations.Figure 3HDAC1 represses AR-mediated transactivation. A, the effect of class I and class II HDACs on AR activity was assessed in transient transfection experiments in COS-7 cells maintained in FCS-containing media. DNA included 50 ng of each HDAC construct, 50 ng of pcDNA3-AR, and 200 ng each of the reporters pPSALuc and pCMV-β-gal per well. Relative luciferase activity was determined. B, cell extracts from Awere immunoblotted with a monoclonal anti-AR antibody to determine relative protein levels of AR in each sample.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6AR and HDAC1 interact in vivoand in vitro. A, using the mammalian two-hybrid system, the potential of an AR-HDAC1 interaction was investigated. COS-7 cells were transfected with 50 ng of pM-AR-DS and 50 ng of the VP16AD fusion constructs pVP16AD-HDAC1, -Tip60, or empty pVP16AD for control, as well as 200 ng of UASTKLuc and pCMV-β-gal per well. Relative luciferase activity was determined.B, COS-7 cells were transiently transfected with 2 μg of pcDNA3-AR and pJB5-FLAG-HDAC1 per 90-mm dish. Cell lysates were immunoprecipitated (IP) with an anti-FLAG antibody and immunoblotted with an anti-AR antibody. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) COS-7 cells were transfected with 3 μg of pcDNA3-AR and 3 μg of pCMV-Tip60 or empty pCMV for control per 90-mm dish. 1 h prior to harvesting, cells were incubated in FCS-containing media supplemented with 100 nm TSA and 1 μm [3H]acetic acid (ICN). Samples were subjected to immunoprecipitation, as described in Ref. 28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, using a polyclonal anti-AR antibody (Santa Cruz). Immunoprecipitates were resolved on a 12% polyacrylamide gel, soaked in Amplify (AmershamBiosciences), and then exposed to x-ray film at −80 °C for 72 h. To determine a direct role for Tip60 HAT activity in AR acetylation, COS-7 cells were transfected with 2 μg of pcDNA3-AR, pCMV-Tip60, or pCMV-Tip60Q377E/G380E per 90-mm dish and lysates were subjected to immunoprecipitation using a polyclonal anti-AR antibody (as described before) and immunoblotting with an anti-acetyllysine antibody (Upstate Biotechnology) to detect the acetylated AR species. To examine a potential AR-HDAC1 interaction, COS-7 cells transfected with 2 μg of pcDNA3-AR and pJB5-FLAG-HDAC1 per 90-mm dish were subjected to immunoprecipitation as before using an anti-FLAG antibody to immunoprecipitate HDAC1-associated complexes and immunoblotting using a polyclonal anti-AR antibody. LNCaP cells were grown on 150-mm dishes in FCS-containing media for 2 days until ∼5 × 106 cells were present. 16 h prior to androgen treatment, cells were transferred to steroid-depleted media (RPMI supplemented with 10% dextran-coated charcoal-stripped FCS). After 16 h, the media was replaced with FCS-stripped media supplemented with or without 10 nm R1881 for the specified time period (see Fig. 8). Following treatment, LNCaP cells were treated with formaldehyde, added directly to culture medium (to a final concentration of 1%), at room temperature for 10 min to cross-link histone proteins to DNA. Soluble chromatin was made as follows: cells were washed and detached by scraping following addition of ice-cold phosphate-buffered saline supplemented with 25 μg/ml leupeptin, 25 μg/ml aprotinin, and 25 μg/ml pepstatin, and pelleted by centrifugation for 4 min at 700 × g. The latter two steps were repeated. The cell pellet was then subjected to immunoprecipitation by resuspending in lysis buffer (50 mmTris (pH 8.1), 1% SDS, 10 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 0.8 μg/ml pepstatin, 0.6 μg/ml leupeptin, and 0.6 μg/ml aprotinin), followed by sonication. Samples were then centrifuged at 13,000 rpm for 10 min and the supernatant was decanted and diluted 10-fold in dilution buffer (25 mm Tris (pH 8.1), 140 mm NaCl, 1% SDS, 3 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.8 μg/ml pepstatin, 0.6 μg/ml leupeptin, and 0.6 μg/ml aprotinin). To pre-clear chromatin solution, 60 μl of salmon sperm DNA/protein A-agarose beads (Upstate Biotechnology) was added to each sample and agitated for 30 min at 4 °C. Beads were pelleted by brief centrifugation and the supernatant was collected. For immunoprecipitation, 2 μg of polyclonal AR antibody, monoclonal HDAC1 antibody (Upstate Biotechnology), or Tip60 polyclonal antibody (see above) were added to 1 ml of the purified chromatin sample and incubated overnight at 4 °C. Immunocomplexes were recovered by adding 60 μl of salmon sperm/protein A-agarose for 1 h at 4 °C with agitation. Beads were washed sequentially for 5 min each in 10 ml of TSE buffers I–III and TE (pH 8), as described previously (36Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (714) Google Scholar). Immunocomplexes were eluted by adding 250 μl of elution buffer (1% SDS and 