©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutually Exclusive Interactions between Factors Binding to Adjacent Sp1 and AT-rich Elements Regulate Gastrin Gene Transcription in Insulinoma Cells (*)

Daniel C. Chung (§) , Stephen J. Brand (¶) , Loyal G. Tillotson (**)

From the (1) Gastrointestinal Unit and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gastrin gene is transiently expressed in fetal pancreatic islets during islet neogenesis but then switched off after birth when islet cells become fully differentiated. Previous studies identified a cis-regulatory sequence between -109 and -75 in the human gastrin promoter which binds islet cell-specific activators and a nonspecific repressor and thus may act as a molecular switch. The present study identified another cis-regulatory sequence (ACACTAAATGAAAGGGCGGGGCAG) which bound two islet nuclear proteins in a mutually exclusive manner, as defined by gel shift competition, methylation interference, and DNase I footprinting assays. The general transactivator Sp1 recognized the downstream GGGCGGGG sequence, but Sp1 binding was prevented when another islet factor bound to the adjacent AT-rich sequence (CTAAATGA). This gastrin AT-rich element is nearly identical to the binding site (ATAAATGA) for the islet-specific transcription factor TF-1. However, the gastrin AT-binding factor appeared to differ from TF-1 in its gel mobility shift pattern. Transfections of rat insulinoma cells revealed that mutations which blocked binding to the AT-rich element but allowed Sp1 binding up-regulated transcriptional activity. These results suggest that the gastrin AT-binding factor blocks transactivation by Sp1 and may have a role in the repression of gastrin transcription seen at the end of islet differentiation.


INTRODUCTION

Pancreatic islet endocrine cells share common evolutionary and developmental origins with gut endocrine cells (1) . This relationship is emphasized by the transient expression of hormones such as gastrin, peptide-YY, and secretin in fetal islets even though these peptides are expressed largely in the luminal gastrointestinal tract in the adult (2, 3, 4) . The transient expression of gastrin may serve to promote islet proliferation and differentiation, which occur concurrently with pancreatic gastrin expression (5, 6, 7, 8) . Although little gastrin is expressed in the adult pancreas, it can be re-expressed when islets undergo neoplastic transformation. Therefore, the molecular mechanisms controlling gastrin gene transcription in islet cells represent an interesting example of developmental control of gene expression.

Transgenic mouse studies have shown that 1300 bp() of 5`-flanking DNA of the human gastrin gene appropriately regulates the transient expression of gastrin in fetal islets (9) . Detailed promoter analysis utilizing cultured gastrin-expressing rat insulinoma cell lines has identified regulatory elements that lie within 200 bp of the transcriptional start site (10, 11, 12) . These include a sequence between -109 and -75 of the gastrin promoter that binds three transcription factors. A transcriptional activator binds to a CACC element at the 5` end of this domain (12) . However, a repressor binds to an immediately adjacent 3` sequence, the Gastrin Negative Element (GNE), which is similar to a repressor binding sequence in the -interferon promoter (11) . Immediately 3` to the GNE sequence is another positive element containing an E-box motif (CANNTG), which has been shown to bind multiple helix-loop-helix transcription factors (13, 14) .

These elements were identified by deletional analysis, a method which may fail to detect other upstream sequences that regulate gastrin transcription. For this reason, we carried out systematic scanning mutagenesis of the 200-bp sequence upstream of the transcriptional start site and identified a new cis-regulatory domain from -163 to -140 which controls gastrin transcription in islet cells. There was mutually exclusive binding of two nuclear proteins to this domain, in which binding of one factor blocked the transcriptional activation by the other. Thus, the gastrin promoter comprises a tandem array of switch-like cis-regulatory elements in which activation can be blocked by factors binding to adjacent sequences. This tandem array of regulatory sequences may provide the molecular mechanism to explain the transient activation and subsequent repression of gastrin transcription in fetal islets.


