(Received for publication, April 21, 1995; and in revised form, September 14, 1995)
From the
Tissue factor (TF) gene expression is rapidly induced in epithelial cells by phorbol 12-myristate 13-acetate and serum. We have shown that this induction is mediated by a novel serum response region (SRR) (-111 to +14 bp) within the human TF promoter. In this study, we characterized cis-acting genetic elements within the SRR that regulated basal and inducible expression of the TF gene in HeLa cells. Gel mobility shift assays using oligonucleotides spanning the entire SRR identified three 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound constitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR-1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids containing the wild-type TF promoter (-111 to +14 bp) or derivatives containing mutations in the three Sp1 and/or EGR-1 sites examined basal and inducible expression. The Sp1 sites mediated basal promoter activity, and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13-acetate or serum. These data indicated that TF gene expression in HeLa cells was regulated by both Sp1 and EGR-1.
Tissue factor (TF) ()is the primary cellular
initiator of the coagulation protease cascades(1) . The TF gene
is expressed in a cell type-specific manner in vivo(2) . For example, TF mRNA is expressed in the upper
stratum granulosum layer of the epidermis but not in the
dermis(3) . In vitro studies defined the TF gene as an
immediate early gene because it is induced in quiescent fibroblasts and
epithelial cells by serum and purified growth factors in the absence of de novo protein
synthesis(4, 5, 6, 7, 8, 9) .
However, the time course of induction of TF mRNA, as well as c-myc mRNA, is delayed compared with the induction of other immediate
early genes, including c-fos and egr-1(4) .
Our previous studies show that serum and phorbol 12-myristate
13-acetate (PMA) induction of the human TF gene is mediated by a region
of the TF promoter (-111 to +14 bp relative to the start
site of transcription) called the serum response region
(SRR)(9, 10) . This SRR does not contain DNA sequences
resembling the serum response element characterized in the promoter of
the c-fos gene and other immediate early
genes(11, 12) . Deletional analysis and mutagenesis of
the SRR failed to identify a single cis-acting regulatory
element that mediated induction (9) in epithelial cells,
suggesting that several distinct regions may act in concert to regulate
inducible TF gene expression.
The SRR of the TF promoter includes a
12-bp motif that contains putative overlapping EGR-1/Sp1 binding
sites(9) . The EGR-1 and Sp1 sites contain six overlapping
nucleotides, suggesting that binding of each transcription factor is
mutually exclusive. Sp1 and EGR-1 both contain three zinc finger motifs
of the Cys-His
subclass that bind to nucleotide
triplets within their respective sites(13, 14) .
Importantly, EGR-1 does not compete Sp1 binding to a consensus Sp1
site(15, 16) . In addition, methylation interference
studies indicate that the two proteins exhibit different contact sites
within a 12-bp DNA sequence containing overlapping EGR-1/Sp1
sites(17) . Similar 12-bp motifs have been reported in the egr-1 gene itself(18) , the murine adenosine deaminase
gene(19) , the human synapsin I gene(20) , and the
homeobox containing gene hox-1.4(21) .
Sp1 is a general transcription factor that activates transcription of a subset of genes containing Sp1 sites(22, 23) . The Sp1 gene is constitutively expressed in HeLa cells(24, 25, 26) . In contrast, the egr-1 gene is rapidly and transiently induced in HeLa cells by serum and PMA(18, 27) . EGR-1 (28) (also known as Zif268, NGF1-A, krox24, and TIS8(29, 30, 31, 32) ) is a nuclear phosphoprotein that binds to a specific DNA sequence, 5`-GCGGGGGCG-3`, in a zinc-dependent manner(16, 17, 33) . EGR-1 has been shown both to activate and repress transcription in transient transfection assays(15, 17, 18, 19) .
In this study, we determined that Sp1 and EGR-1 bound to three 12-bp motifs within the SRR of the TF promoter, each of which contained overlapping EGR-1/Sp1 sites. Sp1 was constitutively expressed, whereas EGR-1 was induced in response to PMA or serum. Functional studies using the cloned wild-type TF promoter and derivatives containing mutations in the Sp1 and/or EGR-1 sites indicated that Sp1 was required for basal TF expression and that both Sp1 and EGR-1 mediated inducible TF expression.
Figure 1: The SRR of the human TF promoter. Wild-type and mutated oligonucleotides spanning a region of the TF promoter between -114 and +14 (designated R1 to R7) were used in gel mobility shift assays. The DNA sequence between -108 and -58 contains three overlapping EGR-1/Sp1 sites(45) . Specific base-pair substitutions used to mutate the EGR-1 and/or Sp1 sites of regions 2 and 3 are indicated in bold. The TATA box consensus element is shown, and the bent arrow indicates the start site of transcription.
