Vol. 11, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1991, p. 2686-2696 0270-7306/91/052686-11$02.00/0 Copyright X 1991, American Society for Microbiology

A GC-Rich Element Confers Epidermal Growth Factor Responsiveness to Transcription from the Gastrin Promoter JUANITA L. MERCHANT, BARBARA DEMEDIUK, AND STEPHEN J. BRAND* Gastrointestinal Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114 Received 6 September 1990/Accepted 12 February 1991 Epidermal growth factor (EGF) and transforming growth factor a are important determinants of mucosal

integrity in the gastrointestinal tract, and they act both directly and indirectly to prevent ulceration in the

stomach. Consistent with this physiological role, EGF stimulates transcription of gastrin, a peptide hormone which regulates gastric acid secretion and mucosal growth. EGF stimulation of gastrin transcription is mediated by a GC-rich gastrin EGF response element (gERE) (GGGGCGGGGTGGGGGG) which lies between -54 and -68 in the human gastrin promoter. The gERE sequence also confers weaker responsiveness to phorbol ester stimulation. The gERE sequence differs from previously described EGF response elements. The gERE DNA sequence specifically interacts with a GH4 DNA-binding protein distinct from previously described transcription factors (Egr-1 and AP2) which bind GC-rich sequences and mediate transcriptional activation by growth factors. Furthermore, the gERE element does not bind the Spl transcription factor even though the gERE sequence contains a high-affinity Spl-binding site (GGCGGG).

ways, onefos dependent the other fos independent (37, 44). Furthermore, different cis regulatory elements mediate the transcriptional responses of different promoters to EGF stimulation (20, 21, 39, 49). EGF stimulation of c-fos transcription is mediated by an AP1 cis regulatory element, whereas EGF stimulation of prolactin gene transcription is mediated by a different cis regulatory sequence (20, 21). EGF regulation of prolactin transcription was studied in GH4 pituitary cells; this study included the identification of specific DNA-binding proteins which bind to the cis-acting EGF response element in the prolactin promoter. Since the gastrin promoter is well expressed in GH4 cells (24), EGF stimulation of gastrin gene transcription can also be analyzed in GH4 cells and compared with EGF regulation of prolactin transcription. A prior study has shown that EGF stimulation of gastrin transcription is dependent on cis regulatory sequences lying upstream from the transcriptional start site of the gastrin gene (24). This study characterizes a distinctive EGF response element in the gastrin promoter comprising a 16-bp GC-rich sequence (GGGGCGGGGTGGGGGG) which includes an Spl consensus site (GGGCGG). The gastrin EGF response element (gERE) also mediates weaker transcriptional activation by phorbol esters (12-0-tetradecanoylphorbol-13-acetate [TPA]). The gERE element binds a specific DNA-binding protein in GH4 cells which does not recognize DNA response elements for other growth factoror TPA-activated transcription factors. Furthermore, the gERE element does not bind purified AP2 (46) or cloned Egr-1 (Krox-24; 11, 59) or Egr-2 (Krox-20; 9), transcription factors which bind GC-rich response elements and mediate growth factor and TPA activation of transcription.

Epidermal growth factor (EGF) and its homolog transforming growth factor a (TGF-a) have physiologically complementary effects on gastric acid secretion and mucosal growth in the stomach. Both EGF and TGF-a directly stimulate mucosal growth and inhibit gastric acid secretion, actions which protect the stomach against ulceration from excessive gastric acid secretion (14, 26, 41, 50). EGF also stimulates gastrin gene transcription (24). The dependence of gastrin gene expression on EGF (TGF-a) stimulation probably represents an important control ensuring that stimulation of gastric acid secretion is coupled to stimulation of mucosal proliferation and regeneration. The peptide product of the gastrin gene is the major hormonal stimulus to gastric acid secretion, and gastrin gene expression is regulated by a complex interaction between regulatory peptides released from paracrine and nerve cells in the antral mucosa (7). Gastrin-expressing endocrine cells are stimulated by the local release of TGF-a which is expressed in the antral mucosa (4, 41). Since all actions of TGF-a have been attributed to interactions with the EGF receptor (43), TGF-a probably acts as a local stimulus to gastrin gene transcription. Paracrine regulation of antral gastrin synthesis by TGF-a would ensure that gastrin synthesis is linked to stimulation of mucosal growth, thus avoiding excessive acid secretion in the absence of adequate stimulation of mucosal cell growth. EGF and TGF-a stimulate the transcription of many genes, including c-myc, c-fos, and c-jun, through signaling mechanisms that are shared by phorbol esters, activators of the protein kinase C signaling pathway (19-21, 39, 44, 49, 53). Multiple mechanisms mediate EGF stimulation of gene transcription. EGF stimulations of c-fos, c-jun, and c-myc transcription are immediate-early responses which are not abolished by inhibition of protein synthesis (25). By contrast, cycloheximide inhibits EGF stimulation of transin gene transcription, which is mediated by at least two path*

MATERIALS AND METHODS Plasmid constructions. (i) Gastrin reporter genes. 40 GASC AT and 82 GASCAT were derived from 1300 GASCAT (24), which was constructed by ligating an EcoRI-PstI fragment of the human gastrin gene (32, 63) upstream of a promoterless chloramphenicol acetyltransferase (CAT) gene in plasmid