0.1m NaHCO3) to beads and subsequently heated for 4 h at 64 °C to reverse formaldehyde-induced cross-links. DNA were then recovered by phenol/chloroform extraction, ethanol precipitation, and resuspended in 50 μl of TE. Semiquantitative PCR was performed with 10 μl of DNA, Bio-Taq DNA polymerase, and [α-32P]dATP, using primers P1F (GTGGAGCTGGATTCTGGG) and P4R (TGGGTACGATCCCCGATT), to amplify the 235 bp of the PSA promoter, encompassing the ARE2 (see Fig. 8). PCR products were resolved, dried, and then exposed to x-ray film for 2–12 h. The AR has been demonstrated to be directly acetylated by p300 and PCAF. Acetylation of the AR was shown to enhance inherent transcriptional activity of the AR, suggesting that acetylation plays a significant role in AR regulation (25Fu M.F. Wang C.G. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). We previously identified Tip60 as a bona fide co-activator for the AR (28Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Considering that Tip60 contains a HAT domain, which has been shown to acetylate free histones H4, H3, and H2A (27Yamamoto T. Horikoshi M. J. Biol. Chem. 1997; 272: 30595-30598Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), we sought to examine if, like p300 and PCAF, Tip60 directly acetylated the AR to increase transcriptional activity. To determine whether the AR is a target for Tip60-mediated acetylation in vivo, COS-7 cells were transiently transfected with wild-type AR and either Tip60 or empty vector for control, and incubated for 1 h in [3H]acetic acid prior to immunoprecipitation with an anti-AR antibody. The level of AR acetylation was determined by measuring [3H]acetate incorporation into the AR protein using autoradiography. Previous work has shown that addition of the HDAC inhibitor TSA greatly enhances AR activity, suggesting that the AR is a potential target for direct deacetylation and down-regulation by HDACs (25Fu M.F. Wang C.G. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). We incorporated 100 nm TSA into our system to block the action of deacetylase enzymes. As shown in Fig.1 A, a, in the presence of Tip60, the level of AR acetylation was increased substantially over that in the absence of Tip60 (compare lanes 1 and 2), indicating that Tip60 may directly acetylate the AR, presumably through the activity of the HAT domain. Our results also indicate that in the absence of overexpressed Tip60, endogenous factors within COS-7 cells have the capacity to induce AR acetylation (lane 1). Whether this modification is through the HAT activity of endogenous Tip60, or other potential HAT-containing proteins, such as p300 and PCAF, remains to be determined. Using Western blotting, incorporating an anti-AR antibody, we confirmed that the difference in acetylation observed was not from variation in transfection efficiencies between the samples (Fig. 1 A,b). To establish that the HAT activity of Tip60 was responsible for directly acetylating the AR, the ability of wild-type Tip60, and a HAT-defective Tip60 mutant (Tip60Q377E/G380E) (25Fu M.F. Wang C.G. Reutens A.T. Wang J. Angeletti R.H. Siconolfi-Baez L. Ogryzko V. Avantaggiati M.L. Pestell R.G. J. Biol. Chem. 2000; 275: 20853-20860Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), to acetylate the AR was investigated. COS-7 cells transiently transfected with full-length AR and wild-type Tip60 or Tip60Q377E/G380E were immunoprecipitated using an anti-AR antibody followed by immunoblotting incorporating an anti-acetyllysine antibody, to compare the levels of AR acetylation in the presence of either wild-type Tip60 or Tip60Q377E/G380E. We figured that if AR hyperacetylation is a result of Tip60-mediated HAT activity, then overexpression of the Tip60 HAT mutant, which lacks a functional HAT domain through the substitution of two residues (Glu-377 and Gly-380) required for acetyl-CoA binding (35Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar), would fail to generate the acetylated form of the AR. As shown in Fig. 1 B, in the absence of wild-type Tip60, no acetylated species of the AR was detected (lane 1), whereas in the presence of wild-type Tip60, the AR was clearly demonstrated to be in an acetylated form (lane 3). In contrast, overexpression of Tip60Q377E/G380E resulted in no change to the acetylation status of the AR (compare lanes 5 and 1), indicating that the HAT-defective Tip60 mutant is unable to acetylate the AR. Together, the data provide evidence that Tip60 is capable of directly
Год издания: 2002
Авторы: Luke Gaughan, Ian R. Logan, Susan Cook, David E. Neal, Craig Robson
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Histone Deacetylase Inhibitors Research, Estrogen and related hormone effects, Prostate Cancer Treatment and Research
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 277
Выпуск: 29
Страницы: 25904–25913