MATERIALS AND METHODS

Plasmid Constructions

Linker scanning mutagenesis was performed utilizing a promoter construct containing 200 bp of 5`-flanking DNA of the human gastrin gene subcloned into the promoterless luciferase vector pXP2, as described previously (12) . 11 linker scanning mutant constructs were generated by substituting a 10-bp cassette specifying adjacent XhoI and ScaI restriction sites (CTCGAGTACT) in sequential 10-bp blocks from -170 to -60 bp utilizing the unique site elimination method (15) . The mutagenic primers contained 13 complementary nucleotides flanking both ends of the 10-bp substitution. The second primer set mutated a unique BglII site in the pXP2 vector. After heat-denaturing at 100 °C and snap-cooling to 0 °C, the plasmid vector with a 1000-fold molar excess of both primers was incubated with T4 DNA polymerase, T4 DNA ligase (both New England Biolabs), and all 4 dNTPs. Newly synthesized double-stranded plasmid constructs were digested with BglII (New England Biolabs) and transformed into the mismatch repair deficient mutSEscherichia coli strain (16) , miniprep DNA was prepared via a modified alkaline lysis procedure (Qiagen), and mutants were selected by digestion again with BglII and re-transformation into E. coli DH5- cells. All mutant constructs were confirmed by sequencing.

Minimal promoter constructs containing the wild-type (WT) or point mutant elements spanning -163 to -140 bp (see legend in Fig. 3 ) were created by ligation of kinased double-stranded synthetic oligonucleotides into the BamHI site of the heterologous thymidine kinase promoter vector pT81 with a luciferase reporter (17) , as described previously (12) . All constructs were sequenced to confirm proper head-to-tail orientation of multimeric inserts.


Figure 3: Characterization of the Sp1 binding domain by mutational analysis and methylation interference assays. A, gel mobility shift assays were performed with Sp1-enriched 38A fraction B and the WT full-length probe in the presence of a 25-fold excess of Sp1 consensus, wild-type, and mutant competitor oligonucleotides. The nucleotide sequences for the plus strand are listed below and the underlined residues indicate the mutated base pairs. B, methylation interference assay performed as described under ``Materials and Methods.'' Lanes 1 and 3 represent free probe and lane 2 represents probe bound to Sp1. The bar at the right represents methylated G and A residues that interfere with Sp1 binding. C, schematic representation of residues important for Sp1 binding to the gastrin promoter ( underlined) as defined by mutational gel-shift analysis ( X) and methylation interference ( O); WT, gatccACAC-T AAA TGA AAG GGC GGG GC AGa ; M 1 , gatccAACAGAAATGAAAGGGCGGGGC-AGa; M2, gatccACACTCCCGGAAAGGG-CG GGG CA Ga ; M 3 , ga tc cAC ACT AA AT-TCCCGGGCGGGGCAGa; M4, gatccACACTAAATGAAA TTTCGGGGCAGa; M5, ga tcc ACA CT AA AT GA AA GC GA GTG GC-AGa; Sp1, ATTCGATCGGGGCGGGGC-GAGC.



Cells

RIN 1046-38A (10, 18) , AR4-2J (19) , Rat 2 (ATCC CRL 1764), Panc-1 (ATCC CRL 1469), and HepG2 (ATCC HB 8065) cells were cultured as described in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2% penicillin-streptomycin (all Bio-Whittaker). The RIN 38A cell line expresses gastrin, as determined by Northern blot hybridization with a rat gastrin Exon III riboprobe (data not shown).

Transfections

RIN 38A cells were transiently transfected with a modified DEAE-dextran protocol followed by a 10% dimethyl sulfoxide shock, as described previously (12) . Luciferase activity was assayed 48 h after transfection with luciferin, ATP, and coenzyme A (Promega) utilizing a monolight luminometer (Analytical Luminescence Laboratory). Transfections were performed at least 3 times and variation between experiments was not greater than 15%.

Nuclear Extracts and Gel Mobility Shift Assays

Nuclear extracts were prepared by the method of Dignam (20) and quantitated utilizing a Bradford protein assay (21) . Partial purification of crude RIN 38A extracts was performed by passage over a phosphocellulose column (Whatman) or a DEAE-Sepharose column (Pharmacia Biotech Inc.) as described previously (22) to partially purify Sp1 and the gastrin AT-binding factor (gAT factor), respectively. Proteins adsorbed to the phosphocellulose or DEAE-Sepharose column were eluted with a 0.5 or 0.25 M KCl solution, respectively. The trace amount of gAT factor that was eluted off the phosphocellulose column was removed by incubation with the AT-rich oligo (gatccCTGACACTAAATGAAAGGGa) at a final concentration of 0.15 µ M prior to passage over the phosphocellulose column again. All samples were then dialyzed against buffer A (20 m M HEPES (pH 8), 10% glycerol, 1.5 m M MgCl, 75 m M KCl, 1 m M dithiothreitol, 0.2 m M phenylmethylsulfonyl fluoride, and a 0.5 µg/ml mixture of aprotinin, antipain, pepstatin A, and leupeptin).

