Direct Acetylation of the Estrogen Receptor α Hinge Region by p300 Regulates Transactivation and Hormone Sensitivity
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m100800200
ISSN1083-351X
AutoresChenguang Wang, Maofu Fu, Ruth Hogue Angeletti, Linda Siconolfi-Baez, Anne T. Reutens, Chris Albanese, Michael P. Lisanti, Benita S. Katzenellenbogen, Shigeaki Kato, Torsten Hopp, Suzanne A.W. Fuqua, Gabriela N. Lopez, Peter J. Kushner, Richard G. Pestell,
Tópico(s)Genomics and Chromatin Dynamics
ResumoRegulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g.p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo.p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERα acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development. Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERα) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERα-regulated gene expression involves interactions with cointegrators (e.g.p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERα is acetylated in vivo.p300, but not P/CAF, selectively and directly acetylated the ERα at lysine residues within the ERα hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERα hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERα acetylation normally suppresses ligand sensitivity. These ERα lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERα acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development. estrogen receptor α activation function mitogen-activated protein kinase MAPK/ERK (extracellular signal-related kinase) kinase CREB (cAMP-response element-binding protein)-binding protein immunoprecipitation histone acetyl transferase high pressure liquid chromatography glutathioneS-transferase trichostatin A matrix-assisted laser desorption/ionization time-of-flight estradiol p300/CBP-associated factor erythroid Kruppel-like factor estrogen response element Nuclear receptors coordinate diverse physiological roles in metabolism and development through ligand-dependent and -independent mechanisms (1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Nuclear receptors form multiprotein complexes with coactivator and corepressor proteins to orchestrate dynamic transcriptional events in response to ligand. In the absence of ligand, nuclear receptors repress transcription through a dominant association with corepressor complexes with histone deacetylase activity (2McKenna J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1650) Google Scholar). Conformational changes induced upon nuclear receptor ligand binding release corepressors, with subsequent transient association of coactivator proteins (2McKenna J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1650) Google Scholar, 3Shang Y. Hu X. DiRenzo J. Lazar M. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar, 4Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Estrogen binds the estrogen receptor (ERα),1 thereby regulating important functions in development and reproduction and in human diseases including breast cancer, cardiovascular disease, osteoporosis, and Alzheimer's disease. The ERα contains domains conserved with other members of the "classical" receptor subclass (termed A—F) and two activation domains, AF (activation function)-1 and AF-2. The two activation domains of ERα contribute synergistically to transcription of target genes. The AF-1 function is both constitutive and induced by mitogen-activated protein kinases (MAPKs) induced by growth factors or oncoproteins (5Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1710) Google Scholar). p300 (6Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and a p300/CBP-binding protein, p68 RNA helicase A (7Endoh H. Maruyama K. Masuhiro Y. Kobayashi Y. Goto M. Tai H. Yanagisawa J. Metzger D. Hashimoto S. Kato S. Mol. Cell. Biol. 1999; 19: 5363-5372Crossref PubMed Google Scholar), also induce AF-1 activity. Thus, p300 binds AF-1 in the absence of ligand (6Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 8Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar) inducing ERα activity 2–3-fold in either reporter or in vitro transcription assays (6Kobayashi Y. Kitamoto T. Masuhiro Y. Watanabe M. Kase T. Metzger D. Yanagisawa J. Kato S. J. Biol. Chem. 2000; 275: 15645-15651Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 8Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar). p300/CBP