(Received for publication, April 25, 1994; and in revised form, December 21, 1994)
From the
Growth factors coordinately regulate a variety of different
genes to stimulate cellular proliferation. In the stomach, gastrin,
epidermal growth factor (EGF), and transforming growth factor- all
mediate gastric mucosal homeostasis by promoting cell renewal. We have
previously shown that EGF and phorbol esters stimulate the human
gastrin promoter through a novel GC-rich DNA element
5`-
GGGGCGGGGTGGGGGG
called gERE
(gastrin EGF response element). In this report, we show that three
factors bind to this element, the transcription factor Sp1 and two fast
migrating complexes designated gastrin EGF response proteins (gERP 1
and 2). To understand how these factors bind and confer EGF
responsiveness, mutations of gERE were tested in vitro for
protein binding and in vivo for promoter activation. Both gel
shift assays and UV cross-linking studies revealed that the factors
bind to overlapping domains, Sp1 to the 5` half-site and gERP 1 and 2
to the 3` half-site. Placing either the 5` or 3` mutations upstream of
a minimal gastrin promoter abolished EGF induction. Therefore both the
5` and 3` domains were required to confer EGF induction. Collectively,
these results demonstrate that complex interactions between Sp1 and
other factors binding to overlapping gERE half-sites confer EGF
responsiveness to the gastrin promoter.
Activation of the epidermal growth factor (EGF) ()receptor by either EGF or transforming growth factor-
initiates a cascade of intracellular events(1, 2) .
These events include the transcriptional activation of various genes
encoding early response factors, receptors, structural proteins, and
hormones. However, despite the number of genes regulated by EGF
receptor activation, few EGF response elements and their corresponding
DNA-binding proteins have been identified. Indeed, the best
characterized EGF responsive elements are the serum response element
found in the c-fos, prolactin, and tyrosine hydroxylase
promoters (3, 4, 5, 6) and the
sis-inducible element residing further upstream of the serum response
element in the c-fos promoter(7) . Moreover, EGF
response elements have been identified in the Moloney murine leukemia
virus long terminal repeat, pS2, and transin
promoters(5, 8, 9) . However, regulatory
proteins capable of binding these elements have not been fully
characterized.
The EGF response element in the human gastrin promoter does not resemble these elements(10) . Instead, the gastrin EGF response element (gERE) is a GC-rich DNA element 5`-GGGGCGGGGTGGGGGG that binds nuclear proteins in both footprinting and gel shift assays(11) . The two overlapping gERE half-sites (GGGGCGGGG and GGGGTGGGGGG) represent binding sites for the transcription factor Sp1(12) ; however, neither element alone is able to confer EGF induction(11) . Sp1 consensus elements bind a 100-kDa zinc finger protein and confer basal activity to the promoters of viral or cellular genes(13, 14) . However, recent studies have shown that Sp1 binding sites also contribute to the inducible expression of growth factors and growth-regulated genes (15, 16, 17, 18) . In the stomach, an increase in Sp1 gene expression occurs in parietal cells during development(19) , which implies that Sp1 gene expression may correlate with gastric epithelial cell growth, maturation, and possibly acid secretion. Furthermore, Sp1 is overexpressed in human gastric carcinomas compared to expression in adjacent histologically normal mucosa(20) . Thus, these findings implicate a potential role for Sp1 in both the developing and neoplastic stomach.
Gastrin is synthesized in the antral G cell of the stomach and is an important growth factor for both the gastrointestinal epithelium and pancreas(21) . Moreover, it is conceivable that Sp1 binding to the gastrin promoter may represent one example of how Sp1 regulates specific gastric genes. Therefore to gain further insight into the molecular mechanisms by which nuclear proteins bind to gERE, we determined whether Sp1 binds this element and second whether Sp1 binding is required for EGF activation of transcription from the gastrin promoter. We show that Sp1 does indeed bind to the 5` domain of gERE and that two other gastrin EGF response proteins (gERP 1 and 2) bind to the 3` domain. Further, we show that the occupation of both overlapping domains, is necessary for EGF induction.
Figure 1:
Transcription factor Sp1 binds to gERE
in the presence of Zn. Sp1 polyclonal antisera was
added to the gel shift assay in the absence (lanes 1-5)
or presence (lanes 8-15) of Zn
. A
P-labeled gERE probe (30,000 cpm/0.1 ng) was used in lanes 1-11. A
P-labeled human
metallothionein IIa Sp1 probe (30,000 cpm/0.1 ng) (13) was used
in gel shift assays containing affinity-purified Sp1 protein (Promega) (lanes 12-15). Lanes 2-5 and 7-11 contained nuclear protein, lanes 12-15 contained Sp1 protein. Lanes 1 and 6 contained
probe alone. Sp1 antisera 2892 (I) was added to the gel shift
assays shown in lanes 3, 9 and 13; preimmune
sera (PI) was added to assays shown in lanes 4, 10 and 14 and heterologous immune sera (HI)
was added to assays shown in lanes 5, 11, and 15. gERP 1 and 2 and Sp1 complexes are indicated with an arrow. The supershifted complexes are indicated
(*).
