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• W1997759424 abstract "Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, andin vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider'sDrosophila line 2, consistent with properties previously attributed to NF-ODC1. Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, andin vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider'sDrosophila line 2, consistent with properties previously attributed to NF-ODC1. Polyamines are essential cations for normal cell growth and differentiation (1Heby O. Differentiation. 1981; 9: 1-20Crossref Scopus (873) Google Scholar, 2Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar). Increased synthesis of these compounds is closely associated with, and necessary for, stimulated cell proliferation and tumor promotion. Tight regulation of polyamine biosynthesis is important as overproduction of these compounds can be toxic to cells (3Heby O. Persson L. Trends Biochem. Sci. 1990; 15: 153-158Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 4Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar). Ornithine decarboxylase (ODC) 1The abbreviations used are: ODCornithine decarboxylaseEMSAelectrophoretic mobility shift assaySL2Schneider's Drosophila line 2TBETris-borate/EDTA electrophoresis bufferGCFGC factorMAZMYC-associated zinc finger proteinTCRT-cell receptor.1The abbreviations used are: ODCornithine decarboxylaseEMSAelectrophoretic mobility shift assaySL2Schneider's Drosophila line 2TBETris-borate/EDTA electrophoresis bufferGCFGC factorMAZMYC-associated zinc finger proteinTCRT-cell receptor. catalyzes a key regulated step in polyamine synthesis, and regulation of ODC activity is a major mechanism for controlling polyamine concentrations within cells. The activity of this enzyme is tightly regulated during normal cell growth and differentiation. An increase in ODC activity is required for reentry of quiescent cells into the cell cycle (2Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar, 5Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3214) Google Scholar, 6Abrahamsen M.S. Morris D.R. Campisi J. Cunningham D.D. Inouye M. Riley M. Perspectives on Cellular Regulation: From Bacteria to Cancer. Wiley-Liss, New York1991: 107-119Google Scholar, 7Law G.L. Li R.S. Morris D.R. Casero R.J. Polyamines: Regulation and Molecular Interaction. R. G. Landes Co., Austin, TX1995: 5-26Google Scholar). Deregulated expression of ODC and the subsequent changes in polyamine concentrations have been associated with several types of tumors (4Morris D.R. J. Cell. Biochem. 1991; 46: 102-105Crossref PubMed Scopus (56) Google Scholar, 8Yoshida M. Hayashi H. Taira M. Isomo K. Cancer Res. 1992; 52: 6671-6675PubMed Google Scholar, 9Black Jr, O. Chang B.K. Cancer Lett. 1982; 1: 87-93Crossref Scopus (8) Google Scholar, 10Seiler N. Sarhan S. Grauffel C. Jones R. Knodgen L. Moulinoux J. Cancer Res. 1990; 50: 5077-5083PubMed Google Scholar). Recent studies indicate that overexpression of oncogenes such as myc (11Pena A. Reddy C.D. Wu S. Hickok N.J. Reddy E.P. Yumet G. Soprano D.R. Soprano K.J. J. Biol. Chem. 1993; 268: 27277-27285Abstract Full Text PDF PubMed Google Scholar, 12Bello-Fernandez C. Packham G. Cleveland J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7804-7808Crossref PubMed Scopus (656) Google Scholar, 13Wagner A.J. Meyers C. Laimins L.A. Hay N. Cell Growth Differ. 1993; 4: 879-883PubMed Google Scholar), ras (14Holtta E. Sistonen L. Alitalo K. J. Biol. Chem. 1988; 263: 4500-4507Abstract Full Text PDF PubMed Google Scholar),fos (15Wrighton C. Busslinger M. Mol. Cell. Biol. 1993; 13: 4657-4669Crossref PubMed Scopus (60) Google Scholar), and mos (16Jaggi R. Friis R. Groner B. J. Steroid Biochem. 