Transcription Factor ZBP-89 Regulates the Activity of the Ornithine Decarboxylase Promoter*

G. Lynn LawDagger , Hideaki Itoh§, David J. Law, Gregory J. MizeDagger , Juanita L. Merchantparallel , and David R. MorrisDagger **

From the Dagger  Department of Biochemistry, University of Washington, Seattle, Washington 98195, the § Department of Biochemistry, Akita University School of Medicine, Akita City 010, Japan, and the parallel  Departments of Internal Medicine and Physiology, University of Michigan and  Howard Hughes Medical Institute, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Polyamines are essential cations for normal cell growth and differentiation (1, 2). 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 (3, 4). Ornithine decarboxylase (ODC)1 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 (2, 5-7). Deregulated expression of ODC and the subsequent changes in polyamine concentrations have been associated with several types of tumors (4, 8-10). Recent studies indicate that overexpression of oncogenes such as myc (11-13), ras (14), fos (15), and mos (16) 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 (17, 18).

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 (6, 7). 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 (7). 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 (19). 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 (19). 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 (20). 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's Drosophila line 2 (SL2) cells, consistent with properties previously attributed to NF-ODC1.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture and Preparation of Nuclear Extracts-- 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 mM L-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. 21, based on the protocol by Dignam and co-workers (22). 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.

Electrophoretic Mobility Shift Assay (EMSA) and Oligonucleotide Sequences-- 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 mM Hepes, 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 [gamma -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) (23). 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'.

Isolation of the NF-ODC1 cDNA-- 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 into Saccharomyces 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 DH5alpha cells. Plasmid DNA was isolated from the transformed bacteria and transformed into the strain containing the mutated target reporter. The Gene TrapperTM cDNA positive selection system (Life Technologies, Inc.) with a SuperscriptTM 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 htbeta : HTB-1, 5'-TCAAGATCGAAGTATGCCTCAC-3'; HTB-2, 5'-GCTCTGAGGAAGATTCTGGGC-3'; and HTB-3A, 5'-TGCCTTCTGAGTCCAGTAAAG-3'.

In Vitro Translations-- 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 the in 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. (20) were used to prepare in vitro transcription/translation products as indicated under "Results."

Enrichment of Activity and Immunoblot Analysis-- 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-ODC1 binding 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 (24). 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, NENTM 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.

Transfection of SL2 Cells-- SL2 cells were transfected using a modification of a previously described method (25). 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. gamma F-gal is an internal control plasmid in which the E. coli beta -galactosidase gene is under the control of the Drosophila melanogaster hsp70 core promoter (26) 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 (27) 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 (29) with PstI and SalI to removing the HTLVIII LTR Sp1 binding site. A double-stranded oligonucleotide containing the ODC Sp1 binding site with PstI 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 (20). To each 60-mm dish, 0.1 µg of gamma F-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-LightTM beta -galactosidase assay (Tropix, Inc., Bedford, MA) and 50 µl of lysate were used to determine the luciferase or chloramphenicol acetyltransferase activity (30, 31). The luciferase or chloramphenicol acetyltransferase activity was normalized to beta -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Characterization of DNA Binding by NF-ODC1-- 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. 19) contained only the NF-ODC1 binding site, which also had low affinity for Sp1 (see below). GCS (formerly ODC53; see Ref. 19) 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. 2A, 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-ODC1 binding (19). 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. 2A, compare lanes 2 and 7). 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. 2A, lane 5). GCS did not compete for NF-ODC1 binding (Fig. 2A, 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. 2A, 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. 2A, 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. 2A, lane 2 with Fig. 3A, lane 2).


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Fig. 1.   DNA sequences of the proximal GC box within the ODC promoter (-123 to -91). The wild type sequence of the ODC proximal GC box is shown. Indicated are the designations and upper strand sequences of double-stranded oligonucleotides used in the study, since these oligonucleotides are referred to throughout the text. The underlined nucleotides denote the Sp1 binding site, and the nucleotides in bold denote the NF-ODC1 binding site. The lowercase letters represent bases that differ from the wild type sequence.


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Fig. 2.   The GC box sequence binds several proteins in Jurkat nuclear extract. A, EMSA was performed, as detailed under "Materials and Methods," using Jurkat nuclear extract (10 µg of total protein/lane). The oligonucleotides used as probes are indicated, as is the molar -fold excess of unlabeled competitors (WT refers to GCWT, N refers to GCN, and S refers to GCS). The reaction in lanes 1 and 6 contained free probe only, with no protein. The four shifted bands of interest are indicated. All the reactions were run on the same gels; only film exposure times differ; lanes 1-5 were exposed for 4 h, and lanes 6-10 were exposed for 12 h. B, EMSA was performed using Jurkat nuclear extract (10 µg of total protein/lane) with GCN as probe. A 100-fold molar excess of unlabeled GCS was added to the reactions to eliminate the radioactive bands of C1, C2, and C4. Lanes 1-4, increasing amounts of the zinc chelator, o-phenanthroline in ethanol; lanes 5-7, 750 µM o-phenanthroline and increasing amounts of Zn2SO4; lane 8, ethanol control. Both o-phenanthroline and Zn2SO4 were added to the reactions prior to the DNA probe.