binding to ERα is also detectable in MCF7 cells in the absence of ligand (4Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). The ligand-dependent transactivation function (AF-2) domain of ERα consists of a conserved carboxyl-terminal helix. The AF-2 domain contributes to ligand-induced activity through further recruitment of coactivator proteins including the p160 family, (SRC-1, TIF2/GRIP1, AIB1/ACTR), the cointegrators (CBP, p300), and p300/CBP-associated factor (P/CAF) (2McKenna J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1650) Google Scholar, 8Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar, 9Hanstein B. Eckner R. DiRenzo J. Halachmi S. Liu H. Searcy B. Kurokawa R. Brown M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11540-11545Crossref PubMed Scopus (341) Google Scholar). The role of p300 as an ERα cointegrator is complex; p300 contributes to ERα induction through several separable subdomains including the histone acetyl transferase (HAT) and the bromodomain (4Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 8Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar, 10Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar), which make separate contacts to distinct domains of the ERα. The enhancement of transcriptional activity by p300/CBP involves several different functions. The cointegrators provide a bridging function, which associates transcription factors with the basal transcription apparatus (11Nakajima T. Uchida C. Anderson S.F. Lee C.-G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Second, p300/CBP provides a scaffold, interacting with numerous transcription factors through dedicated domains to assemble high molecular weight "enhanceosomes" (reviewed in Ref. 12Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Crossref PubMed Scopus (254) Google Scholar). Third, the HAT activity of p300/CBP, which may be either intrinsic or mediated through the recruitment of associated proteins such as P/CAF, contributes to the transcriptional coactivator function. Transcriptional activation in chromatin-containing systems has correlated transcriptional activity with acetylation of specific lysines in the NH2 termini of histones (13Wang L. Liu L. Berger S.L. Genes Dev. 1998; 12: 640-653Crossref PubMed Scopus (220) Google Scholar, 14Kuo M.-H. Zhou J. Jambeck P. Churchill M.E.A. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (398) Google Scholar). Histone acetylation is thought to facilitate binding of transcription factors to specific target DNA sequences by destabilizing nucleosomes bound to the promoter region of a target gene (15Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1547) Google Scholar). In addition, p300/CBP and P/CAF directly acetylate non-histone proteins including a subset of transcription factors and coactivators (p53, EKLF, HMG1(Y), GATA-1, E2F-1, and ACTR (16Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar, 17Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (634) Google Scholar, 18Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 19Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (328) Google Scholar, 20Martinez-Balbas M.A. Bauer U.-M. Nielson S., J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (568) Google Scholar). Transcription factor acetylation by cointegrators has divergent effects. p300/CBP-dependent acetylation enhanced the activity of the tumor suppressor p53 (21Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (653) Google Scholar), the Kruppel-like factor (EKLF) (19Zhang W. Bieker J.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9855-9860Crossref PubMed Scopus (328) Google Scholar), and the erythroid cell differentiation factor, GATA-1 (22Hung H.L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (219) Google Scholar) (reviewed in Ref. 23Berger S.L. Curr. Opin. Cell Biol. 1999; 11: 336-341Crossref PubMed Scopus (141) Google Scholar). In contrast, CBP repressed the transcriptional activity of T cell factor (24Walter L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (326) Google Scholar), and direct acetylation of the coactivator ACTR by p300 contributed to an inhibition of hormone-induced nuclear receptor signaling (4Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Together these studies are consistent with a