From the Department of Biochemistry, University of
Washington, Seattle, Washington 98195, the § Department of
Biochemistry, Akita University School of Medicine, Akita City 010, Japan, and the
Departments of Internal Medicine and
Physiology, University of Michigan and ¶ Howard Hughes Medical
Institute, Ann Arbor, Michigan 48109
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 [-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.
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 DH5 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 ht
: 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.
F-gal is an internal control plasmid in which the E. coli
-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
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
-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
-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.
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RESULTS |
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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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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 ht (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 ht
appears to be a
truncated form of the human ZBP-89 protein. The relationship between
these proteins is detailed further under "Discussion."
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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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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DISCUSSION |
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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 ht 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 ht, T-cell receptor genes (38); and human
NF-ODC1, ODC gene (current study). Two deletions of single
base pairs in the ht
sequence compared with the
ZBP-89/BFCOL1/NF-ODC1 sequence result in an in-frame stop
codon at nucleotide 1753 in the ht
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, ht
appears to be a truncated version of ZBP-89/BFCOL1/NF-ODC1. The mechanism that produces the differences between ZBP-89 and ht
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 ht
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 ht
, 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 V
8.1,
TCR
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-
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-
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-
1(I) site had a much less affinity than
either the
168, Pro-
1(I) or the
180, Pro-
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-
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 V
8.1, the TCR
silencer
I, the
194, Pro-
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.
|
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-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 ht
slightly activated the T-cell receptor gene and inhibited
the silencing effect of the mouse T-cell receptor
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-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.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. R. Tjian for providing
the pPac vectors and BCAT-1, Dr. Pier Paolo Di Nocera for providing the
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.
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FOOTNOTES |
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* 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.
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REFERENCES |
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