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Fig. 3.   Sp1, 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. SS indicates 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.

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. 2B). 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. 3A). 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. 3A, lane 3). Binding of NF-ODC1 was not inhibited by any of these antibodies (Fig. 3A, compare lane 2 with lanes 4-7). The Sp1 band was supershifted as expected when the Sp1 antibody was used (Fig. 3A, lane 7). To determine if C2 or C4 contained proteins that were related to Sp1, visualization of these two proteins was enhanced two ways. GCWT6 (see Fig. 1), which binds NF-ODC1, but none of the other proteins, was used as a competitor, eliminating the NF-ODC1 complex (Fig. 3B, compare lanes 2 and 3). Additionally, anti-Sp1 was added to the reaction so that the Sp1 complex was shifted to a higher location on the gel (Fig. 3B, compare lanes 3 and 4). Not only were C2 and C4 better visualized, but an additional complex was detected, C1b, which was normally hidden by the Sp1 complex. When anti-Sp4 was added to the binding reaction, C1b diminished in intensity but a residual complex remained (Fig. 3B, compare lanes 4 and 5). Doubling the amount of anti-Sp4 did not further reduce the residual band (Fig. 3B, lane 6). Pre-incubation of the control peptide, P4, with the Sp4 antibody eliminated the influence of the antibody on the intensity of C1b (Fig. 3B, compare lanes 5 and 7). When Sp3 antibodies were added to the reaction mixture, C2 and C4 greatly diminished in intensity (Fig. 3B, compare lanes 4 and 8). The control peptide, P3, eliminated the effect (Fig. 3B, compare lanes 4, 8, and 9). Anti-Sp2 caused no change in the banding pattern (data not shown). These results indicate that C2 and C4 are both due to binding of Sp3. In addition, Sp4 appears to be involved in a portion of the C1b complex.

GC factor (GCF; Ref. 32), MYC-associated zinc finger protein (MAZ; Ref. 33), and specific members of the Egr-1 family (34-36) are DNA-binding proteins that are expressed in many different cell types, contain zinc finger domains, act as transcriptional repressors, and specifically bind DNA sequences very similar to the NF-ODC1 binding sequence (Fig. 4A). When double-stranded oligonucleotides containing the GCF or Egr-1 binding sites were used in EMSAs as unlabeled competitors, they did not compete for NF-ODC1 binding (Fig. 4B). Since DNA binding activity of both MAZ and NF-ODC1 have been previously characterized in HeLa nuclear extract (19, 33), supershift experiments with antibodies to MAZ were performed using this type of extract (Fig. 4C). Unlabeled GCS was added to reaction in lanes 2-5 to identify the NF-ODC1 complex. When anti-MAZ was added to the binding reactions containing GCWT as probe, no reduction in NF-ODC1 binding was seen (Fig. 4C, compare lane 2 to lanes 4 and 5). The same experiment was done using Jurkat nuclear extract with the same results; there was no reduction in NF-ODC1 binding with the addition of anti-MAZ (data not shown). As a positive control for the effectiveness of the MAZ antibody, in vitro translated MAZ was used in supershift experiments (Fig. 4C). There was a significant amount of binding activity in the in vitro transcription/translation reaction mixture without any MAZ expression vector added (Fig. 4C, compare lanes 6 and 12). When the MAZ expression vector was added to the in vitro transcription/translation reaction, an additional complex was seen (Fig. 4C, compare lanes 7 and 12). Competition with unlabeled oligonucleotides determined that this complex was specific. When oligonucleotide GCN was added as an unlabeled competitor, the intensity of the MAZ band was diminished significantly (Fig. 4C, compare lanes 7 and 8); the Sp1 binding site (oligonucleotide GCS) competed to a small degree for MAZ binding as previously reported (37)(Fig. 4C, compare lanes 7 and 9) and oligonucleotide E which had no sequence similarity to the MAZ binding site, did not compete for MAZ binding (Fig. 4C, compare lanes 7 and 10). MAZ antibody supershifted the specific band formed by in vitro synthesized MAZ (Fig. 4C, lanes 7 and 11). Taken together, these results indicate that neither GCF, MAZ, nor Egr-1-like proteins are responsible for the NF-ODC1 binding activity.


