p73 Interacts with c-Myc to Regulate Y-box-binding Protein-1 Expression

YB-1 is a member of the cold shock domain family of proteins that is important for signaling DNA damage and cell proliferation. YB-1 is induced by DNA damage and can also recognize cisplatin-modified DNA. In this study we observed a 6-fold increase in the steady-state level of YB-1 mRNA in response to cisplatin exposure in cells of the human cancer cell line KB. We present evidence from cotransfection experiments for a critical role of c-Myc and p73 in the transactivation of the YB-1 promoter. p73 transactivated the YB-1 promoter in experiments with Saos-2 cells, which express c-Myc, but not with HO15.19 cells, which lack c-Myc. In turn, c-Myc transactivated an intact YB-1 promoter but not a YB-1 promoter with a mutant E-box, indicating that the E-box is necessary for the response of the promoter to cisplatin. We also found that p73 interacts with c-Myc in vitro and in vivo. Using deletion mutants we showed that the DNA-binding domain of p73 and the C-terminal region of c-Myc are required for the interaction. Furthermore, p73 stimulated the interaction of Max with c-Myc and promoted binding of the c-Myc-Max complex to its target DNA. Our data suggest that p73 stimulates the transcription of the YB-1 promoter by enhancing recruitment of the c-Myc-Max complex to the E-box. YB-1 is a member of the cold shock domain family of proteins that is important for signaling DNA damage and cell proliferation. YB-1 is induced by DNA damage and can also recognize cisplatin-modified DNA. In this study we observed a 6-fold increase in the steady-state level of YB-1 mRNA in response to cisplatin exposure in cells of the human cancer cell line KB. We present evidence from cotransfection experiments for a critical role of c-Myc and p73 in the transactivation of the YB-1 promoter. p73 transactivated the YB-1 promoter in experiments with Saos-2 cells, which express c-Myc, but not with HO15.19 cells, which lack c-Myc. In turn, c-Myc transactivated an intact YB-1 promoter but not a YB-1 promoter with a mutant E-box, indicating that the E-box is necessary for the response of the promoter to cisplatin. We also found that p73 interacts with c-Myc in vitro and in vivo. Using deletion mutants we showed that the DNA-binding domain of p73 and the C-terminal region of c-Myc are required for the interaction. Furthermore, p73 stimulated the interaction of Max with c-Myc and promoted binding of the c-Myc-Max complex to its target DNA. Our data suggest that p73 stimulates the transcription of the YB-1 promoter by enhancing recruitment of the c-Myc-Max complex to the E-box. Y-box-binding protein-1 glutathione S-transferase hemagglutinin phosphate-buffered saline electrophoretic mobility shift assay YB-1,1 a transcription factor first identified by its ability to bind to the inverted CCAAT box (Y-box), has been implicated in gene transcription, cell proliferation (1Ladomery M. Sommerville J. Bioessays. 1995; 17: 9-11Crossref PubMed Scopus (129) Google Scholar), and cisplatin resistance (2Didier D.K. Schiffenbauer J. Woulfe S.L. Zacheis M. Schwartz B.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7322-732616Crossref PubMed Scopus (358) Google Scholar). YB-1 contains a unique DNA-binding domain, the cold shock domain, that is highly conserved in prokaryotes and eukaryotes (1Ladomery M. Sommerville J. Bioessays. 1995; 17: 9-11Crossref PubMed Scopus (129) Google Scholar, 3Wolffe A.P. Tafuri S. Ranjan M. Familari M. New Biol. 1992; 4: 290-298PubMed Google Scholar, 4Wolffe A.P. Bioessays. 