Induction-independent Recruitment of CREB-binding Protein to the c-fos Serum Response Element through Interactions between the Bromodomain and Elk-1
2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês
10.1074/jbc.m007824200
ISSN1083-351X
AutoresL. Johan Nissen, Jean‐Christophe Gelly, Robert A. Hipskind,
Tópico(s)Chromatin Remodeling and Cancer
ResumoProliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100–1286, and Elk-1. DNA binding and glutathioneS-transferase pull-down assays demonstrate that binding requires Elk-11–212 but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100–1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100–1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation. Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE). Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP). We report a novel interaction between the bromodomain of CBP, amino acids 1100–1286, and Elk-1. DNA binding and glutathioneS-transferase pull-down assays demonstrate that binding requires Elk-11–212 but not the C-terminal transactivation domain. Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site. Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100–1286) interacts via Elk-1 and does not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100–1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively. These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation. mitogen-activated protein kinase calcium- and cAMP-responsive element serum response element v-sis-inducible element protein kinase A cAMP response element binding protein mitogen-activated protein kinase-activated protein kinase signal transducer and activator of transcription serum response factor ternary complex factor extracellular signal-regulated kinase stress-activated protein kinase CREB binding protein amino acid(s) Dulbecco's modified Eagle's medium glutathione S-transferase polyacrylamide gel electrophoresis N-lauroyl sarcosine polymerase chain reaction bovine serum albumin wild type SRF-(90-245) c-fos AP1-like element One response of cells to extracellular stimuli is the activation of signaling pathways that lead to changes in gene expression mediating proliferation, differentiation, and apoptosis. A major messenger system to these downstream events is the various MAPK1 signaling pathways that play a key role in this response by the activation of immediate early genes like the proto-oncogene c-fos (1Karin M. Curr. Opin. Cell Biol. 1994; 6: 415-424Crossref PubMed Scopus (359) Google Scholar). The c-fos promoter contains three major regulatory elements, the CaCRE, the SRE, and the SIE (2Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar, 3Hipskind R.A. Bilbe G. Front. Biosci. 1998; 3: D804-D816Crossref PubMed Scopus (98) Google Scholar). The Ca2+ and cAMP-response element (CaCRE) can mediate activation by cAMP-PKA or Ca2+-calmodulin-dependent kinase signals, which lead to phosphorylation of the transcription factor CREB on Ser-133 (4Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2064) Google Scholar). CREB is also targeted by MAPK cascades through the activation of MAPKAP kinases (5Xing J. Kornhauser J.M. Xia Z. Thiele E.A. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 1946-1955Crossref PubMed Google Scholar, 6Tan Y. Rouse J. Zhang A. Cariati S. Cohen P. Comb M.J. EMBO J. 1996; 15: 4629-4642Crossref PubMed Scopus (568) Google Scholar). The v-sis-inducible element (SIE) is targeted through cytokine- and growth factor-driven STAT1 and -3 activation (7Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3400) Google Scholar), and their activity can be modulated by the MAPKs. The serum response element (SRE) on the c-fos promoter alone is sufficient to confer a signal-dependent activation (8Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (347) Google Scholar). Genomic footprinting studies have shown that a complex is assembled over this promoter before, during, and after induction (9Herrera R.E. Shaw P.E. Nordheim A. Nature. 