Corresponding author. 2686

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pOCAT (51). 1300 GASCAT contains 1,300 bp of gastrin 5'-flanking sequences and the entire first exon (57 bp). 82 GASCAT and 40 GASCAT differ from 1300 GASCAT by having 82 and 40 bp of 5'-flanking DNA and were constructed from 1300 GASCAT by using the endonuclease restriction sites NdeI at -82 and AvaIl at -40, respectively. (ii) Gastrin oligonucleotide reporter genes. Four oligonucleotides, A to D, comprising sequential segments of the human gastrin promoter sequence from transcriptional start site to -82 in the 5'-flanking DNA were synthesized. Oligonucleotides A to D encode the gastrin sequences from +9 to -28, -29 to -53, -54 to -68, and -69 to -82, respectively. All oligonucleotides included BamHI and BglIH sites at their 5' and 3' ends, respectively. The A-CAT reporter gene was constructed by ligating into the BamHI site of pOCAT oligonucleotide A, a 37-bp synthetic oligonucleotide containing 28 bp of 5'-flanking sequence and the first 9 bp of exon 1, which includes the TATA box (-26) and the mRNA initiation site of the human gastrin gene. Other oligonucleotide reporter genes were constructed by ligating oligonucleotides B, C, and D either individually or in combination with oligonucleotide C 5' to oligonucleotide A in the A-CAT plasmid. The 2 x C-cfosCAT construct contains two copies of oligonucleotide C, ligated into the HindIII site of pA'56cfosCAT, 5' to a truncated c-fos promoter containing 56 bp of 5'-flanking DNA (23). Plasmid DNA used for transfections was purified twice by equilibrium cesium chloride-ethidium bromide density gradient centrifugation. Cell culture and DNA transfection. GH4 cells were cultured in Ham's F-10 medium with 12.5% horse serum, 2.5% fetal calf serum, penicillin at 100 ,ug/ml, and streptomycin at 100 pLg/ml (medium) in a humidified atmosphere of 5% C02-95% air. For transient transfections, subconfluent GH4 cells were transfected with a mixture containing DEAE-dextran at 400 ,ug/ml, DNA at 3 ,ug/ml, and 50 mM Tris-HCl in F-10 medium without serum for 15 min at 37°C and were then glycerol shocked (40, 58). EGF was added 24 h after transfection and 24 h before preparation of cell extracts. Gastrin reporter gene activity was measured by assaying CAT enzyme activity (27). Gastrin-CAT activity was normalized to the activity of the cotransfected luciferase gene (1 ,ug/ml) expressed from the thymidine kinase (TK) promoter (pA'109TKLUC; 16). To obtain stable transformants, selected oligonucleotide and GASCAT constructs were cotransfected into GH4 pituitary cells with RSVneo, which expresses the neomycin resistance gene from the Rous sarcoma virus promoter (reporter gene construct/RSVneo ratio, 30: 1), by the calcium phosphate precipitate method (10). Stable transformants were selected by resistance to G418 (400 p.g/ml; Sigma), a neomycin analog. RNase protection analysis. Transcriptional initiation from the gastrin promoter was verified by RNase protection analysis, using total RNA (20 ,ug) extracted from human antrum or pooled stable transformants transfected with the A-CAT and GASCAT constructs (2, 64). Antral gastrin and GASCAT mRNA start sites were identified by using an antisense 32P-labeled RNA probe complementary to exon 1 and 194 nucleotides (nt) of gastrin 5'-flanking DNA. The template for this probe was a 295-bp HincII-PstI fragment containing the 194-bp 5'-flanking DNA and 57-bp exon 1 of the human gastrin gene ligated in antisense orientation downstream of the SP6 promoter in pGeml (Promega). The transcriptional start sites of A-CAT and derived oligonucleotide reporter genes were analyzed by using an SP6 32p_ labeled RNA probe complementary to the 5' 210 nt of the CAT gene (1). The template for this probe was a 295-bp

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EcoRI-PvuII fragment of 2xC.A-CAT (see Fig. 4) ligated into the EcoRI and HinclI sites of pGeml in antisense orientation to the SP6 promoter. This EcoRI-PvuII fragment of 2xC.A-CAT comprises 210 bp of CAT gene sequence and 85 bp of gastrin gene sequence which comprises oligonucleotide 2xC ligated 5' to oligonucleotide A (see Fig. 2C). This RNA probe will map the mRNA start sites for all transcripts arising from the oligonucleotide A-CAT-derived constructs. RNA probes were isolated on a 6% polyacrylamide-7 M urea gel, eluted with 1 M ammonium acetate-10 mM Tris-HCl (pH 7.5)-5 mM EDTA, ethanol precipitated, and then hybridized to 20 pLg of total RNA for 16 h at 45°C in 80% formamide-10 mM PIPES (piperazine-N,N'-bis[2-ethanesulfonic acid]; pH 6.7)-40 mM NaCl-0.2 mM EDTA. RNase digestions were carried out at 25°C for 30 min with a final concentrations of 2 ,ug of RNase A and 0.1 ,ug of RNase T, (Boehringer Mannheim) per ml in 10 mM Tris-HCl (pH 7.5)-300 mM NaCI-5 mM EDTA. Digestions were terminated with 20 p.l of 10% sodium dodecyl sulfate and 10 ,ul of proteinase K (5 mg/ml) for 15 min at 37°C. Protected products were resolved on an 8% polyacrylamide-7 M urea gel after phenol-chloroform extraction and ethanol precipitation. Markers were Klenow-labeled fragments of a HpaII digest of pBR322. Analysis of DNA-GH4 extract protein binding. For DNase footprinting, nuclear extracts were prepared from GH4 cells as described by Dignam et al. (17). Protein concentrations were measured by the Bradford method using bovine serum albumin standards (6) and were stored in aliquots at -80°C. DNase I protection assays (28, 33) were performed with 100 to 200 jig of crude nuclear extracts and a probe containing -194 to +57 bp of the human gastrin gene 32p labeled at the 3' end of the coding strand with Klenow polymerase. Nuclear extracts were preincubated without probe for 10 min on ice in 50 mM Tris-HCl (pH 7.9)-0.1 mM KCl-8% glycerol6 mM MgCl2-1 mM EDTA-1 mM dithiothreitol-1 ,ug of poly(dl-dC) per ml-2% polyvinyl alcohol. The mixture was incubated for another 20 min on ice after the addition of probe (20,000 cpm/ng). Digestion was initiated with the addition of 10 ng of DNase I (Boehringer Mannheim) in 5 mM CaCl2-10 mM MgCl2 at 25°C and terminated after 1 min with 75 ,ll of stop buffer (200 mM NaCl, 20 mM EDTA, 1% sodium dodecyl sulfate, 250 mg of tRNA per ml) prior to phenol-chloroform extraction. Digested DNA was ethanol precipitated and resolved on an 8% polyacrylamide-7 M urea gel with Maxam-Gilbert G and G+A sequencing reactions of the labeled probe. Sequence-specific DNA-protein binding was also determined by gel mobility shift assays. Oligonucleotide probes for gel mobility shift assays were prepared by annealing complementary strands and Klenow end labeling with [32P]dATP. Gel shift mobility assays were performed as previously described (22, 28), using 5 ,ug of GH4 extract and 32P-labeled oligonucleotide probe (10,000 cpm/0.1 to 0.5 ng) in a final volume of 20 p.1 containing 10 mM Tris-HCl (pH 7.9), 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 10%o glycerol, and 0.5 p.g of a 40-bp oligonucleotide as a nonspecific competitor. The mixture was preincubated at 25°C for 10 min with unlabeled competitor and for 10 min with labeled probe and resolved on a 4% nondenaturing polyacrylamide gel (acrylamide/bisacrylamide ratio, 30:1). After preelectrophoresis at 10 V/cm for 2 h, gels were run at 10 V/cm in 45 mM Tris base-45 mM boric acid-i mM EDTA. The gels were dried prior to autoradiography at -800C. Gel mobility shift assays were also used to study gERE

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FIG. 1. EGF stimulation of human gastrin mRNA expression in GH4 cells. (A) Northern blot analysis of human gastrin mRNA levels in 20 ,ug of total RNA isolated from GH4 pituitary cells. Lanes: 1 and 2, GH4 cells, no EGF treatment; 3 and 4, GH4 cells treated with 10 nM EGF for 16 h. The Northern blot was hybridized with a human gastrin cDNA probe. (B) Northern blot analysis of growth hormone mRNA levels. Lanes: 1 and 2, GH4 cells, no EGF treatment; 3 and 4, GH4 cells treated with 10 nM EGF for 16 h. The Northern blot shown in panel A was boiled and rehybridized with a nick-translated GH4 cDNA probe. (C) Northern blot analysis of prolactin mRNA. Lanes: 1 and 2, GH4 cells, no EGF treatment; 3 and 4, GH4 cells treated with 10 nM EGF for 16 h. The Northern blot was hybridized with a nick-translated prolactin cDNA probe.

