©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Rat Ornithine Decarboxylase Promoter Activity by Binding of Transcription Factor Sp1 (*)

(Received for publication, June 2, 1994; and in revised form, September 21, 1994)

Addanki P. Kumar Penny K. Mar Biwei Zhao Raechelle L. Montgomery Dong-Chul Kang Andrew P. Butler (§)

From the University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine biosynthesis. We investigated the transcriptional regulation of the rat ODC gene using transient expression assays. The 5`-flanking region (-1156 to +13) of the ODC gene was sufficient to mediate strong basal expression of a luciferase reporter. Sequences between -345 and -93 contributed to basal promoter activity. This region, containing five potential Sp1 binding sites, was analyzed by electrophoretic mobility shift assays. Three specific DNA-protein complexes were identified using H35 nuclear extracts and the -345/-93 ODC probe. Binding to all three was eliminated by competition with an oligonucleotide containing an Sp1 binding site, but not by a mutant Sp1 oligonucleotide. Preincubation with an antibody against Sp1 supershifted complexes associated with one or more of Sp1 binding sites 1-4 as well as with site 5. DNase I footprinting revealed two protected regions: PR-I (-92 to -130) and PR-II (-304 to -332). PR-I contains a putative binding site for Sp1 that was protected by recombinant Sp1 protein. Transfection studies in Schneider SL2 cells demonstrated that the ODC promoter is trans-activated up to 350-fold by Sp1 and that this trans-activation is dependent on the presence of Sp1 binding sites 1-4. Thus, although the ODC promoter binds multiple nuclear proteins, Sp1 or a related protein appears to be a critical determinant of ODC transcription, possibly through cooperative interactions between Sp1 and additional transcription factors.


INTRODUCTION

Ornithine decarboxylase (ODC; EC 4.1.1.17) (^1)is the first and rate-limiting enzyme of polyamine biosynthesis (for reviews, see (1, 2, 3) ). ODC converts ornithine to putrescine, the precursor for the polyamines, which are necessary for cell growth and differentiation(1, 4) . ODC activity is critical for the G(1)/S transition of the cell cycle(2, 3, 5) . Both ODC mRNA and active enzyme are rapidly and transiently induced following mitogenic stimulation of quiescent cells with growth factors or tumor promoters(6, 7, 8, 9, 10) . Numerous studies have demonstrated that ODC activity and polyamine levels are substantially elevated in transformed cells and tumors(11, 12) . Indeed, it was recently shown that overexpression of ODC may be tumorigenic(13, 14) .

ODC is highly regulated by a variety of mechanisms, including transcription(15, 16, 17) , translation(18) , and enzyme stability(19) . The ODC gene is highly conserved between rat, mouse, and human, both within the coding region and presumptive transcriptional regulatory sequences. Among these three species, the 5`-flanking region has 82% identity within the first 148 bp and only slightly less conservation over the first 380 bp(20, 21, 22, 23) . Computer analysis of the rat ODC sequence indicates potential binding sites for multiple transcription factors, including a TATA box at -33, a CRE-like element at -50, and a possible CAAT box at -84. Three possible IRE are present at -166, -157, and -105(24) . Five consensus Sp1 binding sites are found at -231, -218, -210, -182, and -108 (GC boxes 1-5, respectively). Sequences similar to the consensus binding sites for AP-1 (25) and AP-2 (26) are present at -295 and at -327, respectively. GC boxes 3-5, the TATA box, and the CRE are fully conserved in rat and mouse ODC; GC-box 5 is also conserved in the human ODC gene. However, the importance of these motifs in the regulation of ODC has not been well characterized(22, 23, 27) .

In transient transfection studies using Rat-1 fibroblasts, van Steeg et al.(23) found that 398 bp of ODC upstream from the transcription start site is sufficient for basal promoter activity and that a putative CAAT motif located at -82 contributes to basal expression. Constitutive expression of human ODC also requires sequences within the first 378 bp upstream from the transcription start site, and this region interacts extensively with nuclear proteins(28) . A CRE-like element (CRE2) between -58 and -42 mediates cAMP responsiveness of the mouse ODC promoter, although the associated DNA-binding protein was reported to be distinct from CREB(29) . In contrast, another group reported that CRE2 binds recombinant CREB in vitro and that antibodies to CREB recognize proteins in crude extracts that associate with CRE2(30) . This element may also be involved in basal expression of ODC(30) . Interestingly, deletion of a GC-rich region containing putative binding sites for transcription factors AP-2 and Sp1, and which is just upstream of CRE2, resulted in reduced induction of the ODC promoter by protein kinase A(30) .