1989; 340: 68-70Crossref PubMed Scopus (251) Google Scholar). A complex with similar characteristics can be reproduced on the SRE in vitro by a dimer of serum response factor (SRF) and one molecule of ternary complex factor (TCF) (10Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). The TCFs are encoded by a family of Ets proteins that includes Elk-1, SAP-1a, and a third member variously called NET, ERP, or SAP-2 (11Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Crossref PubMed Scopus (622) Google Scholar). Elk-1 and Sap-1a play a key role in translating signals from kinases into transcriptional activation. The TCFs represent major nuclear targets for the MAPKs ERK, p38, and SAPK. The resulting phosphorylation of TCF plays a major role in the induction of the c-fos gene by a mechanism that remains to be fully resolved (2Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar). SRE-driven transcriptional activation appears to involve coactivators of the CBP/p300-family. CBP has been described to interact with the C-terminal transactivation domains of the TCFs Elk-1 and Sap-1a and with full-length SRF (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar, 14Ramirez S. Ait-Si-Ali S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This involves the CBP region spanning aa 451–721 for TCF (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar) and the N-terminal CBP region spanning aa 1–1097 for SRF (14Ramirez S. Ait-Si-Ali S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Accordingly, CBP increases transcriptional activation by Elk-1, SAP-1a, and SRF in transient transfection assays (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar, 14Ramirez S. Ait-Si-Ali S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 15Kim H.J. Kim J.H. Lee J.W. J. Biol. Chem. 1998; 273: 28564-28567Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Thus, SRE-driven transcriptional activation appears to involve the recruitment of CBP to the ternary complex, as has also been observed for the CREB·CaCRE and STAT·SIE c-fos promoter complexes (16Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1770) Google Scholar, 17Zhang J.J. Vinkemeier U. Gu W. Chakravarti D. Horvath C.M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15092-15096Crossref PubMed Scopus (423) Google Scholar, 18Horvai A.E. Xu L. Korzus E. Brard G. Kalafus D. Mullen T.M. Rose D.W. Rosenfeld M.G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1074-1079Crossref PubMed Scopus (388) Google Scholar). The kinetics of c-fos transcriptional induction suggest that the recruitment of the coactivator is either a very rapid process or that the coactivator is already present on the promoter. Here we have tested the latter possibility, namely that CBP might interact constitutively with the complex assembled over the SRE. We show that the bromodomain of CBP interacts with the TCF Elk-1 in solution and generates an Elk-1-dependent quaternary complex in vitro on the SRE. A similar Elk-1-dependent complex forms on the E74 site bound directly by Elk-1. Moreover, this novel complex represses transcriptional activation of SRE and E74 reporter genes driven by RasV12, whereas activity is restored by including the C- and N-terminal activation domains of CBP. These data suggest a model where the bromodomain anchors CBP to the SRE via TCF in a higher order complex. This would facilitate the rapid transcriptional activation of c-fos mediated by CBP through interaction with transcription factors targeted by signaling-driven phosphorylation. Restriction enzymes were obtained from Life Technologies and New England BioLabs. Antibodies against the DNA binding domain of Gal4 were purchased from CLONTECH and Santa Cruz Biotechnology, Inc., rat monoclonal antibodies to hemagglutinin were from Roche Molecular Biochemicals, and the Elk-1 antibody against the Ets domain (aa 1–82) of Elk-1 (α-Ets) and the SRF antibody (NOP-13) have been described previously (19Zinck R. Hipskind R.A. Pingoud V. Nordheim A. EMBO J. 1993; 12: 2377-2387Crossref PubMed Scopus (143) Google Scholar, 20Hipskind R.A. Rao V.N. Mueller C.G. Reddy E.S. Nordheim A. Nature. 1991; 354: 531-534Crossref PubMed Scopus (350) Google Scholar). Horseradish peroxidase-coupled secondary antibodies were from Sigma-Aldrich. Glutathione-Sepharose, poly (dI-dC)·(dI-dC), ECL-plus nitrocellulose membranes, and the T7 DNA polymerase sequencing kit were purchased from Amersham Pharmacia Biotech, whereas Talon metal-affinity resin came from CLONTECH. Transfast transfection reagent, a TnT-coupled in vitro transcription translation kit, Taq DNA polymerase, and the Dual Luciferase kit were purchased from Promega. Radioisotopes, ECL reagents, and polyvinylidene difluoride were obtained from PerkinElmer Life Sciences. Immobilon-P polyvinylidene difluoride membrane