Gel mobility shift assays were performed by incubating the various extracts with 4 fmol of double-stranded oligonucleotide probe (40,000 cpm) end-labeled with [-P]ATP (DuPont NEN) by T4 polynucleotide kinase (New England Biolabs) in a 20-µl binding reaction containing 25 m M HEPES (pH 7.9), 100 m M KCl, 5 m M MgCl, 1 m M EDTA, 1 m M dithiothreitol, 10% glycerol, and 0.5-1.0 µg of poly(dA-dT)/poly(dA-dT) (Pharmacia) as a nonspecific competitor. The 30-bp wild-type synthetic probe used in this study (gatccACACTAAATGAAAGGGCGGGGCAGa) spans the gastrin promoter from -163 to -140 bp and was polyacrylamide gel-purified. Competition experiments were carried out by preincubating the nuclear extract with a 25-125-fold excess of cold competitor oligonucleotides prior to the addition of probe. For Sp1 supershift assays, extracts were preincubated with varying amounts of a polyclonal Sp1 antibody (Santa Cruz) or control rabbit antiserum (12CA5 polyclonal antisera, gift from Anil K. Rustgi) for 10 min. Zinc chelation assays were performed by preincubating the nuclear extracts in buffer containing either 10 or 25 m M EGTA prior to the addition of probe. EGTA was selected for its ability to efficiently chelate Znbut not Mgcations (23) , the latter being a component of the gel shift assay buffer. All samples were then incubated for 10 min at room temperature prior to loading onto a 0.5 TBE, 4% nondenaturing polyacrylamide gel run at 10 V/cm. For oligonucleotide sequences, upper case letters represent gastrin gene sequence and lower case letters represent BamHI or BglII cloning sites added onto the native sequence.

Methylation Interference and DNase I Footprinting Assays

Methylation interference assays were performed as described previously (24) . The P singly end-labeled double-stranded 30-bp WT oligonucleotide was partially methylated with dimethyl sulfate (Aldrich) and then purified with two rounds of ethanol precipitation. 20,000 cpm of probe was incubated with nuclear extracts in a typical gel shift reaction and run on a 4% nondenaturing polyacrylamide gel as described earlier. An autoradiogram of the gel revealed bands corresponding to Sp1 and gAT binding complexes as well as unbound probe, all of which were excised and electroeluted. After piperidine cleavage, the samples representing bound and free probe were resolved on an 8% sequencing gel.

Probes for footprinting assays were derived from the wild-type gAT-Sp1 pT81 plasmid construct. Probes were 3` Klenow end-labeled on the lower strand with all 4 [-P]dNTPs (DuPont NEN) after restriction digestion with BamHI. After complete excision of the labeled fragment by digestion with SalI, 20,000 cpm of gel-purified probe was incubated with gAT-factor-enriched nuclear extract C for 10 min at room temperature in a 100-µl binding reaction containing 10 m M Tris (pH 8.0), 5 m M MgCl, 1 m M CaCl, 2 m M dithiothreitol, 100 m M KCl, and 3 µg of poly(dA-dT)/poly(dA-dT). The samples were digested with varying amounts of deoxyribonuclease I (Promega) for 2 min at 20 °C and then immediately ethanol precipitated with 5 M ammonium acetate and 2.5 µg of yeast tRNA (Sigma). The digested samples were resolved on an 8% sequencing gel.


RESULTS

Linker Scanning Mutagenesis Identifies Important Gastrin Regulatory Elements Between -160 and -141 bp-To identify additional cis-regulatory elements controlling gastrin transcription in islet cells not seen by 5`-deletional analysis, a series of linker scanning mutations was constructed. Prior deletional analysis demonstrated that 200 bp of 5`-flanking DNA of the human gastrin gene contained the regulatory elements that conferred most of the transcriptional activity in gastrin-expressing RIN 38A insulinoma cells (11, 12) . The linker scanning mutations systematically substituted 10-bp blocks from -170 to -61 bp. The 11 linker scanning mutant constructs were confirmed by direct sequencing and then transiently transfected into 38A islet cells. Reporter gene activity was compared to that of the wild-type gas200 pXP2 vector.