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Fig. 4.   NF-ODC1 binding activity is not due to GCF, MAZ or members of the Egr-1 family. A, DNA binding sites for NF-ODC1, GCF, MAZ, and Egr-1 family are compared. The uppercase, bold letters denote the known minimal binding sites of each protein. B, EMSA was performed as described under "Materials and Methods." Each binding reaction contained radiolabeled GCWT (0.1 pmol) and Jurkat nuclear extract (10 µg of total protein). GCF and Egr-1 are double-stranded oligonucleotides containing the DNA binding site for GCF and the Egr-1 family, respectively (see "Materials and Methods" for oligonucleotide sequences). These unlabeled double-stranded oligonucleotides were added to the indicated binding reactions at the indicated molar -fold excess at the same time as the GCWT probe but before the nuclear extract. All reactions were run on the same gel and exposed to film for same length of time. C, a supershift experiment was performed as detailed under "Materials and Methods." The anti-MAZ was incubated with the protein solution for 1 h before the DNA (probe plus unlabeled competitors) was added to the binding reaction. Radiolabeled GCWT (0.1 pmol/reaction) was the probe in all of the binding reactions, which contained either HeLa cell nuclear extract or the product of a MAZ in vitro transcription/translation reaction. Binding reactions in lanes 1-5 contained 5 µg of total protein/lane of HeLa cell nuclear extract; lane 6 contained no protein; lanes 7-11 contained equal amounts of an in vitro transcription/translation reaction using pMAZHH (a MAZ expression vector); and lane 12 contained the same amount of a control in vitro transcription/translation reaction without vector. To identify the NF-ODC1 complex, unlabeled GCS was added at 75-fold molar excess to reactions in lanes 2-5. Unlabeled competitors as indicated were added to the reactions at the same time as the probe. N refers to GCN, S refers to GCS, and E refers to a double-stranded oligonucleotide that contained the E-box binding site for c-Myc. SS indicates the location of the supershifted complex. The location of the complexes containing NF-ODC1 and MAZ are indicated. All lanes are from the same gel; only film exposure times differ. Lanes 1-5 were exposed for 6 h, and lanes 6-12 were exposed for 2 days.

Identification of a cDNA Encoding NF-ODC1 Binding Activity-- We used a yeast one-hybrid selection method (see "Materials and Methods") to isolate a cDNA encoding the protein responsible for the NF-ODC1 binding activity. We screened a human leukemia cDNA library using three tandem copies of the GCN sequence as the target binding sequence. After screening approximately 1 × 106 independent clones, five independent positive clones were identified, four of which were partially sequenced. One had 88% identity with the mouse interleukin 2 receptor, and the other three had 50-60% identity with known zinc finger DNA-binding proteins over the regions sequenced, but appeared to be cDNAs that coded for as yet unidentified proteins. The further characterization of these four clones is presently under way. The fifth clone, NF6, was sequenced in its entirety and was found to have 98% identity with a cDNA that encodes a human CACCC element-binding protein called htbeta (Ref. 38; accession no. L04282), and 91% identity with both the cDNA for rat ZBP-89, a DNA-binding protein that represses both basal and inducible expression of the gastrin gene (Ref. 20; accession no. U30381), and the cDNA for mouse BFCOL1, a transcription factor that binds to the promoter regions of two mouse type I collagen genes (Ref. 39; accession no. 97184139). ZBP-89 and BFCOL1 are species homologues of each other, and htbeta appears to be a truncated form of the human ZBP-89 protein. The relationship between these proteins is detailed further under "Discussion."

NF6 was not a full-length clone; it started 100 nucleotides downstream of the translational start site of the htbeta /rat ZBP-89/BFCOL1 cDNAs. The "Gene Trapper" method (see "Materials and Methods") was used to identify other hZBP-89 clones from a normal human leukocyte library in order to obtain the complete cDNA sequence. The full-length NF-ODC1 cDNA (accession no. AF039019) contains 2798 nucleotides and is the human homologue of rat ZBP-89/mouse BFCOL1. The sequence of human ZBP-89 is 92% and 96% identical at the nucleotide and amino acid levels, respectively, with both the mouse and rat sequences. In comparison to the sequence of htbeta , the NF-ODC1 sequence is 98% identical at the nucleotide level, and the amino acid sequence is 98% identical to htbeta in the regions in which the reading frames are the same (see "Discussion"). The NF-ODC1 cDNA sequence has the same translational start site as the three other cDNAs and the same termination site as both ZBP-89 and BFCOL1.