was from Millipore. DMEM and Dulbecco's PBS and FBS were purchased from Life Technologies. X-ray films and intensifying screens were purchased from Kodak, and B-Per reagent was purchased from Pierce. Nucleobond AX plasmid purification cartridges and the NucleoSpin extract kit were obtained from Machery-Nagel, and Jetstar plasmid cartridges came from Q-biogene. Protease and phosphatase inhibitors were from Sigma; all other ultra-pure biochemicals were from AppliChem (Darmstadt, Germany). The Gal4-CBP and GST-CBP subclones have been previously described (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar). The Gal4-CBP-(1–1460) construct was generated by insertion of the N-terminal portion of CBP into theSal I and Nar I sites of the Gal4-CBP-(1100–1460) subclone. The Gal4-CBP-(1100–2441) construct was generated by insertion of the C-terminal fragment of CBP into the Bam HI and Nar I sites of the Gal4-CBP-(1100–1460) subclone. The Elk-1 expression vector used for in vitro transcription (aa 1–428) was previously described (21Janknecht R. Nordheim A. Nucleic Acids Res. 1992; 20: 3317-3324Crossref PubMed Scopus (117) Google Scholar). The Elk-1 N-terminal (aa 1–308) and C-terminal aa (Δ14–308) expression vectors for in vitro transcription were generated by digesting the corresponding bacterial expression constructs and subcloning them into the pCal-n vector (Stratagene). The 3xSRE-, 1xEL-, and 3xEL-fos TATA-luciferase reporter genes were kindly provided by G. Bilbe (Novartis Pharma, Basel, Switzerland). The 4xE74-fos TATA-luciferase reporter construct was generated in this vector by inserting anNot I-Spe I fragment containing four tandem copies of the E74 sequence (21Janknecht R. Nordheim A. Nucleic Acids Res. 1992; 20: 3317-3324Crossref PubMed Scopus (117) Google Scholar). The RasV12 and SV40 renilla luciferase expression vectors were kindly provided by A. Philips (Institut de Génétique Moléculaire de Montpellier, Montpellier, France) and a CBPwt expression vector by R. Janknecht (Department of Biochemistry, Mayo Clinic, Rochester, MN). The original mouse CBPwt expression vector used for the GST- and Gal4-CBP constructs (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar) contained a stop codon after aa 1286 leading to truncated proteins from the original clones spanning this region. Therefore the vectors originally described as GST-CBP-(1100–1460) and Gal4-CBP-(1100–1460) actually express a protein containing CBP amino acids 1100–1286 and are labeled accordingly. Plasmids were purified using cartridge systems forin vitro manipulations and by double banding in CsCl for transfections. The oligos 5′-AGTCGAATTCGAGGAGCTACGCCAGGCACTTATGC-3′ (upper strand) and 5′-AGCTAAGCTTCTAAGACTGCATGACAGGGTCAATT-3′ (lower strand) were used to generate a fragment of CBP spanning the bromodomain (aa 1089–1196) flanked by Eco RI and Hin dIII sites to facilitate cloning into expression vectors. The 25-μl PCR reactions contained 2 ng of CBPwt expression vector, 300 nm of each oligonucleotide, 200 nm dNTPs, 1.5 mm MgCl2, the supplier's reaction buffer (Promega), and 5 units of Taq polymerase. After 4 min at 95 °C, amplification was carried out for 24 cycles (0.5 min, 95 °C; 0.5 min, 55 °C; 0.5 min, 72 °C). The fragments were purified using a NucleoSpin extract kit, digested with Eco RI and Hin dIII, and then purified from an agarose gel using the NucleoSpin kit. After cloning into Bluescript KS+ (Stratagene), positive clones were confirmed by sequencing. The bromodomain-encoding fragment was recloned into pGEX-2T-6His and clones screened for expression of the GST-bromo fusion protein, as well as into pCDNA3.1-FLAG. This vector was used to confirm bromodomain expression by coupled in vitro transcription translation (see below). Escherichia coli strain BL21(LysE) was electroporated with the appropriate expression vector. Fresh colonies were used to inoculate 200-ml bacterial cultures, which were grown at 37 °C, and recombinant protein expression induced in exponential phase by adding isopropyl thiogalactoside to 0.1 mm. After 1–4 h at 37 °C, bacteria were collected by centrifugation and lysed in 10 ml of B-Per reagent according to the supplier's recommendations. Insoluble proteins were removed by centrifugation at 27,000 ×g for 15 min at 4 °C. The supernatant was brought to 1 mm imidazole and incubated overnight with 50–200 μl of Talon resin at 4 °C. The mix was