Fig. 1 shows the functional domains identified within the proximal 200 bp of the gastrin promoter which regulate transcription in islet cells. The first includes block mutants 2 and 3, which correspond to an AT-rich sequence CTAAATGAAA (-160 to -151 bp) and an Sp1 consensus binding site GGGCGGGGCA (-150 to -141 bp), neither of which has been described previously as an important regulatory element in the gastrin gene. Mutations in these elements led to a 25-30% fall in transcriptional activity. The second domain is defined by block mutant 6 which mutates the CACC element, known to be a transcriptional activator in 38A cells (12) . There was an augmentation of activity with block mutant 7, corresponding to a de-repression of the inhibitory GNE (11) . The final domain spans blocks 9 and 10, which mutate the previously characterized gastrin E-box (11) and another Sp1 binding site which interacts with the gastrin-epidermal growth factor response element (25) . This linker scanning data corroborates earlier analyses which characterized these aforementioned promoter elements. Prior studies, however, had not identified this gastrin AT-rich (gAT) element and adjacent Sp1 consensus site as important regulatory elements.

Sp1 and a Factor Recognizing the AT-rich Sequence Bind to These Adjacent Gastrin Promoter Elements Independently

Gel mobility shift assays were performed by incubating crude nuclear extracts prepared from RIN 38A insulinoma cells with a 30-bp double-stranded wild-type probe encompassing both new elements (gatccACACTAAATGAAAGGGCGGGGCAGa) in order to characterize the islet nuclear proteins binding to the -160 to -141 sequence. Fig. 2 A shows the presence of 3 binding complexes, labeled Ia, Ib, and II ( lane 1). Complexes Ia and Ib were competed by a 125-fold excess of a consensus Sp1 oligonucleotide, suggesting that they represent Sp1 factor binding to the downstream GC-rich element ( lane 2). Complex II was not affected by competition with the GC-rich Sp1 sequence, indicating that it represents binding by a distinct factor to another sequence. However, complex II was competed away by an oligonucleotide containing only the upstream AT-rich sequence ( lane 3). This AT-rich sequence did not compete for binding of the Sp1-like factor found in complex Ia and Ib, again suggesting the factors recognize discrete sites.

Addition of Sp1 antisera to 38A nuclear extracts in gel mobility shift assays supershifted the slower migrating complex Ia (Fig. 2 B, lanes 2-4), confirming that the Sp1 transcription factor does bind to this sequence. A control antibody did not supershift any complex (Fig. 2 B, lane 5). Since Sp1 binding is dependent upon its zinc finger domains, chelation of zinc abolishes Sp1 binding to DNA (26) . Accordingly, addition of the chelator EGTA to the islet nuclear extracts quantitatively reduced complex Ia and Ib formation but not complex II (Fig. 2 C). Significantly, abolition of Sp1 binding by EGTA enhanced complex II formation, suggesting that there was competition between Sp1 binding and the binding of the gAT factor to the adjacent AT-rich sequence.


Figure 2: Gel mobility shift assays with 38A islet cell extracts. A, crude 38A nuclear extract was incubated with 4 fmol of double-stranded P-end-labeled WT probe (gatccACACTAAATGAAAGGGCGGGGCAGa) with or without a 125-fold excess of cold oligonucleotide corresponding to the consensus Sp1 binding site (ATTCGATCGGGGCGGGGCGAGC) in lane 2 or to the AT-rich domain (gatccCTGACACTAAATGAAAGGGa) in lane 3 prior to electrophoresis on a 4% nondenaturing polyacrylamide gel as described under ``Materials and Methods.'' In the oligonucleotide sequences, the lower case letters represent BamHI or BglII restriction sites. B, Sp1 supershift assay was performed with crude 38A nuclear extract preincubated with 2.0, 1.0, or 0.1 µl of a rabbit polyclonal Sp1 antisera ( lanes 2-4) prior to the addition of WT probe. Lane 5 represents the reaction with 1.0 µl of a control rabbit antisera. C, gel shift assay performed in Zn-deficient conditions. Divalent zinc cations were chelated by preincubating crude 38A nuclear extracts with either 10 or 25 m M EGTA prior to the addition of WT probe. D, gel shift assay with varying titrations of the Sp1-enriched phosphocellulose eluate B and gAT factor-enriched DEAE-Sepharose eluate C using full-length WT probe. The amount of protein in each reaction are: Extract B ( lanes 1-4, 6.7 µg; lane 5, 4.5 µg; lane 6, 2.2 µg; lane 7, 0 µg); Extract C ( lane 1, 0 µg; lane 2, 1.3 µg; lane 3, 2.7 µg; and lanes 4-7, 4.0 µg).