Figure 2: Binding of transcription factors to the SRR. Oligonucleotides (Fig. 1) spanning region 1 (R1), region 2 (R2), region 3 (R3), and region 4 (R4) were radiolabeled and incubated with nuclear extracts from quiescent HeLa cells(-) and cells stimulated with PMA (+) for 1 h as indicated. Protein-DNA complexes were separated from free probe using low ionic strength 6% nondenaturing acrylamide gels (Novex). Complexes I, II, and III formed using regions 1-3 are indicated.
Figure 5: Induction of EGR-1 expression in HeLa cells. A, radiolabeled oligonucleotides spanning regions 2 (R2) and 3 (R3) were incubated with nuclear extracts from quiescent HeLa cells or nuclear extracts from cells stimulated with PMA or serum for the times shown. The Sp1 and EGR-1 complexes are indicated. Complexes were separated using 6% nondenaturing acrylamide gels. B, induction of the 82-kDa EGR-1 protein in PMA-stimulated cells was determined by Western blotting using an anti-EGR-1 polyclonal antibody (36) . Similarly, levels of Sp1 present in these same nuclear extracts were determined by Western blotting using an anti-Sp1 antibody (Santa Cruz Biotechnology). Proteins were separated on an 8-16% SDS-polyacrylamide gel, and the position of the 95- and 105-kDa doublet (40) is shown. C, induction of EGR-1 mRNA. Total RNA was isolated from quiescent serum- and PMA-stimulated (1 h) HeLa cells. RNA (4 µg) was separated by electrophoresis, and EGR-1 mRNA levels were determined by Northern blotting using a radiolabeled EGR-1 cDNA probe. The blot was reprobed to determine glucose-6-phosphate dehydrogenase (G6PDH) mRNA levels as a measure of RNA loading.
Complex I
formed using either the region 1, 2, or 3 probes exhibited the same
mobility ( Fig. 2and Fig. 3D), suggesting that
it may represent binding of the same constitutively expressed protein.
Similarly, complex II formed with the different probes migrated with
the same mobility ( Fig. 2and Fig. 3D),
consistent with binding of the same inducible protein. Region 2
contains the previously identified 12-bp motif that includes
overlapping EGR-1/Sp1 sites (Fig. 1). Inspection of the DNA
sequence of regions 1 and 3 indicated that they each contained a
similar 12-bp motif (Fig. 1). The EGR-1 site in region 2 exactly
matched the proposed EGR-1 consensus binding site and bound large
amounts of the inducible complex II compared with regions 1 and 3. The
oligonucleotides R2-1 and R2-2 (Fig. 1)
exhibited the same pattern of protein binding (data not shown) as the
region 2 (R2) oligonucleotide (Fig. 2), indicating that complex
formation did not involve DNA sequences overlapping with regions 1 and
3. Regions 1 and 3 included putative EGR-1 sites with a C at position 1
instead of a G present in the EGR-1 consensus binding site (16, 17, 33) and bound lower levels of the
inducible complex II compared with region 2.
Figure 3:
Identification of proteins that bind to
the 12-bp motifs in the SRR containing overlapping EGR-1/Sp1 sites. A, the mobility of complexes formed using region 2 (R2) or
region 3 (R3) and nuclear extracts from quiescent HeLa cells or
PMA-stimulated cells (1 h) were compared with that of complexes formed
using oligonucleotides containing either a prototypic Sp1 site or a
prototypic EGR-1 site (see ``Materials and Methods'').
Complexes I, II, and III and the Sp1 and EGR-1 complexes are indicated. B, competition of protein-DNA complexes. Complexes formed
between oligonucleotides spanning region 2 (R2) or region 3 (R3) and
nuclear extracts from quiescent(-) or PMA-stimulated (1 h)
(+) HeLa cells were analyzed by competition with a 50-fold molar
excess of unlabeled oligonucleotides containing prototypic Sp1, AP-1,
or EGR-1 sites (see ``Materials and Methods''). The Sp1
(complex I) and EGR-1 (complex II) complexes are indicated. C,
mutagenesis of the overlapping EGR-1/Sp1 sites in regions 2 and 3.