binding to affinity-purified Spl and AP2 and to bacterial extracts expressing cloned Egr-1 and Egr-2 (designated EGR1 and EGR2). Affinity-purified Spl (34) and AP2 (46) were gifts from Stephen Jackson, Trevor Williams, and Robert Tjian (University of California, Berkeley). Cloned Egr-1 (Zif268, Krox-24; 11, 59) was a gift from Barbara Christy and Daniel Nathans (Johns Hopkins University, Baltimore, Md.), and cloned Egr-2 (Krox-20; 9) was a gift from Johnathan Licht (Dana Farber, Boston, Mass.). Northern (RNA) blot analysis. Total RNA (20 ,ug) extracted from GH4 cells by the method of Cathala et al. (8) was resolved on a 1% agarose denaturing gel and transferred to nitrocellulose. RNA blots were hybridized with a 32P-labeled probe complementary to the coding sequences of human gastrin as described previously (7). RESULTS

Human gastrin gene expression stimulated by EGF. To determine whether EGF stimulates expression of the human gastrin gene, GH4 cells were stably transfected with cloned genomic DNA containing the entire human gastrin gene together with 1.3 kb of 5'-flanking DNA and 2.5 kb of 3'-flanking DNA (32, 63). The cells expressed high levels of gastrin mRNA equivalent to the expression of the prolactin gene (Fig. 1). Treatment with 10 nM EGF increased gastrin mRNA threefold, as determined by scanning densitometry (Fig. 1A). The EGF stimulation did not increase the levels of growth hormone mRNA, indicating that EGF selectively stimulates gene expression in GH4 cells (Fig. 1B). EGF stimulation also increased prolactin mRNA levels threefold (Fig. 1C), stimulation similar to that found in prior studies (20). A GC-rich cis-acting element mediates EGF stimulation of gastrin gene transcription. Prior studies have shown that human gastrin promoter CAT reporter genes are as active as

growth hormone reporter genes when transfected into GH4 pituitary cells but are inactive in nonendocrine cells (24). A gastrin reporter gene containing a gastrin promoter with only 82 bp of 5'-flanking sequence (82 GASCAT) was highly expressed when transfected into GH4 cells (Fig. 2B). RNase

protection analysis indicated that gastrin reporter gene transcripts initiate from the same nucleotide sequence as antral gastrin mRNA (Fig. 3A). The 10-bp difference between antral mRNA and GASCAT mRNA start sites reflects the common 10-bp pGEM1 linker sequence added to the 3' end of the human gastrin exon 1 sequence in both the GASCAT construct and probe template (Fig. 3A). Transcription from 82 GASCAT in GH4 cells is stimulated 3.5-fold by EGF (Fig. 4A), which is equivalent to the stimulation of prolactin transcription in GH4 cells (20). Stimulation of 82 GASCAT transcription by EGF implies that this gastrin promoter sequence contains cis regulatory elements which bind transcription factors activated by EGF stimulation. Since this 82 bp of gastrin gene 5'-flanking sequence does not contain cis regulatory elements previously shown to mediate EGF transcriptional activation (20, 21, 49), the present study was done to identify the cis regulatory elements within this sequence which mediate EGF responsiveness. To analyze the cis regulatory elements in the 82 GASCAT promoter, we synthesized four oligonucleotides (A to D) comprising sequential segments of the 82 bp of gastrin 5'-flanking DNA extending from the transcriptional start site (Fig. 2C). The A-CAT reporter gene (Fig. 2A and C) was constructed by ligating oligonucleotide A 5' to the promoterless CAT gene in plasmid pOCAT (51). The oligonucleotide A sequence contains cis regulatory elements sufficient for basal promoter activity, since the A-CAT reporter gene expressed significant CAT activity when transfected into GH4 cells (Fig. 2B). RNase protection analysis showed that A-CAT initiates transcription from the same nucleotides as transcription from the 82 GASCAT reporter gene (Fig. 3B). In contrast to 82 GASCAT, however, transcription from A-CAT was not stimulated by EGF, implying that oligonucleotide A lacks an EGF response element (Fig. 4A). To identify the EGF response element within the gastrin promoter, oligonucleotides B, C, and D were ligated separately or in different combinations 5' to oligonucleotide A in A-CAT (Fig. 2C and D). When all oligonucleotides were ligated to reconstitute the 82 bp of 5'-flanking DNA sequence (D.C.B.A-CAT), basal activity of the reconstituted sequence was 55% that of 82 GASCAT. This decrease may be a consequence of D.C.B.A-CAT lacking the regulatory sequences identified by Theill et al. (60) within the first exon of the human gastrin gene which activate gastrin transcription in neuroendocrine cells. Ligating oligonucleotide B 5' to oligonucleotide A increased the basal activity of the A-CAT reporter gene (B.A-CAT; Fig. 2D). The effect of oligonucleotide C on basal transcription of A-CAT was more complex and depended on copy number. A single copy of oligonucleotide C reduced basal activity, whereas an A-CAT reporter gene with two copies of oligonucleotide C had the same basal activity as A-CAT (C.A-CAT and 2xC.A-CAT; Fig. 2D). Oligonucleotide D had little effect on basal activity of the A-CAT reporter gene (D.A-CAT; Fig. 2D). The C.B.A-CAT and D.C.A-CAT constructs had basal activity 30 and 42% that of 82 GASCAT, which is approximately the same as the activity of A-CAT (data not shown). Transcription from each of these oligonucleotide constructs was examined for EGF responsiveness after transfection into GH4 cells (Fig. 4A). Only expression from

EGF REGULATION OF GASTRIN TRANSCRIPTION

VOL. 11, 1991

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FIG. 2. (A) Schematic representation of the 82 and 40 GASCAT constructs and the basal gastrin promoter oligonucleotide CAT reporter

gene (A-CAT). 82 GASCAT and 40 GASCAT contain 40 and 82 bp, respectively, of 5'-flanking DNA and the first exon of the human gastrin gene ligated upstream of the promoterless reporter CAT gene (pOCAT). The basal gastrin promoter oligonucleotide reporter gene (A-CAT) was constructed by ligating a 43-bp synthetic oligonucleotide comprising the -28 to +9 segment of the human gastrin gene sequence containing the TATA box and the cluster of transcriptional start sites 5' to the CAT gene in pOCAT. (B) Basal CAT activity expressed by these reporter gene constructs assayed 48 h after transient transfection into GH4 cells. (C) Schematic representation and sequences of gastrin oligonucleotides ligated into CAT reporter gene constructs. Synthetic oligonucleotides correspond to gastrin 5'-flanking sequences from -29 to -48 (oligo B), -53 to -68 (oligo C), and -69 to -82 (oligo D) were ligated either individually or in combination into the BamHI site 5' to oligonucleotide A (-28 to -48) in the A-CAT reporter gene. (D) Basal activities of the gastrin oligonucleotide reporter genes 48 h after transient transfection into GH4 cells, shown as a percentage of the CAT activity expressed by 82 GASCAT. Error bars represent standard errors of the means for at least four experiments performed in duplicate. The 2xC-cfosCAT reporter gene contains two copies of oligonucleotide C (2xC) ligated 5' to a truncated c-fos promoter construct, cfosCAT.

reporter gene constructs containing oligonucleotide C (GG GGCGGGGTGGGGGG) were responsive to EGF stimulation. A single copy of oligonucleotide C increased the EGF responsiveness of the A-CAT reporter gene fivefold (Fig.