The mechanism by which ODC transcription is regulated in response to mitogenic signals or tumor promoters has not been elucidated. Both the murine and human ODC genes may be trans-activated by c-Myc or c-Myc-Max heterodimers(31, 32) . In the case of the murine gene, c-Myc was shown to bind to conserved sequences in the first intron and to regulate expression of reporter plasmids containing this sequence. The human ODC gene, on the other hand, was regulated by c-Myc-Max heterodimers through a 5`-flanking element that is not conserved in the mouse, rat, or hamster ODC genes(32) . Recently, Wrighton and Busslinger (33) provided evidence for the participation of c-Fos in regulating ODC transcription in rat PC12 cells but not in fibroblasts. However, these authors did not provide any information concerning which ODC promoter elements are involved in this response or whether c-Fos binds directly to the ODC promoter. Taken together, these studies suggest that multiple transcription factors may be required to induce ODC expression and that ODC regulation may be cell type-specific(34) .

In this manuscript, we investigated the transcriptional regulation of ODC in rat H35 hepatoma cells. We demonstrate that multiple regions of the 5`-flanking region of rat ODC gene contribute toward its promoter activity. We establish that transcription factor Sp1 or an immunologically related protein in H35 nuclear extracts binds to critical determinants of basal ODC transcription in vitro. Furthermore, Sp1 trans-activates the rat ODC promoter in vivo, and this activation is strictly dependent on the cluster of GC boxes 1-4 of the ODC promoter.


MATERIALS AND METHODS

Reagents

Enzymes were purchased from Boehringer Mannheim, Promega Corp. (Madison, WI), and New England Biolabs (Beverly, MA). Purified recombinant transcription factor Sp1 and double-stranded synthetic oligonucleotides containing transcription factor consensus binding sites were also purchased from Promega. Radionucleotides ([alpha-P]dCTP and [-P]ATP) were purchased from DuPont NEN. Rabbit antisera directed against residues 520-538 of human Sp1 and an oligonucleotide containing a mutant Sp1 binding site were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). TPA was obtained from LC Services (Woburn, MA). Coomassie Blue protein reagent was from Pierce. All other chemicals were from Sigma. Drosophila melanogaster Schneider SL2 cells were obtained from the American Type Culture Collection (Rockville, MD).

Plasmids

The plasmids pGEM-3Z and pGL2-basic (containing the firefly luciferase coding sequence) were obtained from Promega. A cryptic AP-1 site in the SV40 sequences downstream (35) of the luciferase gene was removed by site-directed mutagenesis(36) . The result was confirmed by DNA sequence analysis of both strands, and the BamHI-PflMI fragment containing the site was recloned into the corresponding sites of pGL2-basic. This plasmid, pGL2-m, was the basis for all of the luciferase constructs described in this paper. The plasmid pGL2-TK was derived from pGL2-m by insertion of the minimal herpes simplex virus TK promoter fragment (-105 to +51, containing the TATA element and a single GC box) between the KpnI and BglII sites. The rat ODC genomic clone pODC821 was a gift of Dr. Harry van Steeg(37) . pODClux1m was prepared by insertion of a 1.2-kilobase pair BamHI fragment containing ODC sequences from -1156 to +13 into the BglII site of pGL2-m. Details of the construction will be presented elsewhere. (^2)A fragment of the ODC promoter (-345 to -93) was subcloned into the SmaI site of pGEM-3Z to create pODC 250. Plasmids pODC 250TK, pODC 181TK, and pODC 71TK were made by cloning rat ODC promoter fragments (-345 to -93, -345 to -165, and -164 to -93, respectively) into pGL2-TK digested with XhoI and BglII. All constructs were verified by restriction mapping or DNA sequencing. The expression vector for Sp1 under control of the Drosophila actin promoter, pPacSp1 (38) , and the parental pPac control plasmids were a gift of Dr. Robert Tjian (University of California at Berkeley).

Transfections and Luciferase Assay

Subconfluent Reuber H35 rat hepatoma cells were transfected by electroporation (320 V, 960 µF), using 1 times 10^7 cells, 60 µg of luciferase reporter plasmid, and 10 µg of pRSVbetagal as an internal standard. Following transfection, cells were diluted with complete media and distributed into six 60-mm tissue culture dishes and were harvested 48 h later. The cells were lysed in 250 µl of a buffer containing 100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100, and 1 mM dithiothreitol. Extracts were clarified by centrifugation (10 min, 15,000 times g) and assayed as described below.

SL2 cells were maintained at 27 °C in Schneider's medium and were transfected using the calcium-phosphate precipitation method as described(38, 39) . Cells were seeded at 4 times 10^6/60-mm dish and were transfected 24 h later with 5 µg of luciferase reporter, 0.5 µg of pPacSp1 or parental pPac, and 1 µg of pRSVbetagal. Cells were harvested 48 h after transfection and assayed as described below.