Human ZBP-89 Is the Protein Responsible for the NF-ODC1 DNA Binding Activity-- Several approaches were taken to establish that human ZBP-89 is indeed the protein responsible for the NF-ODC1 binding activity. We developed a four-step purification procedure that used Jurkat nuclear extract as starting material. The steps included an ammonium sulfate precipitation, desalting, fractionation on a Mono Q column, and adsorption to a DNA affinity column made with the GCN double-stranded oligonucleotide (see "Materials and Methods" for details). We used EMSAs to determine the protein fractions that contained NF-ODC1 binding activity and to compare the amount of NF-ODC1 binding activity between the different protein fractions. The first three steps did not result in significant overall enrichment of activity, but they did combine to remove the majority of Sp1 and Sp4 proteins. There was substantial enrichment (34-fold) in NF-ODC1 binding activity in the protein fraction eluted from the DNA affinity column compared with the starting material (data not shown). If human ZBP-89 were responsible for this binding activity, this protein should follow NF-ODC1 binding activity during the enrichment procedure. Equal amounts of total protein from three steps of the purification procedure were analyzed by immunoblot analysis using a polyclonal antibody to rat ZBP-89 (Fig. 5). In vitro translated rat ZBP-89 was analyzed at the same time as a positive control. Rat ZBP-89 ran as a doublet just above the 103-kDa marker (Fig. 5, lane 1), as did the human ZBP-89 in protein fractions from the purification procedure (Fig. 5, lanes 3-5). This was a higher molecular weight than earlier reported, but was a consistent result using this 7.5% acrylamide mini-gel format and the Bio-Rad prestained markers. There was a 37-fold enrichment of the ZBP-89 signal by densitometry between the DNA affinity fraction and the Jurkat nuclear extract. This enrichment in human ZBP-89 paralleled the 34-fold enrichment of NF-ODC1 binding activity in these protein fractions.


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Fig. 5.   Enrichment of NF-ODC1 activity parallels enrichment of ZBP-89 protein. Protein fractions from a NF-ODC1 purification procedure (see "Materials and Methods") and from in vitro transcription/translation reactions were analyzed by immunoblot analysis using anti-ZBP-89. The proteins were separated by SDS-PAGE using a 7.5% acrylamide gel, followed by blotting onto a polyvinylidene fluoride membrane and detection with Phototope-HRP Western blot detection kit using anti-ZBP-89. Lanes 1 and 2 contained 3 µl of the in vitro transcription/translation product of pBKCMV-rat ZBP-89 or the control vector, pBKCMV, respectively. Lanes 3, 4, and 5 contained 10 µg of total protein from Jurkat nuclear extract, the Mono Q fraction containing NF-ODC1 binding activity, or the DNA affinity fraction containing NF-ODC1 activity, respectively. Locations of protein molecular size standards (kDa) are indicated.

The ability of antibodies against ZBP-89 to inhibit NF-ODC1 binding activity was tested. Jurkat nuclear extract was incubated with both anti-Sp1 and anti-Sp3 to both act as a control to show the specificity of the ZBP-89 antibody and to enhance the visualization of the NF-ODC1 complex (Fig. 6, compare lanes 1 and 2). As shown in Fig. 3, neither of these antibodies inhibited the NF-ODC1 binding. When anti-ZBP-89 was added with or without the addition of anti-Sp1 and anti-Sp3, the NF-ODC1 binding to oligonucleotide GCN was abolished (Fig. 6, lanes 3 and 4). We used competition with a truncated form of rat ZBP-89 to show that this inhibition of NF-ODC1 binding was due to a specific interaction with ZBP-89 antibody (Fig. 6). The expression vector, B22, contained a COOH-terminal truncated rat ZBP-89 cDNA. The B22 cDNA encoded a 64-kDa protein that contained the four zinc finger domains and was able to bind to the NF-ODC1 binding site. To detect the competition between B22 and NF-ODC1 for anti-ZBP-89, the antibody could not be in excess. Therefore, one third of the amount of anti-ZBP-89 was used in lanes 8 and 10 as compared with lanes 3 and 4 in Fig. 6. This low level of antibody still strongly inhibited the NF-ODC1 binding activity (Fig. 6, compare lanes 5 and 8). When in vitro translated B22 was added instead of the control translation reaction, NF-ODC1 binding activity was partially restored (Fig. 6, compare lanes 5, 8, and 10) and the B22 binding was greatly reduced (Fig. 6, compare lanes 6 and 10), consistent with competition for the same antibody binding site.


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Fig. 6.   NF-ODC1 binding activity is inhibited by antibodies specific to ZBP-89. In lanes 1-6, 8, and 10, binding reactions containing 2.5 µg of Jurkat nuclear extract were pre-incubated with or without the indicated antibody for 1 h before the addition of radiolabeled GCN (0.1 pmol/reaction). Reactions in lanes 3 and 4 contained 3 µl of anti-ZBP-89, and reactions in lanes 2 and 3 contained 2 µl of both anti-Sp1 and anti-Sp3. In lanes 8 and 10, 1 µl of anti-ZBP-89 was incubated overnight with 7 µl of either B22 in vitro translation reaction or control in vitro translation reaction prior to the incubation with the Jurkat nuclear extract. In contrast, in lanes 5 and 6, 7 µl of either B22 in vitro translation reaction or control in vitro translation reaction was incubated overnight without anti-ZBP-89 prior to the incubation with the Jurkat nuclear extract. In lanes 7 and 9, reactions contained probe and 7 µl of either the control or B22 in vitro translation reaction, respectively; no Jurkat nuclear extract was added. Unlabeled GCS was added at 75-fold molar excess to the reactions in lanes 5-10 to eliminate complexes containing the Sp1 family. SS indicates the supershift complexes. Locations of NF-ODC1, Sp1, Sp3, and B22 complexes are indicated.