4A). Oligonucleotide B conferred only weak EGF responsiveness, and oligonucleotide D alone did not confer EGF responsiveness. Furthermore, neither oligonucleotide B nor oligonucleotide D augmented EGF responsiveness of oligonucleotide C containing A-CAT reporter genes. EGF also stimulated gastrin promoter activity when gastrin reporter genes were stably integrated into the genome of GH4 cells. Expression from stably transfected C.A-CAT was stimulated 8.4-fold by EGF, compared with 4.2-fold for 82 GASCAT stably transfected in GH4 cells. Together these results imply that oligonucleotide C contains a gERE. However, unlike many response elements, the gERE does not enhance basal transcription from the gastrin promoter (Fig. 2D). The gERE could also confer EGF responsiveness to a heterologous promoter. Two copies of the gERE conferred EGF responsiveness on a CAT reporter gene transcribed from an unresponsive truncated c-fos promoter, A'56cfos-

CAT (Fig. 4A). The truncated fos promoter was chosen because it essentially contains only a TATA box as a transcriptional regulatory element and no growth factor response elements (23). A'56cfosCAT has been used as a heterologous promoter in reporter gene constructs characterizing a response element in the major histocompatibility complex gene promoter (38). The activity of A'56cfosCAT was not responsive to EGF stimulation. Interestingly, in the context of the weaker truncated c-fos promoter, two copies of oligonucleotide C substantially increased basal promoter activity (Fig. 2D) as well as conferring EGF responsiveness (Fig. 4A). The gERE was also ligated into the herpesvirus TK promoter construct PUTKAT (51), which contains 200 bp of 5' sequence. Activity of the gERE-PUTKAT (2 x C.ACAT) construct was stimulated 4.3-fold by EGF stimulation (Fig. 4A). This construct was used because prior studies using a 200-bp TK promoter construct in GH4 cells reported no EGF response to the 10 nM EGF stimulation used in this study (20). In this study, however, activity of the PUTKAT reporter gene, which also contains only the -200 TK promoter, was stimulated twofold after EGF incubation of

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transfected GH4 cells (Fig. 4A). Nevertheless, addition of the gERE enhanced the EGF responsiveness of this weakly responsive promoter. The gERE (GGGGCGGGGTGGGGGG) contains a highaffinity DNA binding site for the transcription factor Spl (GGGCGG; 18). However, this Spl-like element alone did not mediate EGF responsiveness, since one copy of the GGGGCGGGG sequence (oligonucleotide C2) did not confer EGF responsiveness on the A-CAT reporter gene (Fig. 4A, C2.A-CAT), nor did the gERE 3' half-site GGGGTG GGG (oligonucleotide Cl) (Fig. 4A, C1.A-CAT). The antisense strand of oligonucleotide Cl, CCCCACCCC, weakly resembles a binding site for AP2, a transcription factor that mediates transcriptional activation by phorbol esters and cyclic AMP (31, 55). Identical motifs in the porcine plasminogen activator and tyrosine aminotransferase genes have been designated AP2 sites even though they deviate from the AP2 consensus sequence CCCCAGGC (55). This result suggests that the active gERE comprises a longer GC-rich sequence than either the individual AP2- and Spl-like halfsites. Although single copies of Cl (GGGGTGGGG) and C2 (GGGGCGGGG) were not effective EGF response elements, a concatemer containing three copies of either oligonucleotide C2 (3xGGGGCGGGGG) or Cl (3xGGGGTGG

GG) conferred partial EGF responsiveness on the A-CAT reporter gene (Fig. 4A, 3xCL.A-CAT and 3xC2.A-CAT). Thus, sequences which differ from the exact gERE sequence can mediate partial EGF responsiveness, implying some degeneracy in the cis-acting sequences recognized by the trans-acting mechanisms activated by EGF stimulation. However, mutations in the gERE motif (GGGGCGTTT TGGGGGG; mutant oligonucleotide C) which disrupted the polyguanine sequence between the pyrimidines abolished EGF responsiveness (mutant C.B.A-CAT; Fig. 4A). EGF stimulation of cells activates the phospholipase C signaling pathway, which liberates the intracellular messengers inositol trisphosphate and diacylglycerol, probably mediated through phosphorylation of phospholipase C by the EGF receptor tyrosine kinase (42, 45, 48). Furthermore, many of the cis regulatory elements mediate transcriptional responses to both EGF and phorbol ester stimulation (20, 21). This finding suggests that EGF stimulates transcription partly through intracellular mediators that are also activated by phorbol esters. Consequently, gastrin reporter genes and derived oligonucleotide constructs were also examined for transcriptional, responsiveness after stimulation of transfected GH4 cells with the phorbol ester TPA. Transcription from 82 GASCAT was stimulated by TPA treatment, but the

EGF REGULATION OF GASTRIN TRANSCRIPTION

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FIG. 4. EGF (A) and TPA (B) stimulation of gastrin reporter activity in GH4 cells. GH4 cells transiently transfected with GASCAT, A-CAT, cfosCAT, or PUTKAT constructs were stimulated for 24 h with 10 nM EGF or 0.1 ,uM TPA. 2xC.A-CAT has two copies of oligonucleotide C ligated 5' to the A-CAT reporter gene. 1 x CL.A-CAT and 3 x CL.A-CAT have one and three copies, respectively, of oligonucleotide Cl (GGGGTGGGG; the 3' half of oligonucleotide C) ligated 5' to the A-CAT reporter gene. 1xC2.A-CAT and 3xC2.A-CAT have one and three copies, respectively, of oligonucleotide C2 (GGGGCGGGG; the 5' half of oligonucleotide C) ligated 5' to the A-CAT reporter gene. The mutant C.BA-CAT construct contains a mutated oligonucleotide C (GGGGCGTTTTGG GGGG) ligated 5' to the B.A-CAT construct. EGF or TPA stimulation of transcription from each construct is shown as the ratio of CAT activity expressed in EGF (A)- or TPA (B)-treated cells to CAT activity in unstimulated cells for each reporter gene construct. Results are means and standard errors for at least four experiments, each containing duplicate plates of transfected cells.