model in which cointegrator proteins, through their acetylation function, are engaged in a dynamic interplay to coordinate both the induction and repression of gene expression. Although transcription factors can serve as substrates for HATs, no direct role for such molecules in hormone signaling had been identified (25Kouzarides T. Curr. Opin. Genet. Dev. 1999; 9: 40-48Crossref PubMed Scopus (587) Google Scholar). Intrinsic HAT activity for histone lysines is shared redundantly by ERα transcriptional regulatory proteins, which include p300, CBP, P/CAF, SRC1, and ACTR (26Kadonaga J.T. Cell. 1998; 92: 307-313Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 27Martinez-Balbas M.A. Bannister A.J. Martin K. Haus-Seuffert P. Meisterernst M. Kouzarides T. EMBO J. 1998; 17: 2886-2893Crossref PubMed Scopus (227) Google Scholar). Redundancy of the HAT function among cointegrators raises the fundamental question of whether alternate substrates to histones may be involved in hormonal signaling. In the current studies we show that the ERα is acetylated in vivo and is directly and selectively acetylated by p300, but not by P/CAF, within the ERα hinge region at conserved lysines in vitro. Substitution mutation established an important role for these acetylated residues in both ligand-dependent and -independent functions, suggesting local conformational changes may regulate interactions between the two activation domains of the ERα. Conservation of the ERα motif acetylated in vitro between a subset of nuclear receptors raises the possibility that direct acetylation may regulate diverse functions of phylogenetically related nuclear receptors. The ERE luciferase reporter gene ERE2TK81 pA3LUC (28Schlegel A. Wang C. Katzenellenbogen B. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1999; 274: 33551-33556Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the Flag-tagged P/CAF mutants (29Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H.Y. Wang J.Y. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), the ERα fusion proteins (30Fan S. Wang J.-A. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar), pcDNA3-HA-p300 (31Albanese C. D'Amico M. Reutens A.T. Fu M. Watanabe G. Lee R.J. Kitsis R.N. Henglein B. Avantaggiati M. Somasundaram K. Thimmapaya B. Pestell R.G. J. Biol. Chem. 1999; 274: 34186-34195Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), the constitutively active MEK1 plasmids, pCMV-ΔN3, pCMV-RΔF (ΔN3-S218E-S222D), and the catalytically inactive mutant MEK1 (K97M) (32Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-969Crossref PubMed Scopus (1259) Google Scholar, 33Watanabe G. Howe A. Lee R.J. Albanese C. Shu I.-W. Karnezis A.N. Zon L. Kyriakis J. Rundell K. Pestell R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12861-12866Crossref PubMed Scopus (196) Google Scholar) were described previously. The ERα mutants were derived by polymerase chain reaction-directed amplification using sequence-specific primers. Both the wild type ERα and ERα mutants were cloned into pCI-neo (Promega, Madison, WI). The integrity of all constructs was confirmed by sequence analysis. Cell culture, DNA transfection, and luciferase assays were performed as previously described (30Fan S. Wang J.-A. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar, 34Watanabe G. Albanese C. Lee R.J. Reutens A. Vairo G. Henglein B. Pestell R.G. Mol. Cell. Biol. 1998; 18: 3212-3222Crossref PubMed Scopus (145) Google Scholar). Cells were incubated in media containing 10% charcoal-stripped fetal bovine serum prior to experimentation using estradiol and transfected by calcium phosphate precipitation or Superfect transfection reagent (Qiagen, Valencia, CA). The medium was changed after 5 h and luciferase activity determined after 24 h. Luciferase activity was normalized for transfection using β-galactosidase reporters or Renilla luciferase as an internal control exactly as described previously (20Martinez-Balbas M.A. Bauer U.