1988; 29: 457-463Crossref PubMed Scopus (11) Google Scholar) result in elevated levels of ODC expression. Importantly, two studies have shown that overexpression of ODC in fibroblasts induces neoplastic transformation and suggest a direct link between deregulation of ODC expression and oncogenesis (17Moshier J.A. Dosescu J. Skunca M. Luk G.D. Cancer Res. 1993; 53: 2618-2622PubMed Google Scholar, 18Auvinen M. Paasinen A. Andersson L.C. Holtta E. Nature. 1992; 360: 355-358Crossref PubMed Scopus (612) Google Scholar). ornithine decarboxylase electrophoretic mobility shift assay Schneider's Drosophila line 2 Tris-borate/EDTA electrophoresis buffer GC factor MYC-associated zinc finger protein T-cell receptor. ornithine decarboxylase electrophoretic mobility shift assay Schneider's Drosophila line 2 Tris-borate/EDTA electrophoresis buffer GC factor MYC-associated zinc finger protein T-cell receptor. Both activation and inhibition of ODC activity is required for precise regulation of ODC levels. A broad spectrum of stimuli, including hormones, growth factors, tumor promoters and oncogenes elevates ODC activity in the cell. In most cases, these increases in activity result from enhanced levels of ODC mRNA (6Abrahamsen M.S. Morris D.R. Campisi J. Cunningham D.D. Inouye M. Riley M. Perspectives on Cellular Regulation: From Bacteria to Cancer. Wiley-Liss, New York1991: 107-119Google Scholar, 7Law G.L. Li R.S. Morris D.R. Casero R.J. Polyamines: Regulation and Molecular Interaction. R. G. Landes Co., Austin, TX1995: 5-26Google Scholar). Several of the DNA elements and protein factors involved in both basal and stimulated activity of the ODC promoter have been identified, including several binding sites for transcription factor Sp1, two binding sites for members of the CREB/ATF family of transcription factors, and binding sites for transcription factors related to c-myc (7Law G.L. Li R.S. Morris D.R. Casero R.J. Polyamines: Regulation and Molecular Interaction. R. G. Landes Co., Austin, TX1995: 5-26Google Scholar). Little is known about DNA elements or protein factors that are involved in repressing ODC transcription. A GC-rich region located at −123 to −91 relative to the transcriptional start site of the ODC promoter seems to be such an element. We have demonstrated that two proteins bind this site in a mutually exclusive manner, Sp1 and NF-ODC1 (19Li R.S. Abrahamsen M.S. Johnson R.R. Morris D.R. J. Biol. Chem. 1994; 269: 7941-7949Abstract Full Text PDF PubMed Google Scholar). Sp1 is a well characterized transcription factor that is found in most eukaryotic cell and is directly involved in both basal and induced expression of many genes. NF-ODC1 has been characterized only through in vitro binding assays. Point mutations that eliminate NF-ODC1 binding, but maintain Sp1 binding, elevate basal activity relative to the wild type promoter (19Li R.S. Abrahamsen M.S. Johnson R.R. Morris D.R. J. Biol. Chem. 1994; 269: 7941-7949Abstract Full Text PDF PubMed Google Scholar). These results suggest that NF-ODC1 functions to repress the transcriptional activity of the ODC gene. The goal of the present study was to identify the protein responsible for NF-ODC1 binding activity. We have used a yeast one-hybrid system to isolate cDNAs that code for NF-ODC1. One of the isolated cDNAs encoded the human homologue of ZBP-89, a known DNA-binding protein that acts to repress both basal and induced expression of the gastrin gene (20Merchant J.L. Iyer G.R. Taylor B.R. Kitchen J.R. Mortensen E.R. Wang Z. Flintoft R.J. Michel J.B. Bassel-Duby R. Mol. Cell. Biol. 1996; 16: 6644-6653Crossref PubMed Scopus (114) Google Scholar). Several lines of evidence, including copurification, demonstrate that ZBP-89 is the protein responsible for the NF-ODC1 binding activity. ZBP-89 represses Sp1 activation of the ODC promoter in Schneider'sDrosophila line 2 (SL2) cells, consistent with properties previously attributed to NF-ODC1. The human Jurkat T-cell line was cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% calf serum, 10 mm Hepes, pH 7.5, and 2 mml-glutamine on 150-mm culture dishes. Jurkat cells were grown in 6-liter spinner flasks from which nuclear extracts were prepared for NF-ODC1 purification. HeLa cells were cultured in Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) and 10% calf serum. SL2 cells (ATCC, Rockville, MD) were grown at 27o, in Shield and Sang M3 insect medium, pH 6.6 (Sigma) and 10% fetal bovine serum, heat-inactivated (Sigma; catalog no. F-3018). All media contained 100 units of penicillin and 100 μg of streptomycin/ml. Nuclear extracts were prepared as outlined in Ref.21Abmayr S.M. Workman J.L. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/Wiley Interscience, New York1991: 12.0.3-12.1.9Google Scholar, based on the protocol by Dignam and co-workers (22Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). Phosphatase inhibitors Na2MoO4 and NaF were added to all buffers at 0.1 mm and 10 mm, respectively. The high salt buffer contained 1.2 m KCl. The following protease inhibitors were added to the buffers immediately before use, at the indicated final concentrations: phenylmethylsulfonyl fluoride (1 mm), pepstatin A (1 μg/ml), leupeptin (1 μg/ml), aprotinin (1 μg/ml), and antipain (5 μg/ml). The nuclear extract was not dialyzed, but stored in appropriate sized aliquots at −70 °C until use. Binding reactions (final volume 20 μl) contained in addition to the protein sample: 0.1 pmol of probe, 200–300 mm KCl, 2 μg of double-stranded poly(dI-dC), and 1.0 μg of sheared salmon sperm DNA in gel shift buffer (20 mmHepes, pH 7.9, 10% glycerol, 6 mm MgCl2, 1 mm EDTA, 100 μm ZnSO4). In experiments utilizing unlabeled double-stranded oligonucleotides as specific competitors, the protein was added to the reaction after the DNA. Binding reactions were incubated for 20 min at 4 °C before loading on a 5% polyacrylamide gel (acrylamide:bisacrylamide ratio of 37.5:1, 0.5× Tris-borate/EDTA electrophoresis buffer (TBE: 45 mm Tris base, 45 mm boric acid, and 1 mm EDTA), 5% glycerol, 3-mm-thick gel) that had been pre-run for 1 h. After running in the cold room at 200 V in 0.5× TBE for 4–6 h and drying, the gel was exposed to film with an intensifying screen for several hours to 2 days as necessary. The probes (1.0–0.5 × 105 cpm/μl and 0.05 pmol/μl) were made by end-labeling double-stranded oligonucleotides with T4 polynucleotide kinase and [γ-32P]ATP. NICK® spin columns (Amersham Pharmacia Biotech) were used to remove non-incorporated isotope. When required, phosphorimage analysis was performed to quantitate signal intensities. Slightly different conditions were used to assay for MAZ binding: 10 μl of 2× MAZ gel shift buffer (0.1% Nonidet P-40, 2% glycerol, 1 μm ZnSO4, 10 mm Tris, pH 7.5, 70 mm KCl, 1 mm dithiothreitol, and 2.5 mm MgCl2) were used per 20-μl reaction, and double-stranded poly(dA-dT) was used instead of double-stranded poly(dI-dC) as a nonspecific competitor. The gels were run in 0.25× TBE at 200 V for 8 h. The MAZ antibody was kindly provided by Kenneth B. Marcu (State University of New York, Stony Brook, NY). In “supershift” experiments, the antibody was added to reaction mixtures containing protein, but no probe. The antibody-protein solution was incubated for 1- 3 h at 4 °C, followed by the addition of the probe and an additional incubation at 4 °C for 20 min before loading the binding reaction onto the gel. Control peptides or antigens were incubated overnight at 4 °C with antibodies prior to use in the binding reactions. The antibodies and control peptides for the Sp1 family of proteins were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibodies were raised against a glutathione S-transferase fusion protein that included amino acids 1–521 of rat ZBP-89 (Rockland, Gilbertsville, PA) (23Taniuchi T. Mortensen E.R. Ferguson A. Greenson J. Merchant J.L. Biochem. Biophys. Res. Commun. 