gene

response was less than observed after EGF stimulation (Fig. 4B). Although the gERE element (oligonucleotide C) alone conferred TPA responsiveness as well as EGF responsiveness, two copies of oligonucleotide C were required to confer significant TPA responsiveness on the unresponsive A-CAT, whereas one copy of oligonucleotide C conferred EGF responsiveness. Two copies of oligonucleotide C also conferred TPA responsiveness on transcription from the heterologous truncated c-fos promoter 2 x C-cfosCAT reporter gene (Fig. 4B). A specific nuclear protein binds to the gERE sequence. To determine whether the gERE specifically bound GH4 nuclear proteins, gastrin gene sequences from -194 to +57 which included the gERE sequence were analyzed by DNase I

(0). footprinting after incubation with crude GH4 nuclear extracts (Fig. 5). Two footprints were observed: footprint I over the sequence -54 5'-GGAC -51 and footprint II over the gERE sequence -66 5'-GGCGGGGTGG -57 (Fig. 5). The specificity of nuclear extract protein binding to the gERE was demonstrated by competition with unlabeled gERE oligonucleotide (GGGGCGGGGTGGGGGG) which abolished the gERE footprint (footprint II) but not footprint I (Fig. 5, lane 6). GH4 nuclear protein binding to the gERE site also induced a specific upstream hypersensitivity site at the -72 guanine residue (Fig. 5) which also disappeared with footprint II after competition with the gERE oligonucleotide sequence (GGGGCGGGGTGGGGGG). The DNA sequence-specific binding of the GH4 gEREbinding protein was further studied by using gel mobility shift assays and competition with different oligonucleotide sequences, including those containing GC-rich binding sites for known transcription factors (Fig. 6A to C). Incubation of GH4 extracts with a labeled gERE probe (GGGGCGGGGT GGGGGG) resulted in a single delayed DNA-protein complex which was abolished by unlabeled gERE sequence (Fig. 6A, lanes 3 and 4). GH4 protein binding to the gERE probe was also abolished by concatamers of the gERE 5' half-site (3 x GGGGCGGGG; oligonucleotide 3 xC2) and the gERE 3' half-site (3xGGGGTGGGG; oligonucleotide 3xCl), which also conferred partial EGF responsiveness (Fig. 6A, lanes 5 to 8). GH4 protein binding to the gERE probe was not displaced by a 100-fold-higher concentration of the mutant gERE oligonucleotide (GGGGCGTITTGGGGGG) (Fig. 6B, lanes 4 and 5). This mutant gERE sequence (mutant C.ACAT) also did not confer EGF responsiveness (Fig. 4A), indicating a correlation between GH4 protein binding to the gERE element and EGF transcriptional responsiveness. In

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FIG. 6. Gel mobility shift assay showing sequence-specific binding of the gERE to DNA-binding proteins in GH4 cell extracts. Binding reaction mixtures were incubated with 0.2 ng of radiolabeled gERE probe and 5 ,ug of GH4 extract at 25°C. Shown are results for probe alone (lane 1), with GH4 extract (lane 2), and with extract and unlabeled oligonucleotide DNA competitor at lOx and lOOx molar excess of probe (A and C, lanes 3 to 8; B, lanes 3 to 7). (D) DNA sequences of competitor oligonucleotides. TH, Tyrosine hydroxylase.

the 5'-flanking DNA of the tyrosine hydroxylase gene (39), the antisense sequence between -112 and -124 (GGCGGGGTGGG) is very similar to the gERE sequence (GGGGCGGGGTGGGGGG). Since the gERE-like sequence is within the tyrosine hydroxylase DNA sequence necessary for EGF transcriptional responsiveness (39), an oligonucleotide containing the tyrosine hydroxylase sequence (GGCGGGGGTGGG; TH oligonucleotide; Fig. 6D) was tested for gERE-binding activity. However, a 100-fold excess of the TH oligonucleotide did not displace GH4 protein binding to the gastrin gERE sequence (Fig. 6B, lanes 6 and 7). The 5' gERE sequence also contains a high-affinity consensus Spl-binding site (GGGGCGGGG), suggesting that transcription factor Spl may bind to the gERE (34). However, GH4 protein binding to the gERE probe was not displaced by a 100-fold excess of an unlabeled metallothionein promoter sequence containing an identical high-affinity Spl sequence, GGGGCGGGG (Fig. 6B, lanes 3 and 4) (34). This result implies that the observed gERE-GH4 protein complex does not contain the Spl transcription factor. Other previously characterized transcription factors also recognize GC-rich DNA regulatory elements, including factors AP2 and Egr-1, which mediate stimulation of gene transcription by growth factors and phorbol esters (12, 13, 31, 46, 59). To determine whether AP2 or Egr-1 binds the gERE sequence, high-affinity AP2- and Egr-1-binding sequences were also used as competitors of gERE binding to the GH4 protein (Fig. 6B). Although the AP2- or Egr-1-binding sites are GC-rich, GH4 protein binding to the gERE probe was not displaced by a 100-fold excess of unlabeled oligonucleotides containing AP2- or Egr-1-binding sites. These observations indicate that the gERE-binding protein in GH4 extracts differs from Spl, AP2, and Egr-1 DNA-binding proteins. However, the presence of uncharacterized components in these crude GH4 extracts may obscure significant interactions. Specifically SPl-, AP2-, and Egrl-like factors may be sequestered or blocked from interacting with the gERE site by competing factors. To conclu-

sively exclude Spl, AP2, and Egr-1 and -2 as potential gERE-binding proteins, gERE binding to these factors was studied by using either affinity-purified Spl and AP2 from HeLa cells or Egr-1 and -2 extracts from bacteria expressing cloned Egr-1 or -2 (Fig. 7). The gERE sequence does not bind affinity-purified Spl (Fig. 7A, lanes 5 to 8). The gERE sequence (100-fold excess) does not displace to Spl binding to the human metallothionein Ila (hMTIIa) gene sequence even though the two sequences have the same GGGGCG GGG Spl consensus site (Fig. 7A, lanes 1 to 4). The gERE sequence also does not bind affinity-purified AP2, and a 100-fold excess of unlabeled gERE does not displace AP2 binding from the AP2-binding site in the hMTIla gene (Fig. 7B). Furthermore, cloned Egr-1 (Krox-24, Zif268; Fig. 7C) and Egr-2 (Krox-20; not shown) expressed in bacterial extracts do not bind the gERE sequence, and a 100-fold excess of unlabeled gERE does not displace Egr-1 binding from the Egr-1-binding site within the Egr-1 (Krox-24) gene (13). Thus, the gERE DNA-binding activity in GH4 extracts is not the result of Spl, AP2, or Egr-1 and -2 binding to the gERE even though these transcription factors bind to GCrich DNA sequences. DISCUSSION Stimulation of gastrin transcription by EGF is mediated by GC-rich sequence designated gERE. Unlike many response elements, the gERE does not activate basal transcription in the context of the gastrin promoter although two copies of the gERE increased basal transcription from a less active truncated cfosCAT reporter gene construct. Although the gERE sequence (GGGGCGGGGTGGGGGG) gave the greatest EGF transcriptional stimulation, a concatemer of the 5' half-site of the gERE, GGGGCGGGG, also mediated EGF transcriptional activation, implying some redundancy in the DNA sequence of the EGF cis regulatory element. However, a single copy of the GGGGCGGGG sequence could not activate EGF transcriptional activation, indicating a