1994; 16: 245-251Crossref PubMed Scopus (326) Google Scholar). YB-1 is mainly cytoplasmic, but it is translocated to the nucleus when cells are treated with anticancer agents (5Koike K. Uchiumi T. Ohga T. Toh S. Wada M. Kohno K. Kuwano M. FEBS Lett. 1997; 417: 390-394Crossref PubMed Scopus (176) Google Scholar). Furthermore, YB-1 mRNA accumulates when cells are treated with UV irradiation or anticancer agents (6Ohga T. Koike K. Ono M. Makino Y. Itagaki Y. Tanimoto M. Kuwano M. Kohno K. Cancer Res. 1996; 56: 4224-4228PubMed Google Scholar). YB-1 can bind to cisplatin-modified DNA and interacts with PCNA (7Ise T. Nagatani G. Imamura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). Cancer chemotherapeutic agents such as cisplatin exert their cytotoxic effect by inducing DNA damage and activating apoptosis (11Eastmann A. Chemistry and Biochemistry of a Leading Anticancer Drug. Wiley VCH, Basel, Switzerland1999: 111-134Google Scholar). The p53 tumor suppressor gene family is central to the pathway that inhibits cell cycle progression following damage (8Gottlieb T.M. Oren M. Biochim. Biophys. Acta. 1996; 1287: 77-102Crossref PubMed Scopus (510) Google Scholar, 9Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6727) Google Scholar, 10Lowe S.W. Endocrinol. Relat. Cancer. 1999; 6: 45-48Crossref PubMed Scopus (88) Google Scholar). It consists of transcription factors that bind to DNA in a sequence-specific manner (12Oren M. Prives C. Biochim. Biophys. Acta. 1996; 1288: 13-19PubMed Google Scholar), and their activation in response to DNA damage elicits apoptosis and cell cycle arrest. We have recently found that YB-1 interacts with p53 and can regulate gene expression (13Okamoto T. Izumi H. Imamura T. Takano H. Ise T. Uchiumi T. Kuwano M. Kohno K. Oncogene. 2000; 19: 6194-6202Crossref PubMed Scopus (134) Google Scholar). We have also identified multiple consensus 5′-CACGTG-3′ sequences, the so-called E-boxes (15Luscher B. Larsson L.G. Oncogene. 1999; 18: 2955-2966Crossref PubMed Scopus (163) Google Scholar) in the YB1 promoter (14Makino Y. Ohga T. Toh S. Koike K. Okumura K. Wada M. Kuwano M. Kohno K. Nucleic Acids Res. 1996; 24: 1873-1878Crossref PubMed Scopus (39) Google Scholar). c-Myc is a transcription factor that has also been found to induce apoptotic cell death under certain conditions and to modulate cellular susceptibility to anticancer agents such as cisplatin (16Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar, 17Evan G.I. Wyllie A.H. Gilbert C.S. Littlewood T.D. Land H. Brooks M. Waters C.M. Penn L.Z. Hancock D.C. Cell. 1992; 69: 119-128Abstract Full Text PDF PubMed Scopus (2771) Google Scholar). Thus, both YB-1 and c-Myc may play key roles in cell proliferation as well as in the DNA damage signaling pathway (18Schmidt E.V. Oncogene. 1999; 18: 2988-2996Crossref PubMed Scopus (313) Google Scholar, 19Soucie E.L. Annis M.G. Sedivy J. Filmus J. Leber B. Andrews D.W. Penn L.Z. Mol. Cell. Biol. 2001; 21: 4725-4736Crossref PubMed Scopus (122) Google Scholar). Interestingly, c-Myc is known to bind to and transactivate via E-boxes (15Luscher B. Larsson L.G. Oncogene. 1999; 18: 2955-2966Crossref PubMed Scopus (163) Google Scholar). The transcriptional apparatus responsible for signaling DNA damage is poorly understood, and YB-1 may provide useful insight into this process. We show that p73, a close relative of p53, interacts with c-Myc and stimulates the E-box binding activity of the c-Myc-Max complex. Both c-Myc and p73 activate YB-1 transcription and may regulate important biological processes via their effect on YB-1 gene expression. Our data may help to account for the dual function of c-Myc in cell proliferation and apoptosis. The KB human epidermoid cancer cell line was grown in modified Eagle's medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum. The Saos-2 human osteosarcoma cell line, the HO15.19 rat fibroblast cell line, and the COS-1 monkey kidney cell line were grown in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% fetal bovine serum. HO15.19 cells were kindly provided by Dr. J. M. Sedivy (Brown University, Boston, MA) (20Adachi S. Obaya A.J. Han Z. Ramos-Desimone N. Wyche J.H. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 4929-4937Crossref PubMed Scopus (87) Google Scholar, 21Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar). The cell lines were maintained in a 5% CO2 atmosphere at 37 °C. Cisplatin was from Sigma. Drugs were added directly to the culture medium at the indicated times. An anti-YB-C antibody to the C-tail domain of YB-1 was generated as described previously (5Koike K. Uchiumi T. Ohga T. Toh S. Wada M. Kohno K. Kuwano M. FEBS Lett. 1997; 417: 390-394Crossref PubMed Scopus (176) Google Scholar). Anti-c-Myc (N262) polyclonal antibody, anti-c-Myc (9E10) monoclonal antibody, and anti-HA (F-7) monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p73α antibody, anti-HA (3F10) (hemagglutinin)-peroxidase, and anti-FLAG (M2) monoclonal antibody were purchased from Wako (Osaka, Japan), Roche Molecular Biochemicals, and Sigma, respectively. The YB-1-Luc and YB-1-m-Luc utilized in the luciferase assays were prepared by digesting pYB-1-CAT (14Makino Y. Ohga T. Toh S. Koike K. Okumura K. Wada M. Kuwano M. Kohno K. Nucleic Acids Res. 1996; 24: 1873-1878Crossref PubMed Scopus (39) Google Scholar). To obtain YB-1-Luc and YB-1-m-Luc, the pYB-1-CAT promoter was amplified by PCR using the following primer pairs; for YB-1-Luc, 5′-AGATCTCTATCACGTGGCTGTTGC-3′ and 5′-AAGCTTCTGCGGCTCCTCCCGGGGTG-3′, and for YB-1-m-Luc, 5′-AGATCTCTATCAGCTGGCTGTTGC-3′ and 5′-AAGCTTCTGCGGCTCCTCCCGGGGTG-3′. The PCR products were cloned into pGEM-T Easy (Promega). To obtain the YB-1-Luc plasmid, aBglII-HindIII fragment including nucleotides −661 to +295 of the YB-1 promoter was ligated into theBglII-HindIII site of vector pGL3-basic (Promega, Madison, WI). To generate the YB-1-m-Luc plasmid, which contains a mutation in the E-box, a BglII-HindIII fragment including nucleotides −661 to +295 of the YB-1 promoter was ligated into the BglII-HindIII sites of vector pGL3-basic. p21-Luc, utilized in the luciferase assay, has been described previously (13Okamoto T. Izumi H. Imamura T. Takano H. Ise T. Uchiumi T. Kuwano M. Kohno K. Oncogene. 2000; 19: 6194-6202Crossref PubMed Scopus (134) Google Scholar). Wild-type c-Myc (full-length), c-Myc Δ383–439, c-Myc Δ347–439 ligated into pcDNA3, and pGEX4T-c-Myc expressing glutathioneS-transferase (GST)1-c-Myc were described previously (22Izumi H. Molander C. Penn L.Z. Ishisaki A. Kohno K. Funa K. J. Cell Sci. 2001; 114: 1533-1544Crossref PubMed Google Scholar). Relevant restriction enzyme sites are shown in Fig.6A. N-terminal hemagglutinin-tagged pcDNA3-HA-p73α, β, δ, and γ expressed in mammalian cells were kindly provided by Dr. G. Melino (University of Rome) (23De Laurenzi V. Costanzo A. Barcaroli D Terrinoni A. Falco M. Annicchiarico-Petruellizz M. Levrero M. Melino G. J. Exp. Med. 1998; 188: 1763-1768Crossref PubMed Scopus (361) Google Scholar). N-terminally truncated p73 was cloned by PCR using the following primer pairs: 5′-ATGACTACATCTGTCATGGCCC-3′ and 5′-TCAGTGGATCTCGGCCTCCGTGAACTCC-3′. The PCR product was cloned into pGEM-T Easy (Promega). The p73 cDNA was cloned into pcDNA for expression in mammalian cells and designated p73ΔN. To obtain GST-p73α, a NheI (filled