(Received for publication, June 2, 1994; and in revised form, September 21, 1994)
From the
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.
Ornithine decarboxylase (ODC; EC 4.1.1.17) ()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
/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.
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
10
/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 pRSV
gal. 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. -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/
-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
-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.
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 TBE (1
TBE = 90 mM Tris, 90 mM H
BO
, 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.
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 -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.
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.
Figure 3:
Competition with synthetic double-stranded
oligonucleotides. A, oligonucleotides containing consensus
binding sites for transcription factors AP-1, AP-2, CREB, NFB, 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.
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.
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.
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.
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, 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-1 and -
3 (48) and the human multidrug resistance gene (49) . Sp1
can also cooperate with other transcription factors, such as
NF
B(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 C 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 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
, and cdc2, which are activated at the G
/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.