1997; 233: 154-160Crossref PubMed Scopus (37) Google Scholar). Anti-ZBP-89 was the IgG fraction from the rabbit antisera. Oligonucleotides were purchased from Life Technologies, Inc. Shown are the upper strand sequences for the double-stranded oligonucleotides used. GCN contained the NF-ODC1 binding site and GCS contained the Sp1 binding site from the ODC promoter. GCWT, 5′-CCACGGAGTCCCCGCCCCTCCCCCGCGCCTCCC-3′; GCN, 5′-GGCCGATGCGCCCCTCCCCCGCGCCGATC-3′; GCS, 5′-GGCCGGATGCCCCGCCCCTCCCGGCC-3′; GCWT6, 5′-CGGAGTATGCACCCCTCCCCCGCGCCTC-3′; GCF, 5′-GCCAACGCCCCGCAACCG-3′; Egr-1, 5′-CGGCCCGCCCCCGCAACCCGAGCC-3′; MAZ, 5′-GATCCCTCCCCTCCCTTCTTTTTC-3′; E box, 5′-GGAAGCAGACCACGTGGTCTGCTTCC-3′. The MATCHMAKER One-Hybrid System from CLONTECH was one method used to isolate the cDNA encoding for the protein responsible for the NF-ODC1 binding activity. The MATCHMAKER One-Hybrid System protocol was used to prepare the target-reporter constructs, to integrate these constructs intoSaccharomyces cerevisiae strainYM4271, to screen the AD fusion library (Human Leukemia MATCHMAKER cDNA Library,CLONTECH), and to isolate plasmid from each candidate clone. Two pairs of oligonucleotides were synthesized (Genset, La Jolla, CA). When annealed, the double-stranded oligonucleotides consisted of either three tandem copies of the wild type NF-ODC1 binding site or three tandem copies of a mutated NF-ODC1 binding site. Wild type oligonucleotides: 5′-AATTCAGCCCCTCCCCCGAAGCCCCTCCCCCGATAGCCCCTCCCCCGTCTAGAGCTACGAG-3′ and 5′-TCGACTCGTAGCTCTAGACGGGGGAGGGGCTATCGGGGGAGGGGCTTCGGGGGAGGGGCTG-3′. Mutated oligonucleotides: 5′-AATTCAGCCCCTCCCAAGAAGCCCCTCCCAAGATAGCCCCTCCCAAGT-3′ and 5′-CTAGACTTGGGAGGGGCTATCTTGGGAGGGGCTTCTTGGGAGGGGCTG-3′. The wild type target site was placed upstream of both the pHis-1 and pLacZi plasmids. The mutated target site was place upstream of pHis-1. The target-reporter constructs were transformed into S. cerevisiae strain YM4271. The two wild type reporter constructs (pHis-1 and pLacZi) were transformed in a consecutive manner to produce a dual reporter strain. The plasmid DNA, isolated from the yeast candidate clones, was transformed into DH5α cells. Plasmid DNA was isolated from the transformed bacteria and transformed into the strain containing the mutated target reporter. The Gene Trapper™ cDNA positive selection system (Life Technologies, Inc.) with a Superscript™ cDNA human leukocyte library (Life Technologies, Inc.) was also used to isolate ZBP-89 cDNAs using the following primers from the NH2-terminal domain of htβ: HTB-1, 5′-TCAAGATCGAAGTATGCCTCAC-3′; HTB-2, 5′-GCTCTGAGGAAGATTCTGGGC-3′; and HTB-3A, 5′-TGCCTTCTGAGTCCAGTAAAG-3′. In vitro coupled transcription/translation reactions were performed using the TNT® coupled reticulocyte lysate system (Promega, Madison, WI). The pET-human ZBP-89 expression vector was constructed by inserting the BamHI/BglII fragment from positive clones into the BamHI site of the pET-3b vector (Novagen, Madison, WI). The MAZ expression vector, MAZHH, (provided by Kenneth B. Marcu, State University of New York, Stony Brook, NY) was used for thein vitro transcription/translation of MAZ. The unmodified pBKCMV (control vector), pBKCMV containing the full-length rat ZBP-89 cDNA or pBKCMV containing the truncated rat ZBP-89 cDNA (designated B22) as described by Merchant et al. (20Merchant J.L. Iyer G.R. Taylor B.R. Kitchen J.R. Mortensen E.R. Wang Z. Flintoft R.J. Michel J.B. Bassel-Duby R. Mol. Cell. Biol. 1996; 16: 6644-6653Crossref PubMed Scopus (114) Google Scholar) were used to prepare in vitro transcription/translation products as indicated under “Results.” Jurkat nuclear extract was precipitated by slowly adding solid ammonium sulfate to a final concentration of 53% saturation. The resulting pellet was resuspended in CB (25 mm Tris, pH 7.9, 10% glycerol, 1 mm dithiothreitol, 5 mm EDTA, 10 mm NaF, 10 mm Na2MoO4, 100 μm ZnSO4, and 0.1% Nonidet P-40). The redissolved protein extract was applied to a P6 (Bio-Rad) desalting column (equilibrated in CB buffer) to remove remaining (NH4)2SO4. The protein fraction from the P6 column was applied to a Mono Q column (Bio-Rad, Hercules, CA), pre-equilibrated in CB, and the bound proteins were eluted using a 0–500 mm KCl gradient in CB. The NF-ODC1binding activity eluted at approximately 350 mm KCl. The fractions containing NF-ODC1 were pooled, diluted to 100 mm KCl with DA buffer (25 mm Hepes, pH 7.6, 12.5 mm MgCl2, 1 mm dithiothreitol, 20% glycerol ,and 0.1% Nonidet P-40), and applied to a DNA affinity column (see below), and the NF-ODC1 activity was eluted from the column with DA buffer containing 600 mm KCl. The following protease inhibitors were added to CB and DA buffers immediately before use, at the indicated final concentrations: phenylmethylsulfonyl fluoride (1 mm), pepstatin A (1 μg/ml), leupeptin (1 μg/ml), aprotinin (1 μg/ml), and antipain (5 μg/ml). The DNA affinity column matrix was made using double-stranded oligonucleotide, GCN (Fig. 1), in which the upper strand was biotinylated at the 5′ end (Genset, La Jolla, CA). The biotinylated GCN was coupled to streptavidin-agarose beads (75 μg of DNA/500 μl of agarose bead) using the procedure and buffers outlined by Ostrowski and Bomsztyk (24Ostrowski J. Bomsztyk K. Nucleic Acids Res. 1993; 21: 1045-1046Crossref PubMed Scopus (15) Google Scholar). EMSAs using GCN as probe were using during this procedure to determine the NF-ODC1-containing protein fractions. For immunoblot analysis, protein samples were fractionated on a 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (PolyScreen, NEN™ Life Science Products) using standard techniques and a mini-gel format. Phototope®-HRP Western blot detection kit and protocol (New England Biolabs, Inc., Beverly, MA) were used for antigen detection. Anti-ZBP-89 (1:1000) was used as the primary antibody. Band intensities were quantitated by densitometry. SL2 cells were transfected using a modification of a previously described method (25Di Nocera P.P. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7095-7098Crossref PubMed Scopus (265) Google Scholar). Cells were plated at 1–2 × 106 cells/60-mm dish, approximately 20 h before transfection. Calcium-phosphate complexes were made by the dropwise addition of the DNA/CaCl2 solution into 2× Hepes-buffered saline while bubbling the mixture. After 20 min at room temperature, the suspension of calcium-phosphate complexes was added dropwise to the culture dishes. The following plasmids were used. γF-gal is an internal control plasmid in which the E. coliβ-galactosidase gene is under the control of the Drosophila melanogaster hsp70 core promoter (26Contursi C. Minchiotti G. Di Nocera P.P. J. Biol. Chem. 1995; 270: 26570-26576Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and was kindly provided by Dr. Pier Paolo Di Nocera (Università degli Studi di Napoli Fecerico II, Napoli, Italy). The pPacSp1 expression plasmid and the parental pPac plasmid, pPacO, have been described previously (27Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1073) Google Scholar) and were kindly provided by Dr. Robert Tjian (University of California at Berkeley, Berkeley, CA). The pOD150WTLuc construct contained the ODC sequence from −133 to +16 in the pGL2-basic vector (Promega, WI) with a modified multiple cloning site. Twenty base pairs (−104 to −84) were removed from pOD150WTLuc with a method for site-directed mutagenesis using the polymerase chain reaction as outlined by Hemsley et al. The sequences of the two primers used were: 5′-AGGGGCGGGGACTCCGTG-3′ and 5′- AACCGATCGCGGCTGGTT-3′. The resulting construct, pOD150M12Luc, retained the entire Sp1 binding site but only 6 out of 11 base pairs of the ZBP-89 binding site. BCAT-S was created by cutting BCAT-1 (29Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Crossref PubMed Scopus (354) Google Scholar) with PstI and SalI to removing the HTLVIII LTR Sp1 binding