Protein concentrations were determined by the method of Bradford(40) . Luciferase activity was assayed in duplicate on extracts containing equal amounts of protein (usually 1-5 µg) using reagents obtained from Promega. beta-Galactosidase activity was determined using a chemiluminescent substrate according to the manufacturer's protocol (Tropix, Bedford, MA). Preliminary experiments were performed to ensure the linearity of both assays. Results were expressed as the ratio of luciferase/beta-galactosidase activity at equal amounts of protein. Results of SL2 transfections were expressed as luciferase activity (relative light units) at equal amounts of protein, since the beta-galactosidase activity was too weak for accurate determination in these cells. Each experiment represents the mean of three dishes and was repeated at least three times, using at least two different plasmid preparations.

Preparation of Nuclear and Cytoplasmic Extracts

H35 cells were grown as described previously(41) . Nuclear and cytoplasmic extracts were made by the method of Dignam (42) with the inclusion of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg ml aprotinin, 10 µg ml leupeptin, and 5 µg ml pepstatin) in each buffer. Nuclear extracts were clarified by centrifugation (17,000 times g for 15 min) and were stored in 50-µl aliquots at -20 °C; they remained stable for at least 2 months. Protein concentration of the extracts was determined as described above.

Electrophoretic Mobility Shift Assays

EMSA were done using standard methods(43) . Plasmid pODC 250 was digested with EcoRI and HindIII, and the -345/-93 fragment was isolated by electroelution(44) . This fragment was labeled with [alpha-P]dCTP using Klenow DNA polymerase I and was purified by phenol-chloroform extractions followed by ethanol precipitation. Double-stranded oligonucleotides were labeled with [-P]ATP using T4 polynucleotide kinase.

Extracts were incubated with the radiolabeled probe in binding buffer (4 mM Tris-HCl, 12 mM HEPES (pH 7.9), 60 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, 12% glycerol) containing about 0.2 ng of the radiolabeled DNA fragment in a final volume of 20 µl for 25 min at room temperature. After incubation, samples were fractionated on a 4% polyacrylamide gel in 0.25 times TBE (1 times TBE = 90 mM Tris, 90 mM H(3)BO(4), 1 mM EDTA) at 4 °C. Following electrophoresis, the gel was dried and autoradiographed using Kodak XAR film at -70 °C with a DuPont Cronex intensifying screen. For competition studies, the radiolabeled probe was mixed with various molar excesses of unlabeled DNA restriction fragments or double-stranded synthetic oligonucleotides, as indicated in the figure legends, for 5 min prior to addition of nuclear extracts. DNA-protein complexes were resolved as described above. In EMSA using antibodies, the nuclear extract was preincubated with either antibody or normal rabbit serum for 20 min on ice prior to DNA probe addition.

DNase I Footprinting

DNase I footprinting was done according to the method of Galas and Schmitz(45) . The single end-labeled ODC promoter fragment from pODC 250 was used in footprinting reactions. Binding reactions were carried out at room temperature for 25 min in a final volume of 30 µl containing 25 µg of nuclear proteins, approximately 1 ng of the labeled DNA fragment, and 0.5 µg of poly(dI-dC)bulletpoly(dI-dC). Following the binding reaction, the DNA-protein complex was digested with 0.2 unit of DNase I for 2 min at room temperature. The digestion was terminated with stop solution (0.45 M sodium acetate, 150 µg ml of yeast tRNA, 0.2 mM EDTA, 500 µg ml of proteinase K, 1.6% SDS) at 37 °C for 30 min. The samples were purified by organic extractions and ethanol precipitation and loaded on an 8% sequencing gel in formamide loading buffer.

Western Blot Analysis

Nuclear extracts were fractionated on 10% SDS-polyacrylamide gels (46) and electrophoretically transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20 (blocking solution) and incubated with primary antibody raised against human Sp1. It was further incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody in blocking solution. Secondary antibody was detected by enhanced chemiluminescence, following the manufacturer's directions (Amersham Corp.).


RESULTS

Expression of ODC Promoter Constructs

In order to determine the relative promoter strength of various portions of the ODC 5`-flanking region, we performed transient expression assays, using the firefly luciferase reporter gene under the control of the rat ODC 5`-flanking region. These experiments revealed multiple regions involved in the regulation of basal ODC expression in H35 hepatoma cells. pODClux1m, containing sequences from -1156 to +13 of the rat ODC gene, conferred strong constitutive luciferase expression (Fig. 1A). Truncation of the promoter to -409 (pODClux2m) and -92 (pODClux2DeltaEm) reduced basal expression to approximately 51 and 21%, respectively (Fig. 1A). The large decrease in expression between pODClux2m and pODClux2DeltaEm indicated that DNA sequences between -409 to -93 contain elements essential for the strong expression of the ODC promoter. To investigate the molecular details of this activity, ODC sequences from -345 to -93 were subcloned upstream of the minimal TK promoter in pGL2-TK. This hybrid ODC 250TK promoter displayed 35% of the basal activity of the full-length ODC promoter (Fig. 1B). The construct pODC 181TK had a slightly reduced basal activity (25% of pODClux1m), whereas the promoter activity of pODC 71TK was less than 5% that observed for pODClux1m or only about 2-fold greater than that of the minimal TK promoter (Fig. 1B). Expression of luciferase activity from pGL2-TK, containing no ODC sequences, was almost undetectable (luciferase/beta-galactosidase, 0.001).