-M. Nielson S., J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (568) Google Scholar). The antibodies used in Western blot analysis were anti-M2 Flag (Sigma), anti-guanine nucleotide dissociation inhibitor (35Lee R.J. Albanese C. Fu M. D'Amico M. Lin B. Watanabe G. Haines G.K.I. Siegel P.M. Hung M.C. Yarden Y. Horowitz J.M. Muller W.J. Pestell R.G. Mol. Cell. Biol. 2000; 20: 672-683Crossref PubMed Scopus (308) Google Scholar), anti-acetyl lysine (16Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar), and GST (B-14) and ERα (H-184) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). In vitro [35S]methionine-labeled proteins were prepared by coupled transcription-translation with a PromegaTNT®-coupled reticulocyte lysate kit (Promega), using 1.0 μg of plasmid DNA in a total of 50 μl. Flag-tagged P/CAF proteins were expressed in Sf9 cells by infecting with recombinant baculovirus and purified using an anti-Flag antibody (Sigma, M2) (36Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2387) Google Scholar). Full-length recombinant baculovirus ERα was obtained from Affinity Bioreagents, Inc. (Golden, CO). Immunoprecipitation histone acetyl transferase (IP-HAT) assays were performed using p300 as described previously (16Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar, 37Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1529) Google Scholar). For immunoprecipitation the protein concentration was adjusted to 1 μg/μl in 500 μl. The relevant antibodies from Santa Cruz Biotechnology (p300, N15) were added (2 μg/500 μg of extract) and incubated at 4 °C for 2 h. A standard HAT assay was performed containing 5 μg of substrate and enzyme, either 200 ng of purified histone acetyl transferase (purified baculovirus p300 or P/CAF) or immunoprecipitated p300 from cultured cells (16Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar, 37Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1529) Google Scholar). The mixture was incubated at 30 °C for 1 h. 90 pmol of [14C]acetyl-CoA reaction was electrophoresed on a SDS-polyacrylamide gel and viewed following autoradiography of the gel. [14C]acetyl incorporation into the substrates was also determined by liquid scintillation counting or filter assays. The interactions between in vitro expressed proteins was performed as described previously (38Neuman E. Ladha M.H. Lin N. Upton T.M. Miller S.J. DiRenzo J. Pestell R.G. Hinds P.W. Dowdy S.F. Brown M. Ewen M.E. Mol. Cell. Biol. 1997; 17: 5338-5347Crossref PubMed Scopus (346) Google Scholar). The in vitro translated protein (15 μl of ERα), 1 μg of rabbit anti-ERα polyclonal antibody (H184, Santa Cruz Biotechnology), and 5 μg of purified Flag-tagged baculovirus-expressed P/CAF were incubated in 300 μl of binding buffer. In vitro acetylation assays were performed as described previously 1(7). Synthetic peptide corresponding to the ERα (ER1, residues 293–310, NH2-PSPLMIKRSKKNSLALSL-OH, and ER2, residues 353–370, NH2-ELVHMINWAKRVPGFVDL-OH) were synthesized by Bio·Synthesis (Lewisville, TX) and purified to 95% purity by HPLC. The peptides were acetylated in vitro by incubation with 5 mm acetyl-CoA and baculovirus-purified Flag-p300 or P/CAF at 30 °C for 2 h. After incubation, acetylated peptides were separated from contaminating p300 by passage through a micron filter (Amicon Inc., Beverly, MA) and further purified by analytical reversed phase HPLC. The reaction products were analyzed with a PE-Biosystems DE-STR MALDI-TOF mass spectrometer. Further analysis by Edman degradation was performed on a PE-Biosystems Procise sequencer. Phenylthiohydantoin-acetyl-lysine was measured by absorbance at 259 nm. The p300/CBP coactivator proteins have been shown to regulate several promoters in a manner dependent upon their histone acetylase activity (25Kouzarides T. Curr. Opin. Genet. Dev. 1999; 9: 40-48Crossref PubMed Scopus (587) Google Scholar), and p300 can both bind and stimulate the activity of the ERα (4Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar, 8Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (289) Google Scholar, 10Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar). In addition, p300/CBP and P/CAF have been shown to acetylate non-core histone-related transcription factors directly through a conserved motif. We assessed whether p300 could acetylate recombinant ERα in vitro. Recombinant p300 acetylated recombinant ERα but did not acetylate GST (Fig.1 A). In contrast, recombinant baculovirus-expressed P/CAF did not acetylate ERα, although it was capable of acetylating histone H3 and itself (Fig. 1 B) as shown previously (39Schiltz R.L. Mizzen C.A. Vassilev A. Cook R.G. Allis C.D. Nakatani Y. J. Biol. Chem. 1999; 