Complexes formed between region 2 (R2) and nuclear extracts from
PMA-stimulated (1 h) HeLa cells were competed with a 50-fold molar
excess of unlabeled oligonucleotides (Fig. 1) containing
wild-type region 2, WT (R2), mutated EGR-1 sites, EGR-1 (R2-2 EGR-1
), and EGR-1
(R2-2 EGR-1
), mutated Sp1 sites, Sp1
(R2-2 Sp1
), or mutated EGR-1 and Sp1 sites,
EGR-1
/Sp1
(R2-2
EGR-1
/Sp1
). The wild-type oligonucleotide
spanning region 3 (WT; R3) and derivatives containing a
mutated Sp1 site, Sp1
(R3 Sp1
), a mutated EGR-1
site, EGR-1
(R3 EGR-1
), or mutated EGR-1 and
Sp1 sites, EGR-1
/Sp1
(R3
EGR-1
/Sp1
), were radiolabeled and incubated
with a nuclear extract from PMA-stimulated (1 h) HeLa cells. The Sp1
(complex I) and EGR-1 (complex II) complexes are indicated. D,
identification of proteins present in the protein-DNA complexes using
specific antibodies. Oligonucleotides spanning regions 2 (R2) and 3
(R3) were incubated with nuclear extracts from PMA-stimulated (1 h)
HeLa cells in the absence or the presence of 5 µl of EGR-1, Sp1, or
AP-2 antibody (Ab) (Santa Cruz Biotechnology). The Sp1
(complex I) and EGR-1 (complex II) complexes are indicated. Complexes
were separated using 6% nondenaturing acrylamide
gels.
Competition studies were performed using various unlabeled oligonucleotides to examine the binding specificity of complex I (designated Sp1), complex II (designated EGR-1), and complex III formed using regions 2 and 3 (Fig. 3B). The Sp1 complex (complex I) and complex III were competed with a Sp1 site but not with an EGR-1 site or AP-1 site. Conversely, the EGR-1 complex (complex II) was competed with an EGR-1 site but not with Sp1 or AP-1 sites. These competition studies further suggested that Sp1 and EGR-1 bound to overlapping EGR-1/Sp1 sites in these regions of the TF promoter.
Next, we analyzed protein binding to oligonucleotides containing
base pair substitutions in the EGR-1 sites and/or the Sp1 sites (Fig. 3C). For region 2, these mutated oligonucleotides (Fig. 1) were used to compete the Sp1 and EGR-1 complexes formed
between the radiolabeled wild-type oligonucleotide, R2, spanning region
2 and nuclear extracts from PMA-stimulated cells (Fig. 3C, lanes 1-6). Unlabeled
wild-type oligonucleotide competed formation of both the Sp1 and EGR-1
complexes. Oligonucleotides containing mutated EGR-1 sites, R2-2
EGR-1 and R2-2 EGR-1
, competed only the
Sp1 complex; an oligonucleotide containing a mutated Sp1 site,
R2-2 Sp1
, competed only the EGR-1 complex; and an
oligonucleotide containing mutations in both sites, R2-2
EGR-1
/Sp1
, did not compete either complex. For
region 3, the wild-type oligonucleotide and mutated derivatives (Fig. 1) were radiolabeled and incubated with nuclear extracts
from PMA-stimulated cells (Fig. 3C, lanes
7-10). Complexes I (Sp1), II (EGR-1), and III were all
observed using wild-type region 3. In contrast, an oligonucleotide
containing a mutated Sp1 site, R3 Sp1
, formed only the
EGR-1 complex, an oligonucleotide containing a mutated EGR-1 site, R3
EGR-1
, formed only the Sp1 complex and the Sp1-related
complex III, and no complexes were observed using an oligonucleotide
containing mutations in both sites, R3
EGR-1
/Sp1
.
Finally, the identity of the proteins present in these complexes was determined using antibodies that specifically recognize the transcription factors EGR-1, Sp1, or AP-2 (Fig. 3D). Indeed, other members of the EGR family can bind to an EGR-1 site(33, 39) , but these transcription factors are not recognized by the EGR-1 antiserum used. The EGR-1 complex (complex II) observed using PMA-stimulated cells, and regions 2 and 3 were abolished by preincubation with an EGR-1-specific antibody but not by Sp1- or AP-2-specific antibodies (Fig. 3D). Similar results were observed using region 1 (data not shown). Conversely, the Sp1 complex (complex I) formed using region 3 was abolished by preincubation with an Sp1-specific antibody but not by EGR-1- or AP-2-specific antibodies (Fig. 3D, lanes 5-8). Therefore, we concluded that complexes I and II formed using oligonucleotides spanning regions 1, 2, and 3 represented binding of Sp1 and EGR-1, respectively. Sp1 was constitutively expressed in HeLa cells, and binding was not increased upon stimulation of the cells. In contrast, EGR-1 binding activity was not detected in nuclear extracts from quiescent cells but was rapidly induced by stimulation with serum and PMA.