site. A double-stranded oligonucleotide containing the ODC Sp1 binding site withPstI and SalI ends was inserted. The sequence of the annealed oligonucleotides were: 5′-GCGGATGCCCCGCCCCGATG-3′ and 5′-TCGACATCGGGGCGGGGCATCCGCTGCA-3′. The Sp1 binding site is underlined. The modified pBKCMV vector and the modified pBKCMV vector containing rat ZBP-89 cDNA (pBKCMV-ZBP-89) have been described previously (20Merchant J.L. Iyer G.R. Taylor B.R. Kitchen J.R. Mortensen E.R. Wang Z. Flintoft R.J. Michel J.B. Bassel-Duby R. Mol. Cell. Biol. 1996; 16: 6644-6653Crossref PubMed Scopus (114) Google Scholar). To each 60-mm dish, 0.1 μg of γF-gal, 5 μg of pOD150WTLuc, and 0.1–0.75 μg of expression vectors were added. The control vectors pPacO and modified pBKCMV were used to keep the total amount of DNA constant. The medium was not changed before or after the addition of DNA complexes, and the cells were harvested 48 h later. The cells were washed two times with phosphate-buffered saline and lysed in Reporter Lysis Buffer (Promega, Madison, WI). Generally, 5 μl of cell lysate were used in the Galacto-Light™ β-galactosidase assay (Tropix, Inc., Bedford, MA) and 50 μl of lysate were used to determine the luciferase or chloramphenicol acetyltransferase activity (30Brasier A.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/Wiley Interscience, New York1991: 9.6.10-9.6.14Google Scholar, 31Abrahamsen M.S. Li R.S. Dietrich-Goetz W. Morris D.R. J. Biol. Chem. 1992; 267: 18866-18873Abstract Full Text PDF PubMed Google Scholar). The luciferase or chloramphenicol acetyltransferase activity was normalized to β-galactosidase activity, and each transfection was done in triplicate. Nuclear extract was harvested, as described above, from SL2 cells transfected with 16 μg of pBKCMV-ZBP-89 and 11 μg of pPacSp1 per 150-mm dish. EMSAs were performed with SL2 nuclear extract using the same protocol as described for Jurkat nuclear extract. We have used EMSAs as a tool to further characterize and identify the protein responsible for the DNA binding activity we called NF-ODC1. Fig. 1 shows the sequence of the −123 to −91 GC box in the proximal promoter in the ODC gene that contains both the Sp1 and NF-ODC1 binding sites and also the sequences of four double-stranded oligonucleotides that we used as probes and competitors in several of the EMSAs detailed in this study. GCWT contained the sequence of the wild type GC box. In GCWT6, the wild type sequence was altered so that Sp1 binding to the oligonucleotide was greatly reduced, but NF-ODC1 binding was unaltered. GCN (formerly ODC543; see Ref. 19Li R.S. Abrahamsen M.S. Johnson R.R. Morris D.R. J. Biol. Chem. 1994; 269: 7941-7949Abstract Full Text PDF PubMed Google Scholar) contained only the NF-ODC1 binding site, which also had low affinity for Sp1 (see below). GCS(formerly ODC53; see Ref. 19Li R.S. Abrahamsen M.S. Johnson R.R. Morris D.R. J. Biol. Chem. 1994; 269: 7941-7949Abstract Full Text PDF PubMed Google Scholar) contained only the Sp1 binding site and did not interact with NF-ODC1. When radiolabeled GCWT was incubated with Jurkat nuclear extracts, a complex band shift pattern resulted (Fig.2 A, lane 2). We previously showed that the first complex (C1) was due to Sp1 binding and the third complex (C3) was the result of NF-ODC1binding (19Li R.S. Abrahamsen M.S. Johnson R.R. Morris D.R. J. Biol. Chem. 1994; 269: 7941-7949Abstract Full Text PDF PubMed Google Scholar). There were two other specific DNA-protein complexes, C2 and C4, in which the identity of the protein component was unknown. When radiolabeled GCS was used as probe, Sp1, C2, and C4 complexes were detected, but there was no NF-ODC1 complex (Fig. 2 A, compare lanes 2 and7). When GCS was used as an unlabeled competitor, no Sp1, C2, or C4 complexes were seen, indicating that GCS had high affinity for Sp1 and the proteins in C2 and C4 (Fig. 2 A, lane 5). GCS did not compete for