Figure 1: Transcriptional activity of rat ODC promoter. A, deletions of the ODC promoter fused to the luciferase reporter (10 µg/dish) were transfected into rat H35 hepatoma cells; approximately 48 h later the cells were harvested and assayed for luciferase and beta-galactosidase, as described under ``Materials and Methods.'' The results shown are mean ± S.E. of 5-13 independent transfections. B, fragments of the ODC promoter were fused to a minimal herpes simplex TK promoter and transfected into H35 cells as described as described for A. The results are the mean ± S.E. of four independent transfections.



Binding of Nuclear Proteins to the ODC Promoter Region

The results in Fig. 1showed that sequences between -345 to -93 contribute strongly to basal ODC transcription. Computer analysis of this region identified potential binding sites for various transcription factors, including AP-1, AP-2, Sp1, and IRE-BP. EMSA was used to characterize protein-DNA interactions in this region. H35 nuclear extracts formed three major shifted bands using the -345/-93 ODC probe (Fig. 2A). None of these complexes were formed with cytoplasmic extracts, and no consistent differences were observed in the EMSA pattern between TPA- or Me(2)SO-treated quiescent cells or with extracts from serum replete, growing cells. (^3)At the highest quantity of nuclear extract used (15 µg), these bands merged into broad, low mobility complexes, indicative of formation of complexes containing additional proteins (Fig. 2A, lane 7). However, even at this protein concentration, the complex was abolished by competition with a 100-fold molar excess of homologous competitor, suggesting that it was not simply a result of nonspecific interactions or protein-protein aggregation.^3 Using 5 µg of extract, a 50-fold molar excess of unlabeled homologous DNA significantly reduced the binding, whereas a 100-fold excess abolished binding (Fig. 2B, lanes 3 and 4). However, even a 100-fold molar excess of a 222-bp heterologous DNA fragment excised from pGEM-3Z did not reduce the formation of the shifted bands, demonstrating the specificity of the interaction (Fig. 2B, lanes 5 and 6).


Figure 2: EMSA using nuclear extracts from rat H35 cells and the -345/-93 ODC probe. A, nuclear extracts prepared were incubated with the end-labeled -345/-93 ODC probe, and the DNA-protein complexes were resolved by electrophoresis on a 4% native polyacrylamide gel as described under ``Materials and Methods.'' Lane 1, no nuclear extract added; lanes 2-7, H35 nuclear extract (2-15 µg, as indicated in the figure). B, the labeled ODC -345/-93 probe was preincubated with a 50- or 100-fold molar excess of unlabeled homologous or heterologous competitor DNA before the addition of 2 µg of H35 nuclear extract. The heterologous fragment was obtained from vector sequences, as described under ``Materials and Methods.'' Lane 1, no nuclear extract; lane 2, no competitor; lanes 3 and 4, 50- or 100-fold molar excess, respectively, of homologous competitor DNA. Lanes 5 and 6, 50- or 100-fold molar excess of heterologous competitor DNA.



Competition Analysis

In view of the numerous potential transcription factor binding sites predicted in this region of the ODC sequence, double-stranded synthetic oligonucleotides containing consensus binding sites for some of these factors were used as unlabeled competitors in EMSA experiments, using the -345/-93 bp ODC probe (see Fig. 3A). A 100-fold excess of consensus AP-1 (lane 3) or NFkappaB binding sites (lane 6) failed to compete. AP-2 (lane 4) and CREB oligonucleotides (lane 5) showed partial competition, but only at 100-fold molar excess. However, competition using an Sp1 oligonucleotide was striking (lane 7) and was significant with as little as a 10-fold molar excess (Fig. 4B, lane 3). A 40-fold molar excess essentially abolished formation of the shifted complexes (Fig. 4B, lane 5). These results demonstrate that H35 nuclear extracts contain factor(s) that specifically recognize the Sp1 binding sites in the ODC promoter.


Figure 3: Competition with synthetic double-stranded oligonucleotides. A, oligonucleotides containing consensus binding sites for transcription factors AP-1, AP-2, CREB, NFkappaB, and Sp1 were preincubated with the -345/-93 ODC probe, as indicated in the figure. Nuclear extracts were added and EMSA was performed as described under ``Materials and Methods.'' Lane 1, no extract; lanes 2-7, H35 nuclear extracts. B, titration of Sp1 consensus competitor. Sp1 oligonucleotide was preincubated with the -345/-93 ODC probe prior to addition of nuclear extract, and EMSA was performed as in A. Lane 1, no added nuclear extract; lanes 2-5, H35 nuclear extracts. The Sp1 consensus binding site oligonucleotide was added to the probe at molar ratios of 0-40 as indicated in the figure.