in)-NotI fragment of pcDNA3-HA-p73α was ligated into theSmaI-NotI site of pGEX4T (AmershamBiosciences). To prepare a FLAG or HA-tagged fusion protein for expression inEscherichia coli, TH-FLAG vector and TH-HA vector were prepared by digesting the pThioHis vector (Invitrogen) withNdeI and Acc65I. It was then self-ligated after T4 DNA polymerase treatment to delete the thioredoxin, and the following double-stranded oligonucleotides were inserted: for FLAG, 5′-ATGGACTACAAGGACGATGATGACAAGGGC-3′, and for HA, 5′-ATGGGTTATCCGTATGATGTTCCTGATTATGCTAGCCTCGGT-3′. To obtain HA-p73α, a NheI (filled in)-NotI fragment of pcDNA3-HA-p73α was ligated into the TH-HA vector. HA-p73α deletion mutants Δ545–636, Δ396–636, Δ313–636, and Δ228–636 were obtained by partial or complete digestion, as appropriate, to delete the TH-HA-p73α fragment from theSdaI, BfmI, EcoO109I, and PsyI sites to the C terminus. Relevant restriction enzyme sites are shown in Fig. 7A. To obtain GST-Max, TH-FLAG-Max, and TH-FLAG-TBP, full-length cDNAs of human Max and TBP were amplified by PCR using the following primer pairs: for Max, 5′-TGAGCGATAACGATGACATCG-3′ and 5′-GCTTAGCTGGCCTCCATCCGG-3′, and for TBP, 5′-GGATCAGAACAACAGCCTGC-3′ and 5′-TTACGTCGTCTTCCTGAATCC-3′. The PCR products were cloned into pGEM-T Easy (Promega). For construction of GST-Max, TH-FLAG-Max, and TH-FLAG-TBP, the cDNA fragments digested with EcoRI were gel-purified and cloned into pGEX-4T and TH-FLAG, respectively. All of the PCR products were sequenced by the ABI 377 DNA sequencing system (PerkinElmer Life Sciences). Total RNA from parental and cisplatin-treated cells was isolated using Sepasol (Nakalai, Tesque, Kyoto, Japan). RNA samples (5 μg/lane) were separated on a 1% formaldehyde-agarose gel and transferred to a Hybond N+filter (Amersham Biosciences) with 10× SSC. Prehybridization and hybridization were performed as described previously (24Furukawa M. Uchiumi T. Nomoto M. Takano H. Morimoto R.I. Naito S. Kuwano M. Kohno K. J. Biol. Chem. 1998; 273: 10550-10555Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Signal intensities were quantified using a bioimaging analyzer (BAS2000; Fuji Film Co., Tokyo, Japan). The nuclear extracts were prepared as described (24Furukawa M. Uchiumi T. Nomoto M. Takano H. Morimoto R.I. Naito S. Kuwano M. Kohno K. J. Biol. Chem. 1998; 273: 10550-10555Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Briefly, 2 × 107 cells were collected in phosphate-buffered saline (PBS), resuspended in 2 ml of ice-cold 10 mm HEPES-KOH, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride and incubated on ice for 15 min. The cells were lysed with Nonidet P-40 to a final concentration of 0.5%, and the lysate was centrifuged at 500 × g for 10 min. The resulting nuclear pellet was resuspended in 300 μl of ice-cold 20 mm HEPES-KOH, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mmEGTA, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride and incubated for 15 min on ice with frequent gentle mixing. Following centrifugation at 21,000 ×g for 5 min at 4 °C in a microcentrifuge, to remove insoluble material, the supernatant (nuclear fraction) was stored at −80 °C until use. The protein concentrations were determined by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). Either nuclear extracts (see Fig.3A) or whole cell extracts (see Fig. 3C) were separated on a 10% SDS-PAGE gel. The proteins were transferred to a polyvinylidene difluoride membrane (Millipore) using a semi-dry blotter. A prestained protein marker was used as a molecular weight standard. The membrane was immunoblotted with anti-c-Myc (N262), anti-Max, anti-p73α, anti-YB-1, and