poured into a column, the resin was washed twice with 5 ml of RJD* buffer (10 mm HEPES, pH 7.9, 5 mm MgCl2, 50 mm NaCl, 17% glycerol, 0.1 mm EDTA, 1 mmdithiothreitol, 0.05% Nonidet P-40) containing 1 mmimidazole and freshly added protease inhibitors (2.5 μg/ml aprotinin, leupeptin, pepstatin, 0.5 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride). Recombinant His-tagged proteins were eluted with two column volumes of RJD* buffer containing 200 mm imidazole, pH 7.6, and stored in aliquots at −70 °C. 35S-Labeled Elk-1 proteins were synthesized from Bluescript KS+ vectors encoding Elk-1-(1–428), Elk-1-(1–307), or Elk-1-(308–428) by coupled in vitro transcription/translation in the presence ofl-[35S]methionine (1000 Ci/mmol) using TnT kits according to the supplier's recommendations. C-terminal truncated proteins were produced identically, using the Elk-1-(1–428) vector digested with the following restriction enzymes: Xba I (Elk-1-(1–428)), Bsm I (Elk- 1-(1–374)), Apa I (Elk-1-(1–253)), Stu I (Elk-1-(1–212)), and Nsp I (Elk-1-(1–122)). Recombinant proteins purified by metal affinity chromatography were directly bound to glutathione-Sepharose beads at 4 °C for 1 h. The beads were then washed three times with a 100-fold excess of RJD*-buffer containing protease inhibitors and stored short-term at 4 °C. 5 μl of the in vitro translated Elk-1 proteins described above were added to 10 μl of a 50% slurry of protein-bearing beads in a total volume of 100 μl of RJD* buffer containing protease inhibitors and incubated with gentle agitation at 4 °C for 4 h. After three washes with 500 μl of RJD*, the beads were collected, resuspended in 20 μl of 5× Laemmli buffer (0.25 m Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromphenol blue) and denatured for 5 min at 95 °C, and the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Proteins were visualized by Coomassie Blue staining, followed by autoradiography of the dried gels. Proteins were subjected to electrophoresis on 6–10% SDS-PAGE minigels. Proteins larger than 100 kDa were transferred to nitrocellulose membranes by immersion blotting, whereas smaller proteins were immobilized on either nitrocellulose or polyvinylidene fluoride membranes by semi-dry transfer. Membranes were blocked with 5% dry milk in TBST (50 mm Tris-HCl, pH 7.5, 140 mm NaCl, 3 mm KCl, and 0.05% Tween 20) and then incubated with the indicated primary antibody diluted 1:1000 into the blocking buffer. After washing in TBST (6 × 5 min), the membranes were incubated with the appropriate secondary antibody coupled to horseradish peroxidase diluted in blocking buffer. The membranes were again washed in TBST as above, and the immune complexes were visualized by enhanced chemiluminescence. NIH3T3 cells were maintained in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin. Cells were seeded to 30% confluency in 6-well plates 16 h prior to transfection. The medium was changed 1 h before transfection, which was performed using a classical Ca2PO4 protocol (22Philips A. Chambeyron S. Lamb N. Vie A. Blanchard J.M. Oncogene. 1999; 18: 6222-6232Crossref PubMed Scopus (36) Google Scholar). The transfection mixes contained 300 ng of Luciferase reporter construct, 1 μg of Gal4-CBP expression vector, 200 ng of RasV12 expression vector, 5 ng of pSV-Renilla Luciferase expression vector, and 3.5 μg of pUC18. These optimal values were determined experimentally several times. Cells were washed in phosphate-buffered salt solution 8–10 h after transfection and maintained in serum-free DMEM for 40 h. Cell extracts were prepared, and luciferase activity was determined following the protocol supplied with the Dual Luciferase kit (Promega). Measurements were made in duplicate using a Berthold Lumat LB950. For protein overexpression, COS-7 cells were transfected using Transfast (Promega) according to the supplier's protocol, and whole cell extracts were prepared as described (23Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). Protein concentrations were determined using Bradford's reagent with BSA as a standard. Protein expression was confirmed using immunoblotting prior to gel retardation analyses. Probes for binding analyses were prepared from subcloned SRE (20Hipskind R.A. Rao V.N. Mueller C.G. Reddy E.S. Nordheim A. Nature. 