A series of double-stranded oligonucleotides was used as competitors in EMSAs to compare the binding specificity of NF-ODC1 and ZBP-89. The sequence changes in these oligonucleotides were placed into the GCN context (Fig. 7B). Fig. 7A shows an EMSA where GCN was radiolabeled and three different oligonucleotides were used as competitors. The ability of the three different double-stranded oligonucleotides to compete for either NF-ODC1 binding or human ZBP-89 binding was compared. The degree of competition by the three oligonucleotides was indistinguishable between Jurkat nuclear extract and human ZBP-89. GCN competed the best, whereas GCN4 did not compete at all. Fig. 7B summarizes the results of EMSAs in which these oligonucleotides and nine additional double-stranded oligonucleotides were used as unlabeled competitors. For each individual oligonucleotide, the relative intensity of the NF-ODC1 signal did not differ significantly from that of the human ZBP-89 signal, indicating that there was no significant difference in the affinity of each individual oligonucleotide to NF-ODC1 compared with human ZBP-89.


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Fig. 7.   Comparison of the binding affinities of NF-ODC1 and human ZBP-89. A, EMSA was performed with 10 µg of total protein/reaction of Jurkat nuclear extract or 2 µl/reaction of in vitro translated human ZBP-89, radiolabeled GCN, and the indicated oligonucleotides as unlabeled competitors at the indicated molar -fold excess (see "Materials and Methods" for details). B, listed are the names and corresponding sequences of the double-stranded oligonucleotides used as competitors in EMSAs as described in A. Phosphorimage analysis was used to determine the signal intensity of the NF-ODC1 complex or the ZBP-89 complex with or without a competitor added to the reaction. The relative signal intensity in the presence of each competitor was then determined by setting the intensity of the signal without competitor at 1.00. The unlabeled competitors were at 15-fold molar excess.

The binding affinities of both NF-ODC1 and ZBP-89 differed between the twelve oligonucleotides. When GCN, GCN7, GCN3, or GCN12 were used as competitors, the signal was decreased by varying degrees. GCN, the wild type sequence, decreased the signal intensity to the largest extent indicating that ZBP-89/NF-ODC1 had lower affinity for GCN7, GCN3, and GCN12 compared with the wild type binding site. In contrast, there was no change in the signal intensities when GCN2, GCN4, GCN8, GCN9, GCN10, GCN11, GCN13, or GCN14 were used as competitors compared with when no competitor was used, suggesting that the five cytidines that were individually altered in these competitors sequences were essential for the binding of ZBP-89/NF-ODC1 to the DNA. These results were based upon EMSAs in which a 15-fold molar excess of oligonucleotides were used to compete radiolabeled GCN. The same result was observed when the competitors were used at a 5-fold molar excess (data not shown). These results, all taken together, strongly suggest that human ZBP-89 is the protein responsible for the NF-ODC1 binding activity.

ZBP-89 Represses Activation of the ODC Promoter by Sp1-- Transient transfections in SL2 cells were used to determine the effect of ZBP-89 expression on ODC promoter activity. EMSAs, with radiolabeled GCN, were performed using nuclear extract isolated from SL2 cells transfected with Sp1 and ZBP-89 expression vectors. Results indicated that both proteins were synthesized and capable of binding DNA (data not shown). SL2 cells were transfected with 5 µg of a luciferase reporter construct containing -133 to +16 of the ODC promoter (pOD150WTLuc) and increasing amounts of a Sp1 expression vector (0.2, 0.5, or 0.75 µg). In addition, either 0.5 µg of a ZBP-89 expression vector or 0.5 µg of the empty expression vector were added to each transfection. Without the addition of ZBP-89 expression vector, the ODC promoter construct was activated by Sp1 (Fig. 8A). ZBP-89 expression repressed the Sp1 activation of the ODC promoter at all three levels of Sp1 expression (Fig. 8A). This repression was also seen with 0.2 µg of ZBP-89 expression vector, but to a lesser degree (data not shown). To determine that the ZBP-89 repression was specific for promoters containing its binding site, the ODC promoter construct, pOD150M12Luc was created. This construct contained a 20-base pair deletion, which resulted in the removal of 6 out of 11 of the base pairs in the ZBP-89 binding site but leaving the Sp1 binding site intact. EMSAs showed that the ODC promoter region in pOD150M12Luc did not bind ZBP-89, but still bound Sp1 (data not shown). In SL2 cells, pOD150M12Luc was activated by Sp1, but instead of repressing this activation, ZBP-89 expression slightly enhanced the Sp1 effect at both 0.5 µg (Fig. 8B) or 0.2 µg (data not shown) of ZBP-89 expression vector. The maximum activation by Sp1 of pOD150M12Luc was generally larger than the maximum activation of pOD150WTLuc, as shown in the experiment depicted in Fig. 8. Sp1 may have higher affinity for pOD150M12Luc than the wild type ODC promoter, but further studies are needed to confirm this hypothesis. A second control plasmid, BCAT-S, was made which contained the ODC Sp1 binding site and the E1b TATA box fused upstream of the chloramphenicol acetyltransferase gene. This construct was also activated by Sp1 expression, but not inhibited by ZBP-89 (data not shown).