Figure 4: DNase I footprinting assay. Nuclear extracts (25 µg) from control or TPA-treated H35 cells were incubated with end-labeled DNA, treated with DNase I, and resolved on an 8% sequencing gel as described under ``Materials and Methods.'' A, footprints using the -345 to -168 probe. B, footprints obtained using the -168 to -93 probe. Lanes 1 and 5, no nuclear extract; lanes 2 and 6, control nuclear extract; lanes 3 and 7, nuclear extract from TPA-treated cells; lanes 4 and 8, recombinant Sp1 protein (1 Promega footprint unit). The regions protected by nuclear extracts (PR-I, -92 to -130; and PR-II, -304 to -332) are shown schematically by the cross-hatched boxes; sequences protected by recombinant Sp1, but not by extracts, are indicated by white boxes.



Footprint Analysis

To precisely localize the protein binding sites, DNase I footprint analysis was performed using crude nuclear extracts and the -345/-93 ODC probe. The nuclear extracts protected two regions (Fig. 4). Protected region I (PR-I) extends from -92 to -130 and protected region II (PR-II) extends from -304 to -332. PR-I includes one putative binding site for Sp1 at -108 and overlaps putative AP-2 and IRE-BP sites at -97. PR-II also includes a weak AP-2 site at -327. Because ODC expression is stimulated by TPA and growth factors, we also performed DNase I footprint analysis using nuclear extracts from TPA-treated H35 cells (Fig. 4, lanes 3 and 7). However, no significant differences between the DNase I footprints of these sequences were noted using extracts from TPA-treated cells. Additional experiments found no significant differences between protein binding in extracts from serum-starved or serum-replete cells.^3

Because the EMSA studies implicated Sp1 as a potential ODC transcription factor, we also investigated the DNase I protection pattern obtained with pure recombinant human Sp1 (Fig. 4, A and B, lanes 4 and 8). Purified Sp1 protected sequences that overlap with PR-I in the nuclear extracts. However, the site protected by Sp1 begins at -101 and extended to -156. Sp1 strongly protected additional sequences from -293 to -202 (containing GC boxes 1-3), whereas protection by crude nuclear extracts was less clear in this region.

Identification of Sp1 in H35 Nuclear Extracts

Both the EMSA results and DNase I footprinting suggested that Sp1 or a related protein could be important for regulation of ODC transcription. Therefore, the presence of Sp1 binding activity in H35 cells was assessed using a radiolabeled Sp1 consensus binding site oligonucleotide as a probe (Fig. 5A). Nuclear extracts from H35 cells formed two broad shifted complexes with the Sp1 oligonucleotide (lane 2). This pattern was eliminated by a 100-fold molar excess of unlabeled Sp1 oligonucleotide (lane 3), but was not affected by a 100-fold molar excess of unlabeled AP-1 oligonucleotide (lane 4), indicating that the interaction is due to a factor with binding specificity similar to Sp1. Recombinant Sp1 produced two major shifted complexes with this probe (lane 5). However, these bands were sharper than those observed with crude extracts, suggesting that the latter complexes may contain other proteins, in addition to Sp1.


Figure 5: Identification of Sp1-related protein in H35 extracts. A, EMSA using the Sp1 consensus binding site oligonucleotide as probe. The Sp1 oligonucleotide was radiolabeled using polynucleotide kinase as described under ``Materials and Methods.'' Nuclear extracts from H35 cells (lanes 2-4) or purified recombinant Sp1 (lane 8) were incubated with the Sp1 probe with or without competitor oligonucleotides in the binding reaction. Lane 1, no nuclear extract; lanes 2 and 5, no competitor; lane 3, 100-fold molar excess of unlabeled Sp1 oligonucleotide; lane 4, 100-fold molar excess of AP-1 consensus oligonucleotide added as competitor. B, Western blot analysis of crude nuclear extracts with Sp1 antibody. SDS-polyacrylamide gel electrophoresis, electrophoretic transfer, and detection using anti-Sp1 polyclonal antibody were performed as described under ``Materials and Methods.'' Lane 1, nuclear extract prepared from control H35 cells; lane 2, nuclear extract from cells treated 3 h with TPA (1.6 µM); lane 3, recombinant human Sp1 protein. Molecular size markers (in kDa) are shown on the right. C, antigenicity of the DNA-protein complex. H35 nuclear extracts were preincubated with the anti-Sp1 antibody for 20 min on ice before addition of the labeled -345/-93 ODC probe. EMSA was then performed as described under ``Materials and Methods.'' Lanes 1, 7, and 11, no nuclear extract; lanes 2-6, 8-10, and 12-14, H35 extracts. Lanes 1-6, -345/-93 probe; lanes 7-10, -345/-168 probe; lanes 11-14, -168/-93 probe. Lanes 2, 8, and 12 contained extracts plus probe only; lane 3 contained consensus Sp1 oligonucleotide as competitor; lane 4 contained a mutant Sp1 binding site oligonucleotide as competitor; lanes 5, 9, and 13 contained nuclear extracts preincubated with normal rabbit serum; lanes 6, 10, and 14 contained nuclear extracts preincubated with anti-Sp1 antibody. Supershifted bands are indicated by arrows.