Complex Ib may also represent binding by Sp1 or an Sp1-like factor because of the identical sequence specificity, sensitivity to zinc chelation, and chromatographic co-purification with Sp1. However, the anti-Sp1 antisera does not apparently supershift the Ib complex. This may be a consequence of the species difference between the 38A extract (rat) and the antigenic specificity of the antibody (human Sp1), or that complex Ib may represent binding by an immunologically distinct member of the Sp1 family (27, 28) . Nevertheless, complex Ib does not represent simultaneous binding of both Sp1 and gAT factors.

The factors binding in complex I and II could be separated physically by ion-exchange chromatography. Crude nuclear preparations from 38A islet cells were first applied to a cation exchange phosphocellulose column. Subsequent elution with 0.5 M KCl yielded extract B, a fraction enriched for Sp1 (complex I) without contamination by the factor in complex II which binds the AT-rich sequence (Fig. 2 D, lane 1). The gAT factor was enriched by passage of the crude nuclear extract over an anion exchange DEAE-Sepharose column. The subsequent 0.25 M KCl eluate (extract C) contained only the AT-binding factor (Fig. 2 D, lane 7). Reconstitution experiments were performed to test whether various stoichiometric mixtures of the two enriched fractions showed any evidence of cooperative interactions (Fig. 2 D, lanes 2-6). In no combination did a new binding complex appear in gel mobility shift assays. While this is suggestive of mutually exclusive binding by the two factors, it is possible that other complexes form which are not stable during electrophoresis.

Sp1 and gAT Factor Binding Domains Are Directly Contiguous

The nucleotides within the -163 to -140 sequence necessary for DNA binding of these two factors were then localized more precisely. First, the nucleotides necessary for DNA binding of the Sp1 factor were defined utilizing gel mobility shift assays with mutant oligonucleotide competitors which sequentially spanned the sequence from -163 to -140 bp (Fig. 3 A, legend). Utilizing the Sp1-enriched 38A nuclear fraction B in a gel shift assay, specific binding residues were identified (Fig. 3 A). Mutant oligonucleotides M1 and M2 competed as well as the wild-type oligonucleotide ( lanes 2-4). Mutant oligonucleotides M3, M4, and M5 were unable to compete, however, indicating that those mutated base pairs interacted with the Sp1 factor ( lanes 5-7). As expected, an Sp1 consensus oligonucleotide competed away all binding activity to the gastrin probe ( lane 8). These binding interactions were directly confirmed in a methylation interference assay by delineating the specific G and A residues in this gastrin promoter sequence that interact with Sp1 (Fig. 3 B). The nucleotides in the sequence necessary for Sp1 binding are summarized in Fig. 3 C, as defined first by competition analysis and then corroborated by methylation interference.

The nucleotides necessary for binding of the gAT factor were also defined by competition with mutant oligonucleotides. The gAT factor binding site lay adjacent to the Sp1 site but did not overlap with nucleotides necessary for Sp1 binding since oligo M2 failed to compete for gAT factor binding (Fig. 4 A, lane 4) but effectively competed Sp1 binding (Fig. 3 A, lane 4). Conversely, oligos M3, M4, and M5 bound the gAT factor but not Sp1 (Fig. 4 A, lanes 5-7). Furthermore, an oligonucleotide containing a consensus Sp1 sequence did not displace gAT factor binding (Fig. 4 A, lane 8). Oligo M1 competed as well as the wild-type (Fig. 4 A, lane 2), indicating that the critical residues for gAT factor binding were mutated in the M2 oligo. Methylation interference assays were unsuccessful probably due to the relative paucity of G residues in the gAT factor binding domain. DNase I footprinting was therefore performed, which demonstrated footprinting of the gAT factor over the AT-rich sequence (Fig. 4 B, lane 1). The close agreement between the nucleotides for binding shown in the competition assays and the residues footprinted (Fig. 4 C) indicate that the AT-rich element is directly adjacent to the GC-rich sequence necessary for Sp1 binding. Such proximity of these two sites makes steric interference the likely mechanism which prevents simultaneous binding of the two factors to the adjacent sequences.