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Fig. 8.   ZBP-89 inhibits activation of the ODC promoter by Sp1. Transient transfections were performed with SL2 cells as detailed under "Materials and Methods." Increasing amounts of pPacSp1 expression vector were added to 5 µg of the ODC construct, pOD150WTLuc (A) or pOD150M12 (B) with 0.5 µg of the modified pBKCMV-ZBP-89 expression vector (open bars) or 0.5 µg of the empty modified pBKCMV vector (black bars). pPac0 vector was added as needed to keep total amount of DNA per transfection constant. Each transfection was done in triplicate. Luciferase activity was normalized to beta -galactosidase activity, and the standard deviation from the mean is presented. Shown in this figure is one of at least three independent experiments performed. The normalized luciferase values differed between experiments, but the trend was the same.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The synthesis and degradation of ODC is highly regulated, and transcriptional regulation is a major route through which the levels of ODC are controlled. The proximal promoter region of the ODC gene contains a GC box that we have shown previously to be involved in both basal and induced transcription. This GC-rich region contains at least three overlapping protein binding sites. Two are sites for known transcription factors, Sp1 and WT1, which we and others have shown to bind in this region and which may participate in regulating transcription of the ODC gene (19, 31, 40, 41).2 Previous results from the use of in vitro binding assays and in vivo promoter studies have suggested the existence of a third protein, NF-ODC1, that also binds in this region and appears to repress transcription of the ODC gene (19). The DNA sequence of the NF-ODC1 binding site in the ODC promoter was used in a yeast one-hybrid screen to identify the human protein responsible for this binding activity. One of the five positive clones obtained had 98% and 92% identity to the cDNAs encoding human htbeta and rat ZBP-89, respectively (20, 38). The full-length cDNA sequence was 92% identical to the rat ZBP-89, indicating that we had cloned the human homologue of rat ZBP-89.

Several different approaches were used to establish that human ZBP-89 is indeed the protein responsible for the NF-ODC1 binding activity. Using in vitro binding as an activity assay, we partially purified the NF-ODC1 activity with the final step being adsorption to a DNA affinity column. Enrichment of the NF-ODC1 binding activity paralleled the enrichment of immunoreactive ZBP-89 protein during this procedure. NF-ODC1 proved remarkably difficult to purify, because of its extreme lability. During our initial characterization of NF-ODC1 binding activity, we found that if protease inhibitors were not used during the preparation of nuclear extract, there were two additional protein complexes with faster mobility associated with the NF-ODC1 binding activity (19). Immunoblot analysis, using antibody to ZBP-89, indicated that NF-ODC1 began to break down dramatically after fractionation on the DNA affinity column even though a complex mixture of protease inhibitors was used throughout the purification procedure (data not shown). A second line of evidence, demonstrating that NF-ODC1 and human ZBP-89 were the same protein, came from the influence of anti-ZBP-89 on NF-ODC1 binding. In binding studies with Jurkat nuclear extract, NF-ODC1 binding was abolished by addition of anti-ZBP-89. EMSAs also showed that the NF-ODC1 from Jurkat nuclear extract and human ZBP-89 from in vitro translation resulted in protein-DNA complexes that migrated to the same location on the gel (data not shown). More importantly, when the binding affinities of 12 different double-stranded oligonucleotides were studied, each individual oligonucleotide had indistinguishable affinities for the two proteins. These three different lines of evidence indicate that ZBP-89 is the protein responsible for the NF-ODC1 binding activity that we have previously characterized. These studies do not however, eliminate the possibility that other related proteins may bind the NF-ODC1 site and function in the transcriptional regulation of the ODC gene.