To confirm the presence of Sp1 protein in H35 cells, we performed Western blot analysis using an anti-Sp1 polyclonal antibody as described under ``Materials and Methods.'' As shown in Fig. 5B, the anti-Sp1 antibody detected a single band of approximately 95 kDa in H35 extracts (lanes 1 and 2), whereas recombinant human Sp1 had an apparent molecular mass of 98 kDa.

Antigenicity of the Complex

As indicated above, oligonucleotides containing the Sp1 binding site competed strongly for the EMSA complexes formed by nuclear extracts with the -345/-93 ODC probe ( Fig. 3and Fig. 5C, lane 3). In striking contrast, a mutant Sp1 oligonucleotide had no effect on complex formation with the -345/-93 ODC fragment (Fig. 5C, lane 4). These results prompted us to determine whether antibody against Sp1 would recognize proteins present in EMSA complexes formed with the -345/-93 ODC probe. Preincubation of the Sp1 antibody with nuclear extracts resulted in a marked reduction in intensity of all three bands seen in the absence of antibody and a new supershifted band (Fig. 5C, lane 6). In contrast, incubation of nuclear extracts with normal rabbit serum had essentially no effect on the band pattern (Fig. 5C, lane 5). Similarly, preincubation of extracts with an antibody directed against c-Jun did not significantly alter the gel shift complexes.^3 In order to determine which of the potential Sp1 binding sites (GC boxes 1-5) interact with Sp1 in H35 extracts, we performed supershift experiments using -345/-168 (containing GC boxes 1-4; lanes 7-10) or -168/-93 (containing GC box 5; lanes 11-14) ODC probes. Using the -345/-168 ODC probe, two major and one minor specific complexes were observed by EMSA (lane 8), whereas with the -168/-93 ODC probe, a single broad complex was routinely obtained. In each case, these bands represent sequence-specific DNA-protein complexes, as judged by complete self-competition and lack of competition with heterologous fragments at 100-fold molar excess.^3 With each probe, normal rabbit serum failed to significantly alter the complexes obtained (lanes 9 and 13; the apparent reduction in the slowest band in lane 9 was not consistent). In contrast, antibody against Sp1 specifically supershifted at least one of the complexes formed on the -345/-168 probe (lane 10), and a significant fraction of the complexes formed on the -168/-93 probe (lane 14). Taken together with the DNase I footprinting results, these findings suggest that the ODC -345/-93 probe binds H35 protein(s) which are immunologically related to Sp1, and that Sp1 interacts with GC box 5 and with one or more of GC boxes 1-4.

trans-Activation of the ODC Promoter by Sp1

The studies above demonstrate that recombinant Sp1 interacts with the ODC promoter in vitro and that complexes between H35 nuclear proteins and the -345/-93 ODC probe, containing five potential Sp1 binding sites, interact with a protein(s) immunologically related to Sp1. To determine if this interaction between Sp1 and the ODC promoter results in functional trans-activation, we performed cotransfection experiments in SL2 cells, which lack significant endogenous Sp1 activity(38) . The results demonstrated a striking dependence of ODC promoter activity on Sp1 (Fig. 6). pODClux1m, containing 5`-flanking sequences of the ODC promoter to -1156 bp, and pODClux2m, which is truncated at -409, were each dramatically activated by cotransfection of pPacSp1 (76- and 356-fold, respectively), relative to the luciferase activity observed with cotransfection of the parent pPac vector or in the absence of expression vector. In contrast, pODClux2DeltaEm, in which sequences 5` from -93 were removed, or pODClux1DeltaEm, in which the five putative Sp1 binding sites between -345 to -93 were deleted, were only weakly trans-activated by pPacSp1 (<10-fold). Furthermore, ODC181TK, containing ODC sequences -345/-168 (four GC boxes: 1-4) fused to a minimal TK promoter was also strongly trans-activated by Sp1 (158-fold). Interestingly, pODClux168, containing the single GC box 5 was only very slightly trans-activated by Sp1 in SL2 cells (3.5-fold increase over pPac control).


Figure 6: trans-Activation of the ODC promoter by Sp1. Schneider SL2 cells were transfected with ODC promoter/luciferase reporter plasmids (5 µg) in the presence of either pPacSp1 expression vector (0.5 µg), or the parental pPac vector (0.5 µg), as indicated in the figure. Cells were harvested 48 h after transfection and assayed for luciferase activity as described under ``Materials and Methods.'' Solid bars, ODC reporter plasmid + parental pPac; diagonally hatched bars, reporter + pPacSp1. Results are the mean ± S.E. of four transfections and are expressed as the luciferase activity normalized to that obtained with pODClux1m in the presence of pPac.