Figure 4: Binding of recombinant Sp1 and EGR-1 proteins to overlapping EGR-1/Sp1 sites in regions 1 and 2 of the SRR. Radiolabeled oligonucleotides (1.25 ng) spanning region 1 (R1) (A) or region 2 (R2) (B) were preincubated with 10 ng of recombinant Sp1 (Promega Corp.) for 20 min before the addition of bacterially expressed EGR-1 as indicated. Reactions were incubated for a further 20 min, and protein-DNA complexes were resolved using low ionic strength 6% nondenaturing acrylamide gels (Novex). Proteins in lanes 5 and 6 were incubated for 20 min. Sp1 and EGR-1 complexes are indicated.
To investigate if stimulation of the
cells induced de novo synthesis of EGR-1 or activated binding
of pre-existing EGR-1 protein by post-translational modification,
levels of EGR-1 protein in nuclear extracts from quiescent and
PMA-stimulated cells were assessed by Western blotting using an EGR-1
polyclonal antibody (Fig. 5B). No EGR-1 protein could
be detected in nuclear extracts from quiescent cells. However, EGR-1
protein expression was induced following stimulation with PMA for 1 h (Fig. 5B). In contrast to the induction of EGR-1
expression, Sp1 protein was constitutively expressed by quiescent HeLa
cells (Fig. 5B). The Sp1-specific antiserum recognized
two polypeptides of 105 and 95 kDa in size, which appear to arise
from differential phosphorylation of Sp1(40) . The slower
migrating species very likely represents the highly phosphorylated form
of Sp1(40) . PMA stimulation of the cells did not increase Sp1
levels and did not change the phosphorylation of Sp1 (Fig. 5B). Similar results were seen with
serum-stimulated extracts (data not shown). To exclude the possibility
that stimulation of HeLa cells induced pre-existing EGR-1 protein that
was present in an inactive cytoplasmic form, we analyzed levels of
EGR-1 protein in cytosolic and nuclear extracts from quiescent PMA- and
serum-stimulated cells. No EGR-1 protein was detected in cytosolic
extracts, but EGR-1 was detected in nuclear extracts from PMA- and
serum-stimulated cells (data not shown), indicating that stimulation of
cells induced de novo synthesis of EGR-1. However, we cannot
exclude the possibility that quiescent HeLa cells contained low levels
of pre-existing EGR-1 that were below the detectable threshold of
Western blotting. Quantitation of EGR-1 protein levels by densitometric
analysis of both gel mobility shift assays and Western blots indicated
that EGR-1 induction by PMA was 2.8 ± 0.5-fold (n = 4) greater than the induction observed with serum.
Finally, EGR-1 mRNA expression was determined by Northern blot. No EGR-1 mRNA was detected in quiescent cells, but EGR-1 mRNA was rapidly induced by serum or PMA stimulation of the cells (Fig. 5C). PMA stimulation resulted in a larger induction of EGR-1 mRNA than serum stimulation, consistent with the relative levels of induction of EGR-1 protein. These results demonstrated that quiescent HeLa cells did not contain detectable levels of EGR-1 mRNA or protein but serum or PMA stimulation rapidly induced egr-1 gene expression with kinetics consistent with the induction of an immediate early gene. In contrast, Sp1 binding activity and protein were constitutively expressed and were not increased by serum or PMA stimulation.