1991; 354: 531-534Crossref PubMed Scopus (350) Google Scholar), EL (10Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar), and E74 sequences (21Janknecht R. Nordheim A. Nucleic Acids Res. 1992; 20: 3317-3324Crossref PubMed Scopus (117) Google Scholar) by Klenow enzyme-catalyzed end labeling of anEco RI-Nar I fragment in the presence of 50 μCi of [α-32P]dATP (3000 Ci/mmol, 10 μCi/μl) and 300 μm dTTP (23Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). The labeled fragments were purified from acrylamide gels by electroelution. The gel retardation experiments were carried out as previously described (23Hipskind R.A. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar). In brief, reactions (7.5 μl) contained 4 fmol of labeled probe, a salt/protein/buffer mix, 2.5 μg of poly (dI-dC)·(dI-dC), recombinant coreSRF-(90–245) with SRE probes, and the proteins indicated in the figures. Where appropriate, 0.5–1 μl of antibodies was added to the reaction before the addition of protein extracts. After incubation for 30 min at room temperature, complexes were resolved on a 5% polyacrylamide gel containing 0.5× TBE at 1 mA/cm for 4 h. Complexes were visualized by autoradiography and phosphorimaging of the dried gel. SRE probes were prepared from the same site described above but cloned into pUC18 at theXba I site. After digestion withHin dIII-Xma I, the upper and lower strands were end-labeled using Klenow enzyme, 300 μm dGTP, and either 50 μCi of [α-32P]dATP (3000 Ci/mmol, 10 μCi/μl) for the Hin dIII end or 50 μCi of [α-32P]dCTP (3000 Ci/mmol, 10 μCi/μl) for theXma I end. The gel-purified fragment was treated for 3.5 min at 22 °C with 1% dimethyl sulfate in 60 mm NaCl, 10 mm Tris-HCl, pH 8.0, 1 mm EDTA. Reactions were stopped as described previously (24Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9014) Google Scholar), and the probes were subjected to several rounds of alcohol precipitation. Binding reactions were prepared and analyzed as for gel retardation analysis except that all components were increased 5-fold. The complexes were visualized by autoradiography of the wet gel. Regions of the gel corresponding to different complexes and free DNA were electrotransferred to DEAE paper (NA45, Schleicher and Schuell) and eluted in 0.5 m NaCl, 0.5% SDS, 20 mm Tris-HCl, pH 8.0, 0.5 μg/ml Proteinase K at 65 °C. The eluted fragments were purified by organic extraction followed by ethanol precipitation. The fragments were incubated for 40 min at 95 °C in freshly made 1 m piperidine, and the piperidine was removed by multiple rounds of lyophilization and resuspension in H2O. The samples were finally resuspended in formamide dye mix (0.3% each bromphenol blue and Xylene Cyanol FF; 10 mm EDTA, pH 7.5, 80% deionized formamide), denatured for 5 min at 95 °C, and analyzed by denaturing electrophoresis in a 10% polyacrylamide-8.5 m urea sequencing gel. The gel was dried, and radioactivity was visualized by autoradiography at −70 °C with an intensifying screen and phosphorimaging as described above. Preparative reactions were carried out as described above but incubated for 30 min at 4 °C. Reactions were brought to 2.5 mm CaCl2, 7.5 mmMgCl2 were attained and incubated with 0.01 unit of DNaseI at 37 °C for 1.5 min. The reaction was terminated by adding 100 μl of 0.01% Sarkosyl, 0.1 m Tris-HCl, pH 8.0, 0.1m NaCl, 0.02 mm EDTA, 0.03 μg/μl calf thymus DNA, and 0.075 μg/μl Proteinase K, incubated an additional 15 min at 37 °C followed by 5 min at 95 °C. Labeled DNA was purified by organic extraction and alcohol precipitation, resuspended in formamide dye mix, denatured again, and analyzed on 10% sequence gels as described above. To investigate the interactions between Elk-1 and CBP in detail, different subdomains of CBP were fused to the Gal4 DNA binding domain for expression in mammalian cells and to glutathione transferase to facilitate purification after overexpression in bacteria (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar). The different expression constructs are presented schematically in Fig.1 A. Immunoblotting with a Gal4-DNA binding domain antibody showed that the various fusion proteins were expressed at different levels after transient transfection of COS-7 cells (Fig. 1 B). Similarly, variable amounts of the various GST fusion proteins were obtained after affinity purification of bacterially expressed proteins (Fig. 1 C,lower panel). These assays were used to determine the functional protein concentration for subsequent experiments. It should be noted