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FIG. 7. Interaction of the gERE with purified or cloned transcription factors Spl (A), AP2 (B), and Egr-1 (C) analyzed by gel mobility shift assay. (A) Lanes: 1, hMTIIa Spl probe alone; 2, Spl probe plus 1 ng of affinity-purified HeLa Spl; 3, probe hMTIIa and Spl as in lane 2 with a 100-fold excess of unlabeled hMTIIa Spl oligonucleotide; 4, as in lane 2 with a 100-fold excess of unlabeled gERE oligonucleotide; 5, gERE probe alone; 6, gERE probe plus 1 ng of HeLa Spl; 7, as in lane 6 with a 100-fold excess of unlabeled gERE oligonucleotide; 8, as in lane 6 with a 100-fold excess of unlabeled hMTIIa Spl oligonucleotide. (B) Lanes: 1, hMTIIa AP2 probe alone; 2, hMTIIa AP2 plus 1 ng of affinity-purified HeLa AP2; 3, same as lane 2 with a 100-fold excess of unlabeled hMTIIa AP2 oligonucleotide as competitor; 4, same as lane 2 with a 100-fold excess of unlabeled gERE oligonucleotide as competitor; 5, gERE probe alone; 6, gERE probe plus 1 ng of affinity-purified HeLa AP2. of control 1 (C) Lanes: 1, EGR1 probe alone; 2, EGR1 probe plussLg bacterial extract; 3, EGR1 probe plus 1 ±g of EGR1-expressing bacterial extract; 4, EGR1 probe plus EGR1 extract with a 100-fold molar excess of unlabeled EGR1 oligonucleotide as competitor; 5, EGR1 probe plus EGR1 extract with a 100-fold molar excess of unlabeled gERE oligonucleotide as competitor; 6, gERE probe; 7, 1 of EGR1-expressing bacterial extract. gERE probe plus ,ug

that the length of GC nucleotide sequence is critical for EGF responsiveness.

The gERE sequence differs from previously described EGF response elements found in the prolactin, Moloney murine leukemia virus long terminal repeat (Moloney LTR), fos, transin, and pS2 promoters (20, 21, 49, 61). These prior studies have shown that EGF stimulates transcription of different genes through different cis regulatory sequences. EGF responsiveness of c-fos transcription is mediated by the AP1 site (TGACTCA) and the flanking serum response element (SRE) in the c-fos promoter (21). However, the EGF response element in the prolactin promoter does not contain a good match to the AP1 motif, implying that the prolactin response is mediated by a distinct transcription factor (20). EGF responsiveness of the tyrosine hydroxylase promoter is mediated by cis regulatory sequences lying in the 272 bp of 5'-flanking DNA upstream of the transcrip-

2693

tional start site. The tyrosine hydroxylase gene sequence between -93 and -81 (GGAGAGGATGCC) is nearly identical to a sequence in the prolactin EGF response element (GGAAGAGGATGCC) (20, 39). Since EGF responsiveness of both promoters was analyzed in GH4 cells, the same DNA-binding protein may interact with EGF response elements in both the prolactin and tyrosine hydroxylase promoters. Although the gERE sequence differs from other cis regulatory elements which mediate EGF responsiveness, the gERE element, like many cis-acting EGF response elements, mediates transcriptional activation by phorbol esters. This presumably reflects the linkage between EGF receptor activation and the C kinase signaling pathway. The EGF receptor tyrosine kinase directly phosphorylates phospholipase C, the enzyme which liberates diacylglycerol, the activator of protein kinase C (42, 45, 48). C kinase stimulation, in turn, activates the transcription factor AP1, which binds to the TGACTCA motif found in many phorbol esterresponsive promoters. The TGACTCA motif also mediates EGF responsiveness in the c-fos promoter, suggesting that transcription factor AP1 is also activated by EGF stimulation (21). However, the prolactin and Moloney LTR promoters have different cis regulatory sequences which also mediate transcriptional responses to both phorbol ester and EGF stimulation (20). These observations are consistent with EGF and phorbol esters stimulating common intracellular signaling events to activate different transcription factors which bind to unrelated cis regulatory sequences. The gERE sequence, which differs from the EGF response elements found in the c-fos, prolactin, and Moloney LTR promoters, also confers transcriptional responsiveness to phorbol ester stimulation as well as to EGF stimulation. However, EGF activation of gastrin transcription is stronger than observed after TPA stimulation, implying either that EGF is more effective than TPA in stimulating C kinase or, more likely, that EGF stimulates gastrin transcription

through additional signaling pathways. The gERE sequence binds specifically to a DNA-binding protein in GH4 extracts, suggesting that stimulation of gastrin transcription by EGF results from modifications or increased expression of this DNA-binding protein induced by EGF stimulation. This observation resembles serum stimulation of c-fos transcription mediated by the SRE (61). In vivo DNA footprinting of unstimulated cells shows that the SRE is already bound by nuclear proteins and that transcriptional activation by serum growth factors does not further increase nuclear protein binding to the SRE (29). This implies that modifications in the non-DNA-binding domains of these nuclear proteins mediate c-fos transcriptional activation (29). The SRE is bound by at least two proteins, SRF (serum response factor) and p62 (57). Although SRF binds the SRE independently, p62 cannot bind DNA alone and must interact with SRF to bind the SRE DNA element (45). Nevertheless, both the SRF and p62 necesprotein components of the SRE-protein complex are growth sary for transcriptional activation of c-fos by serum may factors (57). Similarly, the gERE DNA-binding protein interact with other nuclear proteins to form a complex which activates gastrin transcription. These other proteins, rather than the gERE DNA-binding protein itself, may be the transcription factors directly stimulated by the cellular events that follow EGF receptor activation. Although the gERE sequence contains the GGGGCGGGG sequence which has been identified as a high-affinity Spl site (GGGCGG; 18), Spl is an unlikely transcription factor to

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MERCHANT ET AL.