274: 1189-1192Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Two fundamental types of questions raised by these studies are, first, the relative efficiency of ERα acetylation and, second, whether the failure of P/CAF to acetylate the ERα is due to failed binding or substrate selectivity. To assess the relative efficiency with which p300 acetylates the ERα, a direct comparison was made between equimolar amounts of ERα and histone H3. The products acetylated by increasing amounts of p300 were electrophoresed on a SDS-polyacrylamide gel and the incorporation of [14C]acetyl-CoA assessed (Fig.2 A). The efficiency of incorporation on an equimolar basis was ∼3-fold greater for histone H3 (16 kDa) than ERα (66 kDa) (Fig. 2 B), suggesting ERα is acetylated with substantial efficiency. Thus the ERα is efficiently and selectively acetylated by p300 in vitro. P/CAF has been reported to associate with ERα in vitro(40McMahon C. Suthiphongchai T. DiRenzo J. Ewen M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5382-5387Crossref PubMed Scopus (151) Google Scholar). We examined whether the recombinant P/CAF used in the HAT assays bound to the ERα. As shown in Fig. 2 C, recombinant P/CAF bound with high affinity to ERα, and binding required the HAT domain. Thus, although P/CAF acetylates histone H3 and H4, the failure of P/CAF to acetylate ERα is not due to failed binding. These findings are consistent with the observation that p300 and P/CAF have distinguishable substrate specificities (21Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (653) Google Scholar). To identify the residues required for ERα acetylation in vitro, recombinant GST-ERα fusion fragments were expressed, their integrity was confirmed by Western blotting using a GST antibody, and equal amounts of proteins were assayed in HAT assays using recombinant p300 as a source of HAT activity and the previously described filter assay (16Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2168) Google Scholar). As shown in Fig. 3, Band C, the ERα from residues 282–337 was sufficient to function as a substrate for acetylation by p300. Peptides were synthesized to encompass the two lysine-containing motifs identified within the region of the ERα acetylated in vitro (Fig. 3 D). We identified residues resembling an acetylation motif found in the p53 and GATA-1 transcription factors, which were conserved between species (Fig. 3 D). An additional lysine, residue 362, was identified that had been implicated previously in ligand-regulated ERα function (41Henttu P.M. Kalkhoven E. Parker M.G. Mol. Cell. Biol. 1997; 17: 1832-1839Crossref PubMed Scopus (192) Google Scholar). Polypeptides were synthesized therefore to include residues encoding the consensus acetylation motif ER1-(293–310) (ER1) and a second polypeptide including lysine 366 (ER2-(353–370)) (ER2). HAT assays were performed using recombinant p300 or P/CAF. p300 acetylated the ER1 polypeptide but did not acetylate ER2 (Fig. 3 D). Recombinant P/CAF failed to acetylate either ER polypeptides. Mass analysis of the acetylated ER1 peptide confirmed the presence of two major ions differing by 42 mass units, with the smaller molecular weight product corresponding to the unmodified ER1 peptide and the higher molecular weight component corresponding to the acetylated ER1 product (Fig. 4 A). Followingin vitro acetylation of the ER1 peptide, Edman degradation assays were performed. As only monoacetylated lysine-containing peptides were detected in the samples by MALDI-TOF mass spectrometry, the product analyzed by Edman degradation was a heterogeneous population of polypeptides, each acetylated at a single site (Fig.4 A). These studies demonstrated that lysines 302 and 303 of the ERα were preferentially acetylated by p300 with an additional acetylation site at lysine 299 (Fig. 4 B). To examine the role of histone acetylases in the regulation of ERα activity, an estrogen-responsive luciferase reporter gene was assessed in ERα-deficient cells (MDA MB231). Inhibitors of histone deacetylase(s) trichostatin A (TSA) and sodium butyrate were added to transfected cells for 24 h. TSA induced the ERE-LUC reporter (ERE2TKpA3LUC) 4–6-fold (Fig.5 A). Similarly, sodium butyrate (1 mm) induced ER reporter activity 2-fold (Fig.5 B). To examine the functional consequence of lysines 302 