anti-HA antibodies and then developed by chemiluminescence using an ECL kit according to the manufacturer (Amersham Biosciences). For transient transfections, KB, HO15.19, and Saos-2 cells were plated at a density of 5 × 104cells/well 1 day before transfection. The cells were cotransfected at ∼50% confluence with 0.2 μg of reporter plasmid, 0.2–0.4 μg of expression plasmid, and 0.3 μg of pCH110 (a β-galactosidase expression plasmid; Promega) using 2 μl of SuperFect according to the manufacturer's protocol (Qiagen). Three hours later, the cells were washed twice with PBS and incubated in fresh medium. Each expression plasmid and reporter plasmid was standardized individually to a molar ratio of 1:1, and the total amount of DNA/well was adjusted to 1.0 μg by the addition of a mock DNA plasmid. After 48 h the cells were lysed with 100 μl/well of reporter lysis buffer (Promega). After a brief centrifugation, the luciferase activity in the resulting supernatants was assayed using a Picagene kit (Toyoinki, Tokyo, Japan) as described previously (7Ise T. Nagatani G. Imamura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). Light intensity was measured for 15 s with a luminometer (Dynatech ML 1500, JEOL, Tokyo, Japan). All of the cells were cotransfected with pCH110 as a control for transfection efficiency; β-galactosidase activity was measured using a β-galactosidase enzyme assay system (Promega) and expressed as a ratio of the corrected luciferase activity of cells cotransfected with vector. The results shown are normalized to β-galactosidase activity and are representative of at least three independent experiments. Protein-DNA cross-linking was performed by incubating KB cells with formaldehyde at a final concentration of 1% for 10 min at room temperature. The cells were washed with PBS and collected by centrifugation at 500 ×g for 5 min. They were then lysed in Buffer X (50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 120 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm phenylmethylsulfonyl fluoride) for 15 min on ice. The lysate was sonicated with 10 pulses of 10 s each at 50–60% of maximum power with a sonicator (TAITEC, Tokyo, Japan) equipped with a micro tip to reduce the chromatin fragments to an average size of less than 500 bp. Soluble chromatin was precleared by addition of 10 mg of protein A-Sepharose. An aliquot of precleared chromatin containing 1 × 106 cells was removed and used in the subsequent PCR analysis. The remainder of the chromatin was divided into lots each corresponding to 1 × 106 cells and diluted with Buffer X. The protein-DNA was incubated overnight at 4 °C with 2 μg of antibody, mouse IgG, or rabbit preimmune serum in a final volume of 800 μl. Immune complexes were collected by incubation with 15 μl of protein A/G-agarose for 1 h at 4 °C. Protein A/G-agarose pellets were washed once with 1 ml of Buffer X, once with high salt Buffer X (50 mm Tris-HCl, pH 8.0, 1 mm EDTA, 500 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm phenylmethylsulfonyl fluoride), once with LiCl buffer (10 mm Tris, 1 mm EDTA, 0.25m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, pH 8.1), and twice with 10 mm Tris, 1 mm EDTA, pH 8.0. The immune complexes were then eluted twice with 250 μl of elution buffer (0.1 m NaHCO3, 1% SDS). To reverse protein-DNA cross-linking, The eluted samples were incubated with 0.2m NaCl for 4–5 h at 65 °C. The samples were digested with proteinase K (0.04 mg/ml) for 2 h at 45 °C and then with RNase A (0.02 mg/ml) for 30 min at 37 °C. DNA was purified with phenol:chloroform followed by ethanol precipitation. The