Figure 4: Characterization of gAT factor binding domain by mutational analysis and DNase I footprinting. A, gel mobility shift assays were performed with gAT-factor enriched extract C and full-length WT probe in the presence of a 25-fold excess of the cold mutant oligonucleotides listed in Fig. 3 B, DNase I footprinting assay of gAT factor binding to a 52-bp BamHI/ SalI fragment Klenow labeled on the minus strand. The bar on the right indicates nucleotide residues protected from digestion by DNase I. C, schematic representation of residues critical for gAT factor binding to the gastrin promoter ( underlined) as defined by mutational gel shift analysis ( X) and DNase footprinting ( O).



Binding of the gAT Factor Blocks Transcriptional Activation by Sp1

To determine the functional significance of this mutually exclusive interaction between these 2 factors, heterologous promoter pT81 reporter genes containing the WT and mutant oligonucleotides listed in Fig. 3 A (WT, M1-M5) were designed. These constructs were transfected into the gastrin-expressing islet cell line RIN 38A and luciferase reporter gene activity was measured. A single copy of the -163 to -140 WT gastrin promoter sequence weakly activated transcription (2.3-fold above the basal enhancerless pT81), indicating that the intact bipartite domain functions as a weak activator in islet cells (Fig. 5). Construct M1, which can bind both the gAT and Sp1 factors ( lane 3, Figs. 3 A and 4 A), behaved similarly to the wild-type, weakly activating transcription. However, construct M2, which cannot bind the gAT factor but can bind Sp1 ( lane 4, Figs. 3 A and 4 A), showed a 4.8-fold increase in activity above the wild-type and an 11.0-fold increase over basal, indicating that the gAT factor functions to inhibit the strong transcriptional activation mediated by Sp1. Constructs M3, M4, and M5 which all bind the gAT factor but not Sp1 ( lanes 5-7, Figs. 3 A and 4 A) only weakly activated transcription to levels comparable to the wild-type construct. These findings suggest that in RIN 38A insulinoma cells, the wild-type gastrin promoter preferentially binds the gAT factor, which behaves as a weak activator. Binding of the gAT factor prevents Sp1 binding to the adjacent sequence, blocking the stronger transcriptional activation mediated by Sp1.

The Gastrin-AT (gAT) Factor Binds an AT-rich Motif Recognized by the Islet Cell Transcription Factor TF-1

To further characterize the gAT factor, competition studies were then performed with AT-rich consensus motifs of known transcription factors expressed in the pancreas and gastrointestinal tract. Fig. 6 indicates that there was only partial competition for gAT factor binding with binding sequences derived from the ubiquitous POU-domain factor Oct-1 (29) , the liver and intestinal homeodomain factors HNF-1 and HNF-3 (30) , and the glucagon G2 element (31) . There was minimal competition with elements for the pancreatic islet homeodomain factors isl-1 (32) and STF-1/idx-1 (33, 34) . Only one competitor completely displaced binding, the ``B'' element found in the elastase I promoter which binds the islet transcription factor TF-1 (35) . The 8-bp core binding sequence in the elastase promoter for TF-1 (ATAAATGA) is nearly identical to the gastrin AT-rich element (CTAAATGA) and both include the important binding residues ( underlined) in the gastrin promoter for the gAT factor (CTAAATGA). DNA sequence elements with this core consensus motif are also found within the insulin promoter (GAAATGA) (35) , and an oligonucleotide competitor containing this insulin sequence competed away gAT factor binding from the gastrin probe as effectively as the elastase B oligo (data not shown).

Prior studies defined the TF-1 factor as islet-cell specific because it was detected in 38A islet cells but not in acinar AR42J cells or fibroblast Rat 2 cells (35) . In order to characterize the tissue distribution of the gAT factor, gel shift studies were performed with nuclear extracts from these cell lines utilizing the wild-type gastrin probe. Fig. 7 indicates that the identical gAT factor complex is identifiable in both 38A and AR42J cells but not in Rat 2 cells, suggesting that the gAT factor is therefore distinct from TF-1. These differences in electrophoretic patterns were also observed when the elastase B element and gastrin wild-type probes were run in parallel gel mobility shift assays with 38A and AR42J nuclear extracts (data not shown). Furthermore, a survey of additional cell lines revealed that binding activity was also detected in the ductular pancreatic cell line Panc-1 and weaker activity was observed in the hepatic cell line HepG2.