The slow
migrating complex was supershifted by polyclonal Sp1 antisera
confirming the presence of Sp1 in this band (Fig. 1, lane
9, *). Preimmune (PI) and heterologous immune sera (HI) did not supershift this complex. Affinity-purified Sp1 (lanes 12-15) comigrated with the slow migrating complex
and was also supershifted with Sp1 antisera (lane 13, *). In
the absence of excess Zn, gERP 1 was the predominant
protein bound to gERE (Fig. 1, lanes 2-5). gERP 1 was not supershifted by Sp1 antisera (lane 3)
nor did its binding depend upon Zn
. In addition, a
third complex (gERP 2) that migrated near the probe front
appeared after the addition of exogenous Zn
, but also
was not supershifted by Sp1 antisera. Taken together, these studies
demonstrated that three distinct complexes bind gERE, a slow migrating
complex formed by Sp1 and two fast migrating complexes designated gERP
1 and 2. Two of the factors required Zn
for binding
which suggested that they might differ in their affinity for gERE.
Figure 2:
Quantitative gel shift assays. Gel shift
assays were performed in the presence of Zn using 5
µg of GH
nuclear protein and
250 pM of
radiolabeled gERE probe. Self-competition was performed by adding
increasing concentrations of unlabeled oligonucleotide (1, 2.5, 5, 10,
25, 50, 100, 250, and 500 times the molar concentration of
probe).
Figure 3: Schematic representation of the minimal gastrin promoter construct. The minimal human gastrin promoter construct was created by inserting a 43-bp oligonucleotide cassette with flanking 5` BglII and 3` BamHI restriction sites into the BglII site of a promoterless luciferase vector. Other constructs were created by inserting oligonucleotide cassettes upstream of the gastrin promoter element into a regenerated BglII site or a BamHI site located downstream of luciferase coding elements. Restriction analysis was used to screen for oligonucleotide orientation. wWT and wSp1 represent the WT and Sp1 cassettes inserted in the 3` to 5` direction. Arrows designate the multiple transcriptional start sites of the gastrin promoter previously determined.
Figure 4:
Competition for protein binding to gERE in
gel shift assays. Single and multiple nucleotide changes within the
gERE sequence (shown in Fig. 3) were used to compete for protein
binding to labeled gERE in the absence (upper panel) or
presence (lower panel) of Zn. Gel shift
assays were performed with GH
nuclear proteins as described
in Fig. 1. An excess of oligonucleotide competitor (400 times
the molar concentration of the probe) was added to the assay mixture 10
min prior to the addition of radiolabeled gERE. Sp1 and gERP 1 and 2
complexes are indicated.
Figure 5:
Gel shift assay using the Split WT
element. The WT (gERE) element or 30-bp Split WT element
(GGGGCGGGGTTTTTTGGGTGGGGGG) was Klenow end-labeled and incubated with
GH nuclear protein in the presence of Zn
. Lanes 1-3 contain radiolabeled WT gERE. Lane 2 contains 200 times molar excess of unlabeled WT gERE; lane 3 contains 200 times the molar excess of unlabeled Split WT element. Lane 4 contains the radiolabeled Split WT element without
competitor.
UV cross-linking confirmed that Sp1 binds to the 5` domain of
gERE; whereas gERP 1 and 2 bind to the 3` domain (Fig. 6). This
experiment was performed by substituting bromodeoxyuridine for thymine
in the gERE and M1 sense strands. Nuclear extracts and
affinity-purified Sp1 were then incubated with these radiolabeled
probes. The DNA-protein mixture was exposed to UV light prior to
resolving the mixture on an SDS-polyacrylamide gel. The M5 and Sp1
elements were used as competitors to demonstrate the specificity of
binding. Three major complexes cross-linked to the M1 probe
corresponding to Sp1 at 100 kDa, gERP 1 at
45 kDa, and gERP 2
at
25 kDa. However, since thymines are not present in the 5`
domain of the WT element, neither affinity-purified Sp1 nor Sp1
contained in the extracts cross-linked to this probe. Furthermore, a
higher molecular mass complex indicative of an Sp1/gERP complex was not
detected.
Figure 6:
UV cross-linking of Sp1 and gERP 1 and 2.
Bromodeoxyuridine was substituted for thymines in the sense strand of
the M1 and gERE sequences. After hybridizing to the complementary
strand, these double-stranded oligonucleotide cassettes were Klenow
end-labeled. GH nuclear protein (lanes 1 and 3-5) or affinity-purified Sp1 protein (lanes 2 and 6) was incubated in Zn
-containing
buffer with probe alone or 100
the molar excess of unlabeled
oligonucleotide before exposing to UV light (254 nm) for 5 min.
DNA-protein complexes were resolved by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography. Lanes 1-4 contain the M1 probe; lanes 5 and 6 contain WT
probe. Nucleotide differences between the mutations and the WT sequence
are indicated. The Sp1 consensus sequence is underlined.