ZBP-89 protein has now been identified, including isolation of cDNAs encoding the protein, in four different promoter systems and three species: rat ZBP-89, gastrin gene (20); mouse BFCOL1, type I collagen genes (39); human htbeta , T-cell receptor genes (38); and human NF-ODC1, ODC gene (current study). Two deletions of single base pairs in the htbeta sequence compared with the ZBP-89/BFCOL1/NF-ODC1 sequence result in an in-frame stop codon at nucleotide 1753 in the htbeta cDNA, resulting in a polypeptide with a predicted mass of 49 kDa. In contrast, the first in-frame stop codon in the ZBP-89/BFCOL1/NF-ODC1 sequence occurs 1018 nucleotides downstream, resulting in a polypeptide with the predicted mass of 89 kDa (for details, see Ref. 20). Analysis by Southern blot indicates only one gene (20, 38). Therefore, htbeta appears to be a truncated version of ZBP-89/BFCOL1/NF-ODC1. The mechanism that produces the differences between ZBP-89 and htbeta is not known, but alternative splicing would not appear to explain the single-base deletions. A major band at around 49 kDa, which would represent the htbeta protein, has not been detected in any of the immunochemical studies of ZBP-89. There are several potential translational start sites in all of the cDNAs, and doublets have been seen upon gel electrophoresis of both the in vitro and in vivo translated ZBP-89/BFCOL1/NF-ODC1 (current study and Refs. 20 and 39). Amino acid sequence analysis indicates that this protein has several distinct motifs including four C2H2 Krüppel-type zinc finger motifs (making up the DNA binding domain), one acidic and two basic regions and, in all but htbeta , a serine-rich region in the carboxyl terminus. The sequence homology to known proteins and the corresponding deduced functions of these motifs have been discussed previously (20, 38, 39).

Our previous studies determined that the NF-ODC1 binding site in the ODC promoter is GCCCCTCCCCC. Methylation of any of the guanine residues on either strand of DNA interfered with NF-ODC1 binding to some degree, and nuclease protection experiments showed that the 5' half of the NF-ODC1 binding site overlaps with the 3' half of the Sp1 site in the ODC promoter (19). The additional binding studies reported here showed that 5 C/G pairs out of the 11 base pairs in the NF-ODC1 binding site were required for binding of ZBP-89, and a significant decrease in the affinity for ZBP-89 was seen when four additional nucleotides were individually mutated. These results suggest a consensus binding site for ZBP-89: gccCCtxCxCC, where the uppercase C represent the five essential cytidines, the lowercase letters represent residues that are involved in binding but not essential, and x represents residues that have not been mutated (Fig. 9). All of the ZBP-89 binding sites on the genes encoding the type I collagens (-180, Pro-a2(I); -168, Pro-a1(I); and -194, Pro-a1(I)), and the T-cell receptor (TCR Vbeta 8.1, TCR alpha  silencer I), contain these five essential cytidine residues (Fig. 9). The binding site on the gastrin promoter contains four out of the five essential cytidine residues. Importantly, the results of other binding studies with various mutations within these sites are consistent with the importance of these residues (20, 39). There must be additional requirements beyond the five essential cytidines since several other characterized binding sites contain this motif, but do not in most cases compete for ZBP-89 binding (compare, for example, the binding sites for Egr-1, MAZ, GCF, and Sp1; Figs. 1 and 4). Interestingly, except for the gastrin and the -194, Pro-alpha 1(I) sites, all of the ZBP-89 binding sites contain in addition to the five essential cytidines, five consecutive cytidine residues preceded by either a T or an A (see Fig. 9). Both the gastrin and -194, Pro-alpha 1(I) sites appear to have significantly lower affinity for ZBP-89. In EMSAs using either in vivo or in vitro translated humans ZBP-89, there was no competition by the gastrin binding site at either 5- or 15-fold molar excess with the ODC binding site (data not shown). Hasegawa and co-workers (39) showed that the -194, Pro-alpha 1(I) site had a much less affinity than either the -168, Pro-alpha 1(I) or the -180, Pro-alpha 2(I) site for BFCOL1. Consistent with these observations, the Sp1 binding site competes significantly with the gastrin site for ZBP-89 binding, but does not compete with either the ODC or -180, Pro-alpha 2(I) site for ZBP-89 binding (current study and Refs. 20 and 39). Relative to the ODC gene, the ZBP-89 binding site in the TCR Vbeta 8.1, the TCR alpha  silencer I, the -194, Pro-alpha 1(I), and the gastrin promoters are inverted. The importance of the orientation of the ZBP-89 binding site relative to the transcriptional start site is not known.


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Fig. 9.   The suggested consensus sequence for the ZBP-89 binding site is shown. Bold and underlined letters represent nucleotides that were required for ZBP-89 interaction with its binding site in the ODC gene. The lowercase letters in the consensus site represent nucleotides that when altered lowered the affinity to ZBP-89 but did not abolish binding, and x represents residues that were not tested. The known ZBP-89 binding sites in the promoters of the ODC gene, the type I collagen genes (Pro-alpha 2(I); -168, Pro-alpha 1(I); and -194, Pro-alpha 1(I)), the T-cell receptor genes (TCR Vbeta 8.1, TCR alpha  silencer I), and the gastrin gene are aligned with the consensus sequence. The asterisk denotes sequences that are inverted in relationship to the transcriptional start site when compared with the binding site in the ODC promoter.