Figure 6:
Functional analysis of the human TF
promoter in HeLa cells. A, plasmids used in this study are
shown (see ``Materials and Methods''). The EGR-1 sites are
indicated by the hatched boxes, and the Sp1 sites are
indicated by the filled boxes. B, plasmids (10
µg) containing the wild-type TF promoter (-111 to +14
bp) and mutated derivatives were transfected into HeLa cells. After 53
h of serum-starvation in 0.5% serum, luciferase activity was determined
as a measure of basal TF promoter activity. C, transfected
cells were serum-starved in 0.5% serum for 48 h before adding PMA (50
ng/ml) for 5 h. Luciferase activity expressed by each plasmid in the
presence and the absence of PMA stimulation was used to calculate the
fold induction. D, similarly, transfected cells were
serum-starved in 0.5% serum for 48 h before the addition of 20% serum
supplemented with epidermal growth factor (10 ng/ml), insulin-like
growth factor-1 (10 ng/ml), and basic fibroblast growth factor (10
ng/ml) (Collaborative Biomedical Products, Bedford, MA) for 5 h. The
reduced level of serum induction using pTF(Sp1)Luc and
pTF(EGR-1
)Luc were not statistically significant, whereas
the reduction observed with pTF(EGR-1
/Sp1
)Luc
was statistically significant (p < 0.05). E, TF
promoter plasmids (8 µg) were cotransfected into HeLa cells with
pSVKrox-24 (8 µg), which expresses EGR-1(17) , or with
pUC19 (8 µg) as a control. Cells were serum-starved for 53 h before
assaying luciferase activity in the presence and the absence of EGR-1
protein to calculate the fold induction. In each case (C and D) data are shown minus the background of the vector control
(pGL2-Basic, Promega Corp.). In each experiment, values were determined
from triplicate wells of a 6-well plate and corrected for transfection
efficiency (see ``Materials and Methods''). Results from at
least four independent experiments are shown (means ±
S.E.).
Further studies examined if expression of EGR-1
alone could transactivate the TF promoter in the presence of endogenous
Sp1 (Fig. 6E). Plasmids containing the wild-type TF
promoter and mutated derivatives were cotransfected with a plasmid,
pSVKrox-24, that expresses EGR-1(17) . The wild-type promoter
in pTF(-111 to +14)Luc was transactivated by EGR-1. The
level of transactivation was increased by mutation of the Sp1 sites in
a similar manner to the results observed using PMA. However, mutation
of the EGR-1 sites in pTF(EGR-1)Luc and in
pTF(EGR-1
/Sp1
)Luc abolished transactivation
mediated by EGR-1. Therefore, EGR-1 acted as a transcriptional
activator of the TF gene in HeLa cells.
This study demonstrated that three 12-bp motifs within the SRR, each of which contained overlapping EGR-1/Sp1 sites, mediated both PMA and serum induction of the TF gene. Sp1 was constitutively expressed in unstimulated cells, whereas de novo synthesis of EGR-1 was rapidly induced by PMA and serum stimulation. Functional analysis of plasmids containing mutated 12-bp motifs suggested the presence of two transcriptional components requiring either intact Sp1 sites or intact EGR-1 sites, which both contributed to maximal induction of the TF promoter in HeLa cells.
Previous studies defined the TF gene as an immediate early gene(4, 5, 6, 7, 9) . The data presented here suggested that transcriptional activation of the TF gene in HeLa cells by PMA or serum was in fact composed of both an immediate early component and a delayed early component. We propose that the immediate early component of TF gene induction requires Sp1 sites. Specific mutation of the EGR-1 sites reduced but did not abolish induction by serum or PMA, suggesting that this residual activity represented the immediate early component. Further mutation of the Sp1 sites completely abolished induction. Sp1 was constitutively expressed by HeLa cells, consistent with a proposed role in immediate early induction. However, induction of the TF promoter may be directly or indirectly mediated by Sp1. The transcriptional activity of Sp1 may be increased by some form of post-translational modification, such as phosphorylation, although our Western blots did not detect a change in the ratio of the 95- and 105-kDa forms of Sp1. Nevertheless, we cannot exclude the possibility that PMA and serum induce phosphorylation of Sp1 without detectable changes in electrophoretic mobility. Alternatively, PMA and serum may activate pre-existing coactivators that interact with both Sp1 bound to the SRR and the basal transcriptional machinery to mediate transcriptional activation(41) . Another possible explanation for these results is that another as yet unidentified transcription factor is required for the immediate early induction of the TF gene. This protein may bind to the SRR at a site(s) distinct from the 12-bp motifs and possibly interact with Sp1 to mediate activation of the TF gene.