mediate transcriptional activation by EGF. Pure Spl does not bind the gERE sequence avidly, even though Spl binds strongly to an identical GGGGCGGGG sequence in the hMTIIa sequence (34). Apparently the sequences flanking the Spl site in the gERE sequence do not support highaffinity Spl binding, implying that the gERE-binding protein identified here is distinct from Spl. Recent studies have characterized transcription factors other than Spl which bind to the GGGCGG Spl motif. The HeLa transcription factor LSF (late simian virus 40 factor) stimulates transcription from the simian virus 40 late promoter by binding GC motifs also bound by Spl (30). HeLa cells also express an inducible metal-activated transcription factor, MTF, which specifically activates the human methallothionein Ta promoter by binding a metal response element that contains a consensus Spl-binding motif (GGGCGG) within the metal response element sequence (56, 62). However, DNA sequences flanking the GGGCGG motif also contribute to MTF binding specificity since an identical Spl-binding site in the herpes simplex virus promoter does not bind MTF (62). Comparisons of MTF and Spl methylation interference patterns also revealed differences between MTF and Spl binding to guanine contacts with the GGGCGG motif. The EGF receptor promoter also has extended GC-rich regulatory elements similar to the gERE which bind the positive transcription factor ETF and the repressor GCF (35, 36). ETF was shown to stimulate transcription only from promoters lacking TATA boxes and did not activate transcription from promoters with TATA boxes (35). Similar ETFbinding sites are found in the actin promoter (35). Since EGF stimulates both actin and EGF receptor gene expression (5, 18), examination of the ETF-binding sites in these promoters as potential EGF response elements may be of interest. Other transcription factors that both recognize GC-rich DNA elements and also meditate transcriptional activation by growth factors have also been identified. Like Spl, Egr-1 (also known as Zif268 or Krox-24) and Egr-2 (Krox-20) are members of the zinc finger class of transcription factors and bind to a GC-rich consensus motif (GCGGGGGCG) (9, 11, 59). The transcription protein AP2 (31, 46) recognizes the GC-rich consensus sequence CCCCAGGC and is activated by increases in protein kinase A and C activity-initiated cyclic AMP and phorbol ester stimulation. Although the consensus sequences recognized by these factors differ from the gERE sequence, studies of Oct 1 binding have shown that specific DNA recognition sequences for a given transcription factor can be highly degenerate (3). However, it is improbable that AP2 or Egr-1 binds to the gERE sequence to mediate EGF activation of gastrin transcription. Binding of the GH4 DNA-binding protein to the gERE sequence is not displaced by competition with unlabeled DNA containing AP2- and Egr-l-binding sites. Furthermore, analysis of gERE DNA binding to either affinity-purified AP2 or cloned Egr-1 and -2 proteins failed to show significant interaction between the gERE sequence and these factors. Therefore, EGF activation of gastrin gene transcription is probably mediated by a hitherto uncharacterized DNA-binding protein specifically recognizing the gastrin EGF cis regulatory element. ACKNOWLEDGMENTS We thank Stephen Jackson and Trevor Williams for advice and assistance in this study. We are grateful to Lee Kaplan, Andrew Leiter, and Dona Chikaraishi for critically reading the manuscript and for helpful comments. Thanks are also extended to Stephen Jackson, Trevor Williams, and Robert Tjian (University of Califor-

MOL. CELL. BIOL.

nia, Berkeley) for the gift of affinity-purified Spl and AP2; Barbara Christy and Daniel Nathans (Johns Hopkins University, Baltimore, Md.) for cloned Zif268, and Johnathan Licht (Dana Farber, Boston, Mass.) for cloned Egr-2 (Krox-20). J.L.M. was supported by a fellowship from the Robert Wood Johnson Foundation. This research was supported by grants from the NIH to S.J.B. REFERENCES 1. Alton, N. K., and D. Vapnek. 1979. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature

(London) 282:864-869. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1989. Current protocols in molecular biology, vol. 1 and 2. Greene Publishing Associates and Wiley-Interscience, Brooklyn, N.Y. 3. Baumruker, T., R. Strum, and W. Herr. 1988. OBP100 binds remarkably degenerate octamer motifs through specific interactions with flanking sequences. Genes Dev. 2:1400-1413. 4. Beauchamp, R. D., J. A. Bernard, C. M. McCutchen, J. A. Cherner, and R. J. Coffey, Jr. 1989. Localization of transforming growth factor a and its receptor in gastric mucosal cells. J. Clin. Invest. 84:1017-123. 5. Bjorge, J. D., A. J. Paterson, and J. E. Kudlow. 1989. Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor a in a breast cancer cell line. J. Biol. Chem. 264:4021-4027. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 7. Brand, S. J., and D. Stone. 1988. Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria. J. Clin. Invest. 82:1059-1066. 8. Cathala, G., J. F. Savouret, B. Mendez, B. L. West, M. Karin, J. A. Martial, and J. D. Baxter. 1983. A method for isolation of intact translationally active ribonucleic acid. DNA 2:329-335. 9. Chavier, P., M. Zerial, P. Lemair, J. Almendral, R. Bravo, and P. Charnay. 1988. A gene encoding a protein with zinc fingers is activated during GO/Gl transition in cultured cells. EMBO J. 7:29-35. 10. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752. 11. Christy, B., and D. Nathans. 1988. A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with zinc finger sequences. Proc. Natl. Acad. Sci. USA 85:7857-7861. 12. Christy, B., and D. Nathans. 1989. Functional serum response elements upstream of the growth factor-inducible gene zi#268. Mol. Cell. Biol. 9:4889-4895. 13. Christy, B., and D. Nathans. 1989. DNA binding site of the growth factor-inducible protein Zif268. Proc. Natl. Acad. Sci. USA 86:8737-8741. 14. Dembinski, A. B., and L. R. Johnson. 1985. Effect of epidermal growth factor on the development of rat gastric mucosa. Endocrinology 116:90-94. 15. Derynck, R. 1988. Transforming growth factor a. Cell 54:593595. 16. De-wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725-737. 17. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1488. 18. Dynan, W. S., and R. Tjian. 1983. The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 early promoter. Cell 35:79-87. 19. Elder, P. K., L. J. Schmidt, T. Ono, and M. J. Getz. 1984. Specific stimulation of actin gene transcription by epidermal growth factor and cyclohexamide. Proc. Natl. Acad. Sci. USA 81:7476-7480. 20. Elsholtz, H. P., H. J. Mangalam, E. Potter, V. R. Albert, S.

VOL.

21.

22. 23. 24. 25.

26. 27. 28. 29.

30. 31. 32.

33. 34.

35. 36. 37.

38.

39. 40.

41.