Figure 7: Promoter activity of gERE constructs. A, the basal activity of each construct was expressed as a percent of normalized luciferase activity determined after transfection of the human metallothionein IIa Sp1-containing construct. B, the relative induction of each construct by 10 nM EGF is shown. The constructs are labeled according to the oligonucleotide sequence inserted upstream of ALuc (see Fig. 3). The wWT-ALuc and wSp1-ALuc represent the WT gERE and Sp1 elements in the 3` to 5` orientation (see Fig. 3). The mean ± S.E. of five experiments performed in duplicate is shown. p values <0.05 were considered significant (*).
Only the elements that competed for all three factors conferred EGF induction (see Fig. 7B, WT-, M1-, M2-ALuc). The mutated 3` domain that competed for Sp1 (M6-ALuc) conferred high promoter activity with or without EGF. In contrast, the mutated 5` domain conferred low promoter activity with or without EGF (M5-ALuc). However, the Split WT element was an exception. Although it competed for all three factors, it did not confer EGF induction. Thus some overlap between the two domains was required to create a functional EGF response element. This observation raised the possibility that the orientation of these half-sites might affect promoter inducibility (Fig. 7B, Split WT-ALuc).
The present study shows that a DNA element in the human gastrin promoter is composed of two overlapping domains that cooperate to confer EGF responsiveness; a 5` domain that binds Sp1 and a 3` domain that binds gERP 1 and 2. Moreover, these factors bind gERE with similar relative affinities. However, further studies will be required to establish the precise molecular mechanisms by which the factors become activated and confer induction.
One attractive possibility is
that Sp1 and the gERP proteins switch the promoter from a basal to an
activated state by alternate binding or alternate activation of the
constitutively bound proteins. Support for this hypothesis was
strengthened by showing that constructs containing mutations of the 5`
domain (M5) resulted in low promoter activity (20%), whereas
mutations of the 3` domain resulted in high promoter activity (100%).
In either instance, these constructs did not confer EGF induction.
Thus, promoter activity in the setting of exclusive occupation of
either the 5` or 3` domains differed by a factor of five. This
corresponded to the same incremental change conferred by the WT
element. However, EGF did not stimulate an increase in factor binding, (
)implying that alternate activation of constitutively bound
factors may indeed regulate promoter induction. This might occur
through protein-protein interactions or covalent modifications, e.g. phosphorylation. Furthermore, intranuclear divalent
cation concentrations might also regulate DNA-protein interactions
since two of the factors (Sp1 and gERP 2) required specific
concentrations of Zn
to bind. It was difficult to
determine whether factor binding reflected cooperative interactions
because the appearance of an additional slow migrating complex was not
observed in gel shift assays or by UV cross-linking. Nevertheless, such
complexes may not be either stable or abundant enough to be detected.
Growth factor induction without significant changes in factor binding
also has been observed for the serum response factor binding to the
serum response element in the c-fos promoter(28, 29, 30) .
Many promoters contain GC-rich elements that are capable of binding Sp1 in addition to other DNA binding proteins. Therefore, how the cell coordinately regulates genes through DNA elements capable of binding multiple proteins requires further study. Given the abundance of cellular Sp1, to coordinately regulate genes containing Sp1 binding sites, the access of Sp1 protein to these promoters must be regulated to ensure some specificity. Possible mechanisms include regulating the level of Sp1 activation by phosphorylation or glycosylation(31, 32) , regulating the affinity of Sp1 for DNA(33) , and regulating its nuclear levels (14) or its concentration relative to other transcription factors occupying adjacent or overlapping GC-rich sites(16) . Many of these adjacent or overlapping elements represent binding sites for other regulatory proteins that under appropriate conditions are able to successfully exclude Sp1 from binding(28, 34, 35) . Since Sp1 binds in the major groove, it cannot bind to tandem sites that are less than 10 bp away; therefore, other factors may bind to the adjacent repetitive GC-rich elements(14) . In the c-myc promoter, several zinc finger proteins including Sp1 and ZF87 bind tandem GC-rich elements to regulate basal c-myc expression(36, 37) . In the SV40 promoter, directly repeated GC-rich sites are recognized by both Sp1 and LSF(38) . Although abundant in most cell lines, Sp1 expression varies substantially in different tissues during development(19) . Consequently when Sp1 levels are low, factors other than Sp1 may occupy Sp1 consensus elements or successfully out-compete Sp1 for its binding site. These other factors include the Sp1-related factors, e.g. SPR-2, -3, and -4, that bind to the same DNA element, but differ in their transactivation domains and tissue specific expression(39, 40) .
In summary, transcription factors binding to the 5` and 3` domains of gERE, including Sp1, are required to confer EGF responsiveness to the gastrin promoter perhaps by regulating factor abundance, access to DNA or the level of phosphorylation. Analysis of cloned gERP 1 and 2 should help to discern whether they cooperate or compete with Sp1 in order to bind gERE and activate transcription. Moreover, it will be important to determine whether Sp1 and gERP function in a similar manner on other promoters given the numerous putative Sp1 sites residing in the promoters of a variety of genes.