The delayed early component of TF gene induction required EGR-1 sites. Mutation of the EGR-1 sites reduced PMA and serum induction of the TF promoter. Moreover, the wild-type TF promoter, but not plasmids containing mutations in the EGR-1 sites, was transactivated by over-expression of EGR-1 in quiescent HeLa cells. Induction of TF mRNA is maximal 2 h after PMA or serum stimulation (9) and is delayed compared with c-fos, egr-1, and other immediate early genes. This delayed induction is consistent with a role for newly synthesized EGR-1 in the regulation of the TF promoter. Previously, we demonstrated that TF mRNA was induced in HeLa cells in the presence of the protein synthesis inhibitor, cycloheximide(9) . However, cycloheximide can both induce TF gene transcription and stabilize TF mRNA(42) , which could compensate for the loss of the delayed early component due to inhibition of de novo synthesis of EGR-1. Recently, it was demonstrated that transcriptional activation of nur77, which was also originally identified as a serum-inducible immediate early gene, is mediated by both an immediate early component and an EGR-1-dependent delayed early component(43) . In addition, EGR-1 may be involved in the regulation of the c-myc gene, which contains an EGR-1 site (17) and also exhibits delayed induction(4) . Thus, the rapid expression of EGR-1 in serum and PMA stimulated cells may contribute to the transcriptional activation of a several genes previously classified as immediate early genes.
Transcriptional
activation of the TF gene required the Sp1 and EGR-1 sites because
mutation of all six sites within the SRR abolished both PMA and serum
induction. However, several differences were noted between PMA and
serum. For instance, serum was a less potent inducer of the wild-type
TF promoter in this vector system than PMA, which may be due, in part,
to the relative levels of EGR-1 induced by these agonists. In addition,
PMA stimulation phosphorylates EGR-1 more efficiently than serum
stimulation, ()and highly phosphorylated forms of EGR-1 bind
to DNA with increased affinity (36) and may have increased
transcriptional activity. Mutation of the Sp1 sites reduced serum
induction but increased PMA induction. This would abolish Sp1
competition for the 12-bp motifs, again suggesting that EGR-1 induced
by PMA may be a more potent transcriptional activator than EGR-1
induced by serum. These differences may reflect activation of different
signaling pathways. Previously, we showed that the signaling mechanisms
for serum and PMA stimulation are distinct(9) . TF mRNA
induction by both agonists required intracellular calcium mobilization,
whereas inhibition of protein kinase C abolished induction of the TF
gene by PMA but had no effect on induction by serum(9) .
Nevertheless, our data showed that the same three 12-bp motifs within
the SRR mediated PMA and serum induction of the TF promoter.
Comparison of the DNA sequences of the human, murine, and porcine TF promoters is shown in Fig. 7. The Sp1 and EGR-1 sites in regions 1, 2, and 3 are indicated. The Sp1 site in the 12-bp motif in region 1 is completely conserved, whereas the 5` triplet of the EGR-1 site is not conserved. The consensus EGR-1 sites in the 12-bp motifs in region 2 from the human and porcine TF promoters contain a single nucleotide substitution in the murine TF promoter. The porcine TF promoter did not contain the 12-bp motif in region 3, which is consistent with our previous results indicating that two of the three 12-bp motifs within the human promoter are sufficient for PMA and serum induction(9) . The spacing of the two 12-bp motifs in regions 1 and 2 is precisely conserved in all three TF promoters, suggesting that protein-protein interactions may be important in the assembly of transcription factors on this region.
Figure 7: Conservation of overlapping EGR-1 and Sp1 sites in the human(45) , mouse(46) , and porcine (47) TF promoters. Numbering is from the human TF sequence(45) . Sp1 sites are shown as filled boxes, and EGR-1 sites are shown as open boxes. Bases conserved in all three species are boxed.
Our data suggest a model for PMA and serum induction of the TF gene in which Sp1 is constitutively expressed by uninduced cells and binds to all three sites within the promoter to regulate basal expression of the TF gene. We speculate that in PMA- or serum-stimulated cells Sp1 is either post-translationally modified or interacts with pre-existing, activated coactivators to mediate immediate early induction of the TF gene in the absence of de novo protein synthesis. Alternatively, other members of the Sp1 multigene family may be involved in TF gene regulation(25, 26) . For instance, Sp3 binds to a GC box and is present at low levels in HeLa cells(44) . However, Sp3 represses Sp1-mediated activation via competition for Sp1 binding sites (44) . In stimulated HeLa cells, the ability of Sp1 and Sp3 to bind DNA may be changed in favor of Sp1, permitting immediate early activation of the TF gene. In addition, stimulation of the cells rapidly induces expression of EGR-1, which binds to the three EGR-1 sites in the SRR to mediate a delayed early response and achieve maximal transcriptional activation of the TF gene. The ability of EGR-1 to compete Sp1 binding to the 12-bp motifs within the SRR may be determined by both the levels of EGR-1 protein and their degree of phosphorylation. Thus, these overlapping EGR-1/Sp1 sites within the three 12-bp motifs may function to control the level and pattern of TF expression in different cell types exposed to a variety of agonists.