DISCUSSION

The transient expression analysis of ODC promoter activity presented here generally agrees with previous work by van Steeg et al.(23) and Moshier et al.(28) , who showed that 5`-flanking sequences within 398 bp of the transcription start site are important for basal activity of the rat and human ODC genes, respectively. However, we also observed significant contributions toward ODC promoter activity from sequences upstream from -410 (see Fig. 1A). This difference may be cell type-dependent, since we did not observe it in Rat-2 fibroblasts,^2 which are similar to the Rat-1 cells used by van Steeg et al.(23) . The GC-rich ODC sequences from -345/-93 contributed approximately 25-35% of the activity of the intact promoter (Fig. 1, A and B). Although this region contains potential binding sites for numerous transcription factors, our results indicate that the five GC boxes in this region may be key determinants of ODC promoter activity.

These results strongly suggest that Sp1 or an immunologically related protein binds to and activates the ODC promoter for the following reasons: (i) complexes between H35 nuclear proteins and the ODC sequence were abolished by competition with an Sp1 consensus binding site oligonucleotide but not by a mutant Sp1 oligonucleotide (Fig. 3B and Fig. 5C); (ii) antibody against Sp1 specifically recognized protein(s) associated with sequences containing GC boxes 1-4, and with GC box 5, whereas normal rabbit serum did not (Fig. 5C); (iii) purified Sp1 protein bound to the -345/-93 ODC probe and partially protected sequences that were also protected against DNase I digestion by H35 extracts (Fig. 4); (iv) ODC promoter constructs containing GC boxes 1-4 were strongly trans-activated by cotransfected Sp1 in SL2 cells; deletion of these sites dramatically decreased the effect of Sp1 (Fig. 6). Previous reports presented circumstantial evidence based on oligonucleotide competition or footprinting that Sp1 might bind to the ODC promoter(28, 29, 30) . However, none of these investigators analyzed the nature of the proteins bound to these sequences or their functional role in ODC transcription.

Recombinant Sp1 protein formed two major complexes with the -345/-93 ODC probe (Fig. 5A, lane 5), whereas nuclear extracts formed at least three distinct complexes. All three complexes observed with nuclear proteins were eliminated by competition with an Sp1 oligonucleotide, but not by a mutant Sp1 oligonucleotide (see Fig. 5C). The additional bands seen with extract could be due to binding of modified forms of Sp1 or to the presence of additional proteins in the complexes. The latter possibility is likely, since extracts contain numerous transcription factors and co-activators. DNase I footprinting revealed that H35 nuclear extracts protect two broad regions between -92 to -130 (PR-I) and -304 to -332 (PR-II). PR-I contains GC box 5, which is conserved between mouse, rat, and human, and therefore likely to be important in ODC regulation(20, 22, 23) . Purified Sp1 also protects from -101 to -156, suggesting that Sp1 in nuclear extracts is at least partially responsible for PR-I. PR-II was not protected by recombinant Sp1 and is not predicted to contain strong Sp1 binding sites. Identification of the other components requires additional studies. Recombinant Sp1 protected adjacent sequences (-202 to -293) containing GC boxes 1-3. Although crude nuclear extracts did not clearly protect this region, partial protection was observed. Use of the purified Sp1 may lead to a higher site occupancy than is feasible with the concentrations of extract protein used. The observation that the Sp1 consensus oligonucleotide abolishes essentially all of the complexes observed with the -345/-93 fragment, not just those seen with recombinant Sp1, suggests that Sp1 may be required for formation of a multiprotein complex on the ODC promoter. This model is also consistent with the dramatic affect of cotransfected Sp1 on ODC promoter activity in SL2 cells (Fig. 6). Although these results indicate that all five GC boxes may bind Sp1 in extracts, results of the SL2 cotransfections (Fig. 6) suggest that GC boxes 1-4 may be functionally more important. This is also consistent with H35 transfection results, since pODC181TK (containing GC boxes 1-4) was far more active than pODC71TK, containing only GC box 5 (Fig. 1B).

Sp1 interacts with many cellular and viral promoters(47) . Although originally associated with constitutive transcription, Sp1 also regulates several inducible genes, including transforming growth factor-beta1 and -beta3 (48) and the human multidrug resistance gene (49) . Sp1 can also cooperate with other transcription factors, such as NFkappaB(50) . Extracellular signals may modulate trans-activation through Sp1 sites in at least three ways: (i) by influencing the expression of Sp1 protein(51, 52) , (ii) by altering the DNA binding affinity of Sp1(53) , or (iii) by altering the trans-activation potential of Sp1(54) . Other factors that may also recognize the Sp1 consensus sequence include Sp2, Sp3, and Sp4, which share homology with Sp1(55, 56) , and basic transcription element-binding protein, which is not closely related to Sp1(57) . Western blot analysis of H35 extracts using anti-Sp1 detected a single band with approximate molecular mass of 95 kDa, approximately that obtained for pure Sp1 protein (Fig. 5B, (51) and (58) ). The predicted molecular mass for rat Sp1 is 81 kDa(57) ; however, both the mobility and activity of Sp1 are influenced by phosphorylation and glycosylation(51, 59) . Sp2 and Sp3 have molecular masses of 80 and 100 kDa, respectively(56) . Although our results strongly suggest that Sp1 directly activates the ODC promoter, it remains possible that a closely related factor may be responsible.