ZBP-89 is an ubiquitous protein found in most cell types and tissues examined to date (19, 20, 23, 38, 39) and has been shown to be overexpressed in gastric cancer (23). The function of ZBP-89 in transcriptional regulation is currently under investigation. A general scheme has emerged; ZBP-89 binds to a GC-rich region that is within the first 200 base pairs downstream of the transcriptional start site of the gene. The ZBP-89 binding site is overlapping or adjacent to other known transcription factors, in particular, Sp1. Mutations in the ZBP-89 binding site result in significant changes in the promoter activity. In earlier studies, we showed that the activity of the ODC promoter increased in several cell lines when the NF-ODC1 site was mutated (19). In GH4 cells, a cell line derived from a rat pituitary tumor, ZBP-89 represses both basal and EGF-stimulated promoter activity of the gastrin gene, with no effect on expression of a control promoter construct (20). When the BFCOL1 site was mutated in the promoter of the type 1 collagen genes, the promoter activity increased 3-4-fold in transient transfection experiments (43, 44). However, Hasegawa and co-workers (39) did not detect an effect of BFCOL1 on the pro-alpha 2(I) collagen promoter in transient co-transfection experiments in BALB/c 3T3 fibroblasts or S194 B cells. They did show that a fusion polypeptide between the COOH terminus of BFCOL1 and the yeast Gal4 DNA-binding domain activated a reporter construct, suggesting that the COOH terminus contains a domain with transactivating potential in yeast. Wang and co-workers (38) showed that htbeta slightly activated the T-cell receptor gene and inhibited the silencing effect of the mouse T-cell receptor alpha  gene silencer in HeLa cells. The endogenous level of ZBP-89 and other relevant transcription factors such as Sp1 may be too high in the BALB/c 3T3, S194 B, or HeLa cells to see a significant effect of additional ZBP-89. In this study, we used SL2 cells for in vivo promoter analysis. This insect cell line has an advantage over mammalian cell lines in that there are no detectable levels of the Sp1-like activity (27) or NF-ODC1 activity (data not shown). Transient expression of Sp1 in SL2 cells increased the ODC promoter activity in a dose-dependent manner with the maximum increase depending on the region of ODC promoter used (40).3 All three of the constructs used in this study contained a Sp1 binding site and were stimulated by Sp1 expression. ZBP-89 expression repressed the Sp1 activation only of the ODC wild type promoter construct, which contained the ZBP-89 binding site. This repression was not seen with the constructs that did not contained the ZBP-89 binding site, BCAT-S or pOD150M12Luc.

The limited functional studies done to date suggest that ZBP-89 has the potential to either repress or stimulate transcriptional activity, depending on the particular promoter. This duality is seen with other transcription factors, and there are several paradigms that explain this phenomenon, including competition with different transcriptional factors for DNA binding, interference with the activity of DNA-bound activators, alternative splicing, alternative translational initiation, and positional effects of binding sites (42, 45, 46). As mentioned earlier, all of the known ZBP-89 binding sites either overlap or are in close proximity to binding sites of other transacting proteins. These include binding sites for Sp1, Sp3, Sp4, and WT1 on the ODC gene (current study, Ref. 41, and Footnote 2), for Sp1 and Krox on the pro-alpha 2(I) gene (39), and for Sp1 on the gastrin gene (20). ZBP-89 may repress transcription by competing with these other factors for DNA binding, thereby decreasing the activation. Studies have shown, for both the gastrin and ODC promoters, that the Sp1 and ZBP-89 binding sites overlap and that these two factors bind in a mutually exclusive manner (19, 20). However, more detailed studies must be undertaken to differentiate between this mechanism and at least two other possibilities: ZBP-89 may act as an active repressor by directly inhibiting transcriptional initiation, or ZBP-89 may mask or quench the activity of other factors through protein-protein interactions.

    ACKNOWLEDGEMENTS

We are grateful to Dr. R. Tjian for providing the pPac vectors and BCAT-1, Dr. Pier Paolo Di Nocera for providing the gamma F-gal vector, and Drs. K. Marcu and Amanda J. Patel for providing the MAZ antibody, the MAZHH expression plasmid, and helpful advice on this study. We thank Dr. Karol Bomsztyk for valuable technical advice.

    FOOTNOTES

* This work was supported in part by United States Public Health Service Grants DE08229 (to D. R. M.) and DK45729 (to J. L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039019.

** To whom correspondence should be addressed: Dept. of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350. Fax: 206-543-4822; E-mail: dmorris{at}u.washington.edu.

The abbreviations used are: ODC, ornithine decarboxylase; EMSA, electrophoretic mobility shift assay; SL2, Schneider's Drosophila line 2TBE, Tris-borate/EDTA electrophoresis bufferGCF, GC factorMAZ, MYC-associated zinc finger proteinTCR, T-cell receptor.

2 R. S. Li, G. L. Law, R. A. Seifert, P. J. Romaniuk, and D. R. Morris, submitted for publication.

3 G. L. Law and D. R. Morris, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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