NF-ODC1 binding (Fig. 2 A, compare lanes 2 and 5). When needed, GCS was used in EMSAs as an unlabeled competitor to unambiguously identify the NF-ODC1-containing complex. Using either radioactive GCWT or GCS probe, unlabeled GCN also competed for binding to Sp1 and the proteins in C2 and C4 complexes, albeit less efficiently than GCWT at 30-fold molar excess or GCS at 50-fold molar excess (Fig. 2 A, compare lanes 3–5 and also lanes 8–10). These results indicated that Sp1 not only had the capacity to bind with high affinity to the Sp1 consensus site found in the wild type ODC promoter sequence, GCWT, but also interacted at lower affinity with the NF-ODC1 site. GCN competed efficiently with GCWT for formation of the NF-ODC1 complex (Fig. 2 A, compare lanes 2, 3, and 4), indicating that GCN bound to NF-ODC1 with relatively high affinity. As needed, radiolabeled GCN was used as probe in EMSAs, which resulted in significantly reduced band intensities for C2, C4, and Sp1 complexes (compare Fig. 2 A, lane 2with Fig. 3 A, lane 2).Figure 3Sp1, Sp3, and Sp4 bind to the GC box of the ODC promoter. Supershift experiments were performed as detailed under “Materials and Methods.” The indicated antibody was added to the binding reaction containing Jurkat nuclear extract, and the mixture was incubated for 3 h at 4 °C followed by an additional 20-min incubation with 0.1 pmol of the indicated probe. SSindicates the location of supershifted bands. The location of complexes containing Sp1, C1b, C2, C4, and NF-ODC1 are indicated. A, GCN was used as probe; 10 μg/lane Jurkat nuclear extract and 1 μg of each indicated antibody was used as indicated. B, GCWT was used as probe and the binding reactions contained either 5 (lanes 2–7) or 2.5 μg of total protein/lane (lanes 8 and 9) of Jurkat nuclear extract. Lane 1 contained no protein. Additional reagents were added to some of the binding reactions:lanes 3–9, 30-fold molar excess of GCWT6; lanes 4–9, 1 μg of anti-Sp1; lanes 5, 6, and 7 contained 1, 2, and 1 μg of anti-Sp4 respectively; lane 7, 1.2 μg of Sp4 control peptide (P4); lanes 8 and 9, 0.1 μg of anti-Sp3; lane 9, 1 μg of Sp3 control peptide (P3). All reactions were run on the same gel and exposed to film for same length of time.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The DNA binding domains of many proteins that bind to GC-rich regions contain zinc finger motifs. To determine if the interaction between NF-ODC1 and DNA required zinc, EMSAs were performed with the zinc chelator, o-phenanthroline. To eliminate the radioactive bands of C1, C2, and C4, unlabeled GCS was added to each binding reaction. Increasing the amount of zinc chelator resulted in decreasing amounts of the NF-ODC1 complex (Fig.2 B). When zinc was added back to the reaction, in the form of Zn2SO4, NF-ODC1 binding was restored. These results indicate that NF-ODC1 binding is dependent on the presence of zinc. To determine if the complexes formed with the GC-box contained known members of the Sp1 family of transcription factors, specific antibodies were employed (Fig. 3). Antibodies to Sp1, Sp2, Sp3, and Sp4 were added separately to binding reactions containing GCN as probe and Jurkat nuclear extract (Fig. 3 A). As expected, NF-ODC1 binds strongly to this probe, while Sp1 gives a weak signal. GCS was used as an unlabeled competitor to eliminate Sp1 binding and to identify the NF-ODC1 complex (Fig. 3 A, lane 3). Binding of NF-ODC1was not inhibited by any of these antibodies (Fig. 3 A, compare lane 2 with lanes 4–7). The Sp1 band was supershifted as expected when the Sp1 antibody was used (Fig.3 A, lane 7). To determine" @default.
• W1997759424 created "2016-06-24" @default.
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• W1997759424 title "Transcription Factor ZBP-89 Regulates the Activity of the Ornithine Decarboxylase Promoter" @default.
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