and 303 in ERα function, point mutation of the ERα acetylation sites was performed. The ER-responsive reporter was assessed in ERα-deficient cells (MDA MB231 and HeLa). Activity was assessed through normalization to the internal standard β−galactosidase reporter. The 2-fold induction of wild type ERα by sodium butyrate was abolished by the ER(K302A/K303A) mutant (Fig. 5 C). The abundance of the ERαK302A/K303Amutant was similar to ERα wild type in cultured cells (Fig.5 D). HeLa cells were transfected with either wild type ERα or mutants of the acetylation site and assessed for ERE activity. The wild type ERα was induced 3-fold by the addition of TSA in a dose-dependent manner (Fig. 5 E). Both the alanine and threonine substitutions failed to respond to TSA (Fig.5 E). Together these findings suggest that direct ERα acetylation contributes to induction by histone deacetylase inhibitors. To investigate further the in vivoconsequence of the ERα acetylation site, point mutation substitutions were introduced into the wild type ERα at the lysine residues acetylated in vitro. It was reasoned that the acetylation of a lysine, a positively charged, hydrophobic residue, is thought to both reduce its charge and increase its polarity. If acetylation augments activity through increasing the polarity or reducing the charge, a mutation of the two ERα lysines to polar residues, ER(K302Q/K303Q), may function as an activating mutant. The introduction of a large positively charged amino acid with a significant side chain (ER(K302R/K303R) might be anticipated to mimic acetylation if increasing polarity is of greater importance. Substitution of lysine to alanine, (ER(K302A/A303A)) or another small hydrophobic threonine residue (ER(K302T/K303T)) was anticipated to result in a loss of function. If the post-translational modification of acetylation itself were important in regulating ERα activity, the substitution of the lysine residues with any of these other residues would be expected to have a similar effect. The results of these studies are shown in Fig. 6. The mutant ERα proteins were expressed equally in transfected cells (data not shown). HeLa cells were transfected with either wild type ERα or mutants of the acetylation site and assessed for their ability to regulate the activity of a synthetic ERE in the absence of ligand. Assessment was made of the AF-1 function mediated by MAPK signaling. Growth factors induce ligand-independent activity of the ERα through activation of MAPK (5Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1710) Google Scholar) and the p160 coactivator AIB1 (also named RAC3, ACTR, or SRC3) (42Font de Mora J. Brown M. Mol. Cell. Biol. 2000; 20: 5041-5047Crossref PubMed Scopus (400) Google Scholar). p160 proteins bind p300 (43Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1262) Google Scholar) and contact both the AF-1 and AF-2 of the ERα (44Webb P. Nguyen P. Shinsako J. Anderson C. Feng W. Nguyen M.P. Chen D. Huang S.M. Subramanian S. McKinerney E. Katzenellenbogen B.S. Stallcup M.R. Kushner P.I. Mol. Endocrinol. 1998; 12: 1605-1618Crossref PubMed Scopus (0) Google Scholar, 45Tremblay A. Tremblay G.B. Labrie F. Giguere V. Mol. Cell. 1999; 3: 513-519Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). To determine whether the lysine substitutions within the ERα hinge regulated MAPK-dependent ERα activity, constitutively activated MEK1 (ΔN3, ΔN3-S218E-S222D) were coexpressed with the ERα mutants (Fig. 6 A). The wild type ERα was induced 3.5-fold by activated MEK1 but was not significantly induced by the catalytically defective MEK1 (K97 m). The basal activity of the ERα(K302A/K303A) mutant was reduced 2.5-fold; however, the magnitude of induction by activated MEK1 was not significantly changed for any of the mutants (Fig. 6 A). The finding that the ERα acetylation mutants are not altered in their responsiveness to MAPK activation suggests the mechanisms governing ligand-induced ERα activity through the ERα acetylation site are distinct from those governed by ACTR. In previous studies of ERα activity in HeLa cells using a similar reporter assay, estradiol (10−8m) induced ERE-dependent luciferase activity 2-fold (41Henttu P.M. Kalkhoven E. Parker M.G. Mol. Cell. Biol. 1997; 17: 1832-1839Crossref Pub
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