purified DNA was resuspended in 10 μl of H2O. Aliquots of 2 μl of serial dilutions were analyzed by PCR with the appropriate primer pairs. The YB-1 promoter primers were 5′-AGATCTCTATCACGTGGCTGTTGC-3′ and 5′-AAGCTTATCAGTCCTCCATTCTCATTGG-3′. The HMG1 promoter primers were 5′-GCCTAGTTGGCATTCTCGTA-3′ and 5′-GCTCTGGGAACACTCCCACCC-3′. Amplification was performed for a predetermined optimal number of cycles. PCR products were separated by electrophoresis on 2% agarose gels, which were stained with ethidium bromide. COS-1 cells were seeded in 6-well plates at a density of 1 × 105 cells/well. The following day the cells were cotransfected with 1.5 μg of c-Myc and HA-p73α expression plasmids along with 6 μl of SuperFect according to the manufacturer's protocol (Qiagen). 3 h following transfection, the cells were washed with PBS, and the medium was replaced with fresh medium. After 48 h the cells were washed twice with PBS and lysed in Buffer X. After incubating for 30 min on ice, the lysates were centrifuged at 21,000 × g for 10 min at 4 °C. The supernatants (1 mg) were incubated for 60 min at 4 °C with 2 μg of mouse IgG, preimmune rabbit IgG, anti-HA (F-7) antibody, anti-c-Myc (N262) antibody, or anti-p73α antibody, in each case together with 10 μl of protein A/G-agarose (Qiagen), and the beads were washed three times with binding buffer. Nuclear extracts of control and cisplatin-treated KB cells were also incubated with anti-c-Myc antibody. The immunoprecipitated samples and starting material were separated on a 10% SDS gel and transferred onto a polyvinylidene difluoride membrane. The membranes were immunoblotted with anti-c-Myc (N262) antibody, anti-HA-peroxidase, or anti-p73α antibody and developed by chemiluminescence as described above. Expression of recombinant GST, HA, and FLAG fusion proteins was induced by 1 mmisopropyl-1-thio-β-d-galactopyranoside (Roche Molecular Biochemicals) for 1 h at 25 °C as described previously (7Ise T. Nagatani G. Imamura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). The bacteria were collected by centrifugation at 3,000 × gfor 10 min at 4 °C. The cells were lysed in Buffer X and subsequently sonicated for 10 s at 4 °C. After incubating on ice for 30 min, the lysates were centrifuged at 21,000 ×g for 15 min at 4 °C, and the supernatants were stored at −80 °C until use. GST and full-length GST-c-Myc were immobilized on glutathione-Sepharose beads for 1 h at 4 °C. After the immobilized GST fusion proteins had been washed three times with Buffer X, soluble HA-p73α, or its deletion mutants, FLAG-Max, and FLAG-TBP fusion proteins were added with 1 mm dithiothreitol and further incubated for 3.5 h at 4 °C. The binding samples were washed three times with Buffer X and separated on a 10% gel by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, immunoblotted with anti-HA-peroxidase, and developed by chemiluminescence as described above. To prepare purified GST fusion proteins, GST-Myc and GST-Max immobilized on glutathione-Sepharose beads were eluted with elution buffer containing 50 mm Tris-HCl, pH 8.0, and 20 mm reduced glutathione according to the manufacturer's protocol (Amersham Biosciences). The concentrations of the GST fusion proteins were equalized with GST elution buffer. HA-p73α protein was made in a coupled transcription and translation system (Promega). Briefly, 0.5 μg of DNA was added directly to 20 μl of transcription and translation rabbit reticulocyte lysate with 0.5 μl of methionine, and the reactions were carried out at 30 °C for 90 min. The translated products were stored at −80 °C until use. Oligonucleotides from the E-box and p53 consensus binding sites were used as probes. The sequences were: YB-1 E-box (E-box Y), 5′-GGCCCTCTCTATCACGTGGCTGTTGC-3′; E-box consensus (E-box C), 5′-GGTCAGACCACGTGGTCGGG-3′, and Y-box, 5′-GGTGAGGCTGATTGGCTGGGCAGGA-3′ (13Okamoto T. Izumi H. Imamura T. Takano H. Ise T. Uchiumi T. Kuwano M. Kohno K. Oncogene. 