11, 1991

Supowit, R. M. Evans, and M. G. Rosenfeld. 1986. Two different cis-active elements transfer the transcriptional effects of both EGF and phorbol esters. Science 234:1552-1557. Fisch, T. M., R. Prywes, and R. G. Roeder. 1989. An APlbinding site in the c-fos gene can mediate induction by epidermal growth factor and 12-O-tetradecanoyl phorbol-13-acetate. Mol. Cell. Biol. 9:1327-1331. Freid, M., and D. M. Crothers. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9:6505-6525. Gilman, M. Z., R. N. Wilson, and R. A. Weinberg. 1986. Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression. Mol. Cell. Biol. 6:4305-4316. Godley, J. M., and S. J. Brand. 1989. Regulation of the gastrin promoter by epidermal growth factor and neuropeptides. Proc. Natl. Acad. Sci. USA 86:3036-3040. Greenberg, M. E., A. L. Hermanowski, and E. Ziff. 1986. Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription. Mol. Cell. Biol. 6:1050-1057. Gregory, H., S. Walsh, and C. R. Hopkins. 1979. The identification of urogastrone in serum, saliva, and gastric juice. Gastroenterology 77:313-318. Gorman, C. M., L. F. Moffat, and B. W. Howard. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. Henningbausen, L., and H. Lubon. 1987. Interactions of protein with DNA in vitro. Methods Enzymol. 152:725-728. Herrera, R. E., P. E. Shaw, and A. Nordhein. 1989. Occupation of the c-fos serum response element in vivo by a multi-protein complex is unaltered by growth factor induction. Nature (London) 340:68-70. Huang, H., R. Sundseth, and U. Hansen. 1990. Transcription factor LSF binds two variant bipartite sites within the SV40 late promoter. Genes Dev. 4:287-298. Imagawa, M., R. Chiu, and M. Karin. 1987. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251-260. Ito, R., K. Sato, T. Helmer, G. Jay, and K. Agarwal. 1984. Structural analysis of the gene encoding human gastrin: the large intron contains an Alu sequence. Proc. Natl. Acad. Sci. USA 81:4662-4666. Jones, K. A., K. R. Yamamoto, and R. Tjian. 1985. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell 42:559-572. Kadonaga, J. T., K. R. Carner, F. R. Maslarz, and R. Tjian. 1987. Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. Cell 51:107901090. Kageyama, R., G. T. Merlino, and I. Pastan. 1989. Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box. J. Biol. Chem. 24:15508-15514. Kageyama, R., and I. Pastan. 1989. Molecular cloning and characterization of a human DNA binding factor that represses transcription. Cell 59:815-825. Kerr, L. D., J. T. Holt, and L. M. Matrisan. 1988. Growth factors regulate transin gene expression by c-fos dependent pathways and c-fos independent pathways. Science 242:14241427. Lenardo, M., A. K. Rustgi, A. R. Schieveila, and R. Bernards. 1989. N-myc suppresses MHC class I antigen expression by decreasing binding of an enhancer activating protein. EMBO J. 8:3351-3355. Lewis, E. J., and D. M. Chikaraishi. 1987. Regulated expression of the tyrosine hydroxylase gene by epidermal growth factor. Mol. Cell. Biol. 7:3332-3336. Lopata, M., D. Cleveland, and B. Soliner-Webb. 1984. High level transient expression of a chloramphenicol acetyltransferase gene by DEAE dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 12:5705-5717. Maiden, L. T., U. Novak, and A. W. Burgess. 1989. Expression of transforming growth factor alpha messenger RNA in the

EGF REGULATION OF GASTRIN TRANSCRIPTION

42.

43.

44.

45.

46. 47. 48.

49.

50.

51. 52.

53. 54.

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normal and neoplastic gastro-intestinal tract. Int. J. Cancer 43:380-384. Margolis, B., S. G. Rhee, S. Felder, M. Mervic, R. Lyall, A. Levitzki, A. Ullrich, Z. Zilberstein, and J. Schlessinger. 1989. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell 57:11011107. Massague, J. 1983. Epidermal growth factor-like transforming growth factor I. Isolation, chemical, characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells. J. Biol. Chem. 258:13606-13613. Matrisian, L. M., P. Leroy, C. Ruhlmann, M.-C. Gesnel, and R. Breathnach. 1986. Isolation of the oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts. Mol. Cell. Biol. 6:1679-1686. Meisenhelder, J., P. Shu, S. G. Rhee, and T. Hunter. 1989. Phospholipase C is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57:11091122. Mitchell, P. J., C. Wang, and R. Tjian. 1987. Positive and negative regulation of transcription in vitro: enhancer-binding protein AP2 is inhibited by SV40 T antigen. Cell 50:847-861. Muller, R., R. Bravo, J. Burkhardt, and T. Curren. 1984. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature (London) 312:716-720. Nishibe, S., M. I. Walk, S. G. Rhee, and G. Carpenter. 1989. Tyrosine phosphorylation of phospholiase C-II in vitro by the epidermal growth factor receptor. J. Biol. Chem. 264:1033510338. Nunez, A. M., M. Berry, J. L. Imler, and P. Chambon. 1989. The 5' flanking region of the pS2 gene contains a complex enhancer region responsive to estrogens epidermal growth factor, a tumor promoter TPA, c Ha ras oncoprotein and the c jun protein. EMBO J. 8:823-829. Olsen, P. S., S. S. Poulsen, K. Therkelsen, and E. Nexo. 1986. Oral administration of synthetic human urogastrone promotes healing of chronic duodenal ulcers in rats. Gastroenterology 90:911-917. Prost, E., and D. D. Moore. 1986. CAT vectors for analysis of eukaryotic promoters and enhancers. Gene 45:107-111. Prywes, R., and R. G. Roeder. 1986. Inducible binding of a factor to the c-fos enhancer. Cell 47:777-784. Quentin, B., and R. Breathnach. 1988. Epidermal growth factor stimulates transcription of the c-jun proto-oncogene in rat fibroblasts. Nature (London) 334:538-539. Rhodes, J. A., J. P. Tam, U. Finke, M. Saunders, J. Bernanke, W. Silen, and R. A. Murphy. 1986. Transforming growth factor a inhibits secretion of gastric acid. Proc. Natl. Acad. Sci. USA

83:3844-3846. 55. Roesler, W. J., G. R. Vendenbark, and R. W. Hanson. 1988. Cyclic AMP and the induction of eukaryotic gene transcription. J. Biol. Chem. 263:9063-9066. 56. Serfling, E., A. Lubbe, K. Dorsch-Hasler, and W. Schaffner. 1985. Metal-dependent SV40 viruses containing inducible enhancers from the upstream region of metallothionein genes. EMBO J. 4:3851-3859. 57. Shaw, P. E., H. Schroter, and A. Nordheim. 1989. The ability of a ternary complex to form over the serum response element correlates with serum inducibility of the human c-fos promoter. Cell 56:563-572. 58. Sompayrac, L. M., and K. J. Danna. 1981. Efficient infection of monkey cells with DNA of simian virus 40. Proc. Natl. Acad. Sci. USA 78:7575-7578. 59. Sukhatme, V. P., X. Cao, L. C. Chang, C. Tsai-Morris, D. Stamenkovich, P. C. P. Ferreira, D. R. Cohen, S. A. Edwards, T. B. Shows, T. Curran, M. M. LeBeau, and E. D. Adamson. 1988. A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization. Cell 53:37-43. 60. Theffil, L. E., 0. Wiborg, and J. Vuust. 1987. Cell specific expression of the human gastrin gene: evidence for a control element located downstream of the TATA box. Mol. Cell. Biol. 7:4329-4336.

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61. Treisman, R. 1986. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell 46:567-574. 62. Westin, G., and W. Schaffner. 1988. A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J. 7:3763-3770.

MOL. CELL. BIOL.

63. Wiborg,

0. L., L. Berglund, E. Boel, K. Norris, F. Norris, J. Rehfeld, K. A. Marcker, and J. Vuust. 1984. Structure of a human gastrin gene. Proc. Natl. Acad. Sci. USA 81:1067-1069. 64. Zinn, K., D. DiMaio, and T. Maniatis. 1983. Identification of two distinct regulatory regions adjacent to the human ,-interferon gene. Cell 34:865-879.