that the fusion proteins encoded by Gal4-CBP-(1100–1460) (12Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar,13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar) are truncated due to the presence of a stop codon after aa 1286 in the original CBP expression vector. Therefore, this protein contains the bromodomain of CBP but not the histone acetyltransferase domain immediately downstream and will subsequently be labeled CPB-(1100–1286). We first tested for solution interactions between various regions of CBP and Elk-1. Recombinant, bacterially produced GST-CBP fusion proteins were immobilized on glutathione-agarose beads and incubated with 35S-labeled Elk-1 (aa 1–428) produced by coupled in vitro transcription/translation (Fig.1 C). Two versions of Elk-1 are produced in vitro, the smaller of which arises from internal initiation of translation at Met-55 (also see below) (25Vanhoutte, P., Nissen, J. L., Brugg, B., Della Gaspera, B., Besson, M. J., Hipskind, R. A., and Caboche, J. (2000)J. Biol. Chem., in pressGoogle Scholar). Both full-length and N-terminal truncated Elk-1 bound to CBP-(451–721), which has previously been shown to interact with the C-terminal activation domain of Elk-1 (13Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (173) Google Scholar). Full-length and N-terminal truncated Elk-1 also interacted with CPB-(1100–1286), which spans the bromodomain, and CBP-(1460–1891), which contains most of the histone acetyl transferase domain. The increased yield of the truncated version of Elk-1 versus that of full-length Elk-1, particularly in respect to their relative levels in the reaction input (left lane), suggests that these two CBP domains bound preferentially to N-terminal truncated Elk-1 in solution. Neither version of Elk-1 interacted with GST alone (Fig. 1 C) or with other domains of CBP (not shown), nor did we detect any binding of SRF produced in vitro to CBP (not shown). We then investigated the solution interaction between the bromodomain of CBP and different portions of Elk-1. To this end, in vitro translated 35S-labeled full-length (aa 1–428), N-terminal (aa 1–307), and C-terminal (aa 308–428) Elk-1 were used as above (Fig. 1 D). Similar to full-length Elk-1, the N-terminal construct gave rise to two translation products in vitro, unlike the C-terminal construct. This further supports the notion that the smaller form of Elk-1 arises from internal initiation of translation and not premature termination. Once again, full-length Elk-1 bound to the bromodomain of CBP in solution, as did Elk(NT), with some preference shown for the truncated version (compare bound lanes to input). CBP-(1100–1286) also interacted within vitro-translated C-terminal domain of Elk-1. Interestingly this interaction appears to be independent of its activation by phosphorylation, because Elk-(308–428) is not phosphorylated during in vitro translation. In contrast to these interactions, none of these proteins bound to GST alone (Fig.1 D). Thus we can detect complexes between multiple domains of CBP, in particular the bromodomain, and Elk-1 in solution. To confirm that the bromodomain alone (CBP aa 1089–1196) accounts for binding to Elk-1, an expression vector for GST-CBP-(1089–1196) was generated by PCR. This protein showed interactions with Elk(FL) and Elk(NT) that were indistinguishable from CBP-(1100–1286) (Fig.1 E) and in fact showed binding to progressively truncated versions of Elk until the region between amino acids 212 and 122 was deleted (Fig. 1 E). This indicates that the region in Elk-1 spanning aa 122 to 212 is important for interaction in solution with the CBP bromodomain. The interactions in solution between the bromodomain of CBP and the TCF Elk-1 led us to test for similar interactions in the context of DNA-bound complexes, using the E74 Ets sequence to which Elk-1 binds directly as a monomer (21Janknecht R. Nordheim A. Nucleic Acids Res. 1992; 20: 3317-3324Crossref PubMed Scopus (117) Google Scholar). To this end we employed extracts from COS-7 cells, transiently transfected with expression vectors for different Gal4-CBP fusions, and analyzed complex formation on a 32P-labeled E74 oligonucleotide. Endogenous Elk-1 forms a characteristic complex in nontransfected cell extracts and those containing Gal4-CBP-(1–451) (Fig. 2 A, lanes 1 and 2). However, cell extracts con
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