2000; 19: 6194-6202Crossref PubMed Scopus (134) Google Scholar). The E-box probe was labeled with [γ-32P]ATP using T4 polynucleotide kinase and purified on a 15% polyacrylamide gel in 1× TBE buffer. EMSA was performed as described previously (7Ise T. Nagatani G. Imamura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). Briefly, eluted GST fusion proteins and 4 ng of radiolabeled oligonucleotides were mixed in reaction buffer containing 25 mm HEPES, pH 7.5, 50 mm KCl, 0.5 mmEDTA, 5% glycerol, 10 mm dithiothreitol, 0.1% Nonidet P-40, and 0.5 mg/ml bovine serum albumin and incubated for 20 min at 20 °C (26Kitaura H. Shinshi M. Uchikoshi Y. Ono T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2000; 275: 10477-10483Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 27Takayama M. Taira T. Iguchi-Ariga S.M. Ariga H. FEBS Lett. 2000; 477: 43-48Crossref PubMed Scopus (16) Google Scholar). The binding reactions were analyzed on a 4% polyacrylamide gel in 0.5× TBE buffer, followed by autoradiography as described previously (7Ise T. Nagatani G. Imamura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). For the competition experiments, preincubation was performed in the presence of 10× or 40× unlabeled competitor DNA for 15 min at 20 °C before the addition of radiolabeled oligonucleotides. We first examined YB-1 gene expression in the in the human cancer cell line KB and found that YB-1mRNA increased in response to cisplatin treatment (Fig. 1A). We tested whether cisplatin could induce luciferase expression from reporter constructs in cells that express high levels of c-Myc and p73α. Significant induction of luciferase activity was observed when intact YB-1 Luc was transfected into KB cells but not when YB-1-m-Luc, whose promoter contains a mutant E-box, was introduced (Fig. 1, Band C). The E-box that is proximal to the transcriptional start site of the YB-1 promoter has the nucleotide sequence 5′-CACGTG-3′ that is bound with high affinity by c-Myc (28Amati B. Dalton S. Brooks M.W. Littlewood T.D. Evan G.I. Land H. Nature. 1992; 359: 423-426Crossref PubMed Scopus (380) Google Scholar, 29Amin C. Wagner A.J. Hay N. Mol. Cell. Biol. 1993; 13: 383-390Crossref PubMed Scopus (120) Google Scholar, 30Gu W. Cechova K. Tassi V. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2935-2939Crossref PubMed Scopus (133) Google Scholar, 31Kretzner L. Blackwood E.M. Eisenman R.N. Nature. 1992; 359: 426-429Crossref PubMed Scopus (380) Google Scholar). It therefore seemed possible that c-Myc expression might affect the outcome of such reporter assays. To show that c-Myc binds specifically to the YB-1 promoter in vivo, we utilized a chromatin immunoprecipitation assay. PCR amplication of the YB-1 promoter was carried out with DNA extracted from the immunocomplex obtained with anti-c-Myc antibody. Fig. 2shows that the YB-1 promoter sequence was significantly enriched in this complex. In controls, no HMG1 promoter sequence was detected in the immunocomplex (Fig. 2B), and enrichment of the YB-1 promoter sequence was not observed with preimmune serum or mouse IgG.FIG. 2c-Myc binds to the YB-1 promoter in vivo.A, chromatin immunoprecipitation experiments with KB cells. Formaldehyde cross-linked chromatin was isolated from KB cell. Chromatin was immunoprecipitated with antibody to c-Myc and preimmune or