Although regulation of ODC occurs at multiple levels, it has been convincingly demonstrated that transcriptional regulation makes an important contribution to changes in ODC levels in response to mitogens (15, 16, 17) or cyclic nucleotides(34) . In this study, we have shown that rat ODC sequences from -345 to -93 are critical for high levels of expression, but that sequences from -92/+13 retain about 20-25% of the activity of the intact 5`-flanking region. While this manuscript was in preparation, Verma and co-workers (60, 61) reported that human ODC sequences between -42 and +72 are trans-activated by cotransfected protein kinase Calpha catalytic domain or by treatment with TPA. Since these sequences of mammalian ODC genes lack canonical AP-1, AP-2, or serum-responsive elements, the details of this activation remain uncertain. In this regard, we have found that rat ODC sequences -92/+13 also mediate increased promoter activity in response to TPA, serum, or proto-oncogenes. It will be of interest to determine if the GC-rich region studied in this report cooperates in mitogenic stimulation of the ODC promoter. Although the EMSA complexes formed with the -345/-93 probe were present irrespective of TPA treatment of the cells, TPA may affect post-translational modification of one or more factors, increasing interactions with other components of the transcriptional machinery, without affecting the DNA binding activity.

ODC is also a marker for progression of quiescent cells through G(1) and into S phase of the cell cycle. It will therefore be important to establish which elements of the ODC promoter may play a role in cell cycle-dependent expression. We have noted low affinity binding sites for E2F at positions -126, -160, -229, and -275 of the ODC gene, and the PR-I footprint contains a sequence similar to the E2F consensus binding site(62) , suggesting possible involvement of an E2F-like protein in this complex. E2F regulates growth-associated genes such as c-myc, dihydrofolate reductase, DNA polymerase alpha, and cdc2, which are activated at the G(1)/S boundary(63) . In addition, the RB control element of several RB-regulated genes binds Sp1 and Sp1 mediates RB regulation through these elements(54) . In that some of the ODC Sp1 sites overlap putative E2F sites, it is tempting to speculate that cooperative or competitive interactions between Sp1 and E2F might be involved in the regulation of ODC.

In summary, the results presented here indicate that the ODC promoter interacts with Sp1 or an immunologically related protein. Sp1 appears to be essential for formation of all three DNA-protein complexes observed in EMSA with the -345/-93 probe, although footprinting indicates binding of additional transcription factors as well, suggesting interactions between Sp1 and other factors. Finally, we have demonstrated that Sp1 dramatically trans-activates the ODC promoter in vivo. Experiments to test the functional significance of these Sp1 binding sites in the growth-regulated transcription of ODC are in progress. Although the precise role of Sp1 toward ODC regulation in response to tumor promoters or during cell cycle progression remains to be elucidated, we propose that Sp1 binds specifically to the Sp1 consensus binding sites of the 5`-flanking region and affects transcription of ODC through protein-protein interactions with additional transcription factors.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant CA-46629 from the National Cancer Institute (to A. P. B.) and by American Cancer Society Grant SIG-19. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 512-237-9461; Fax: 512-237-2437; butler{at}odin.mda.uth.tmc.edu.

(^1)
The abbreviations used are: ODC, ornithine decarboxylase; CRE, cAMP response element; CREB, CRE-binding protein; EMSA, electrophoretic mobility shift assay; IRE, insulin response element; RB, retinoblastoma gene product; TPA, 12-O-tetradecanoylphorbol-13-acetate; TK, thymidine kinase.

(^2)
P. K. Mar and A. P. Butler, manuscript in preparation.

(^3)
A. P. Kumar and A. P. Butler, unpublished observations.

(^4)
A. P. Butler, D.-C. Kang, and B. Zhao, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Harry van Steeg for the gift of the ODC genomic clone and Dr. Robert Tjian, University of California, Berkeley, CA for the gift of the Sp1 expression vector. We would also like to thank our colleagues at Science Park, particularly Dr. Michael MacLeod and Dr. Rodney Nairn, for helpful discussions and Dr. Michael LaBate for many suggestions and for his expert assistance with DNA sequence analysis. We also acknowledge the help of Judy Ing and Carol Hildman in preparing the figures and manuscript, respectively.


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