monoclonal IgG. Immunoprecipitated DNA was purified and analyzed by PCR, together with placenta DNA, using primers specific for the YB-1 promoter. The amounts of placenta DNA used as a positive control were 3, 10, 30, and 90 ng (right toleft). Immunoprecipitated DNA was diluted to 1/27, 1/9, and 1/3 (right to left). Amplification products were electrophoresed in 2.0% agarose gels containing ethidium bromide.Lane M, DNA ladder mix marker (MBI, Fermentas, Lithuania). The arrowhead indicates 725 bp of the YB-1 promoter sequence. B, chromatin immunoprecipitation was carried out as described above. Immunoprecipitated DNA was purified and analyzed, together with placenta DNA, by PCR, using primers specific for the HMG1 promoter. The arrowhead indicates 441 bp of the HMG1 promoter sequence.View Large Image Figure ViewerDownload (PPT) Western blots with c-Myc antiserum indicated that the level of c-Myc protein increased dramatically after cisplatin treatment (Fig.3A). Cisplatin treatment has been shown to stabilize p73 protein (32Gong J.G. Costanzo A. Yang H.Q. Melino G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; 399: 806-809Crossref PubMed Scopus (833) Google Scholar), and as expected, treatment of KB cells with cisplatin increased p73 protein levels (Fig.3A). We also examined whether overexpression of p73 can induce the expression of endogenous YB-1 in a c-Myc-dependent manner. As expected, a significant increase of endogenous YB-1 was observed when p73 was overexpressed in Saos-2 cells (Fig. 3C) but not in rat HO15.19/c-Myc null cells (19Soucie E.L. Annis M.G. Sedivy J. Filmus J. Leber B. Andrews D.W. Penn L.Z. Mol. Cell. Biol. 2001; 21: 4725-4736Crossref PubMed Scopus (122) Google Scholar, 20Adachi S. Obaya A.J. Han Z. Ramos-Desimone N. Wyche J.H. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 4929-4937Crossref PubMed Scopus (87) Google Scholar, 21Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar) (data not shown). Because the induction of p73 after treatment with cisplatin correlates with the induction of c-Myc, we tested the effect of p73 and c-Myc on YB-1 promoter activity. To determine whether p73 or c-Myc is responsible for the YB-1 promoter activity, we utilized two cell lines: Saos-2/p53 null cells, which express low levels of p73α (data not shown), and HO15.19/c-myc null cells (19Soucie E.L. Annis M.G. Sedivy J. Filmus J. Leber B. Andrews D.W. Penn L.Z. Mol. Cell. Biol. 2001; 21: 4725-4736Crossref PubMed Scopus (122) Google Scholar, 20Adachi S. Obaya A.J. Han Z. Ramos-Desimone N. Wyche J.H. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 4929-4937Crossref PubMed Scopus (87) Google Scholar, 21Mateyak M.K. Obaya A.J. Adachi S. Sedivy J.M. Cell Growth Differ. 1997; 8: 1039-1048PubMed Google Scholar). The SV40 promoter-Luc plasmid (P2) served as a control, and we transected the YB-1 promoter-luciferase reporter, together with increasing amounts of p73 or c-Myc expression vectors, into these cell lines. c-Myc activated the intact YB-1 promoter but not the reporter with an E-box mutation in HO15.19 cells (Fig. 4A). Interestingly, p73 was able to increase YB-1 promoter activity in Saos-2 cells (Fig. 3B) but not in HO15.19 cells (Fig.4B). Because there is no p73-binding site in the YB-1 promoter, this suggests that p73 regulates expression indirectly by promoting binding of c-Myc to the E-box. To examine whether p73 interacts with c-Myc, we performed coimmunoprecipitation assays. Because p73α is expressed at low levels in tumor cell lines (33Ikawa S. Nakagawara A. Ikawa Y. Cell Death Differ. 1999; 6: 1154-1161Crossref PubMed Scopus (136) Google Scholar), we tra