DISCUSSION

This study has identified additional cis-regulatory elements within the gastrin promoter lying between -163 and -140, upstream of previously characterized regulatory sequences (11, 14, 25) . The proximal 200 base pairs of the gastrin promoter contain multiple positive and negative cis-regulatory sequences, and scanning mutagenesis studies indicate that no single element is essential for transcription but rather that transcriptional activity is dependent upon the combined action of the CACC, E-box, and proximal and distal Sp1 positive cis-regulatory elements. Adjacent to each of these positive sites are negative cis-regulatory elements which modulate the activity of these activating elements. Transactivation by the CACC and E-box binding factor is antagonized by a factor binding to the GNE (11) . Sp1 transactivation via the proximal Sp1 site (-68) is inhibited by factors interacting with the adjacent epidermal growth factor-response element (36) . Similarly, the present study demonstrated that Sp1 transactivation at the distal Sp1 site (-150) is inhibited by a competing factor binding to an adjacent AT-rich site.

Although the gAT factor has not been fully characterized, it binds a TAAT motif recognized by many homeodomain transcription factors (37, 38) . Homeodomain factors often regulate tissue-specific gene expression and in islet cells, isl-1 and STF-1/idx-1 are well characterized pancreatic endocrine homeodomain factors which regulate insulin and somatostatin gene expression, respectively (32, 33, 34) . However, the gAT factor displayed minimal affinity for these homeodomain factor binding sites in gel mobility shift assays. Rather, the gAT factor recognized a sequence bound by the islet cell nuclear protein TF-1. Although their sequence specificity is similar, the gAT binding factor differs from TF-1 in electrophoretic mobility and cell specificity in gel shift assays. Interestingly, TF-1 was first identified as a factor which binds the B cis-regulatory element (ATAAATGA) in the enhancer of the elastase I gene (35) . Although the elastase gene is specifically expressed in pancreatic exocrine cells, a transgene comprising multiple copies of the isolated TF-1 element is expressed only in islet -cells (35) . The islet cell factors binding to the TF-1 sequence also activate insulin gene transcription via a TF-1-like element found within the insulin enhancer (35) . Multiple copies of both the isolated elastase and insulin TF-1 elements strongly activate islet-specific transcription of a basal promoter reporter gene in RIN 38A cells (35) . In contrast, 5 tandem copies of the gastrin-AT element subcloned into the minimal promoter construct pT81-Luc activated transcription only a modest 2-fold above basal levels in RIN 38A cells (data not shown). Collectively, these results indicate that the gAT factor is not TF-1 even though both bind similar DNA sequences. Further characterization of both TF-1 and the gAT binding factor will be necessary to more precisely define their relationship.

Transactivation of gastrin transcription by Sp1 appears to be attenuated by binding of the gAT factor to the adjacent AT-rich sequence. Down-regulation of Sp1 transactivation by steric effects of neighboring transcription factors has been shown to be important in regulating transcription of other genes. In the collagen (I) gene promoter, the GC-rich Sp1 binding site directly overlaps with the CCAAT box binding site such that Sp1 and nuclear factor-1 interactions are mutually exclusive (39, 40) . Although each factor can activate the collagen gene independently, nuclear factor-1 functions as an inhibitor of Sp1-mediated transcriptional activation. Other examples of transcriptional regulation mediated by the displacement of Sp1 by competing factors include the ornithine decarboxylase gene (41) and the -globin gene (42) . A more complex interaction has been described between Sp1 and the pituitary-specific homeodomain factor Pit-1 in the regulation of the growth hormone gene (43, 44) . Binding studies indicate that their interactions are also mutually exclusive. Although they do not bind simultaneously, maximal growth hormone gene activity occurs when the binding sites for both factors are intact, indicating that transcriptional regulation is mediated by sequential rather than simultaneous binding of the two factors.

Activation of gastrin transcription in islet cells is strongly dependent upon the ubiquitous transactivator Sp1 even though gastrin gene expression is highly cell-specific and temporally restricted to the neonatal period. Although negative regulation is probably a highly important determinant of this tissue and temporal specificity, Sp1 levels can vary dramatically in different developmental stages (45) . In the mouse, it is expressed at highest levels in tissues undergoing final differentiation, such as in spermatids or hematopoietic cells. Lower levels are seen in terminally differentiated cells. Within the gastrointestinal tract, expression in the stomach is high at the time of weaning and persists throughout adult life (45) . Since there is continuous differentiation of gut endocrine cells from the population of endodermal stem cells throughout adult life, the concurrent high level of Sp1 expression may be relevant. The developmental expression of Sp1 within the pancreas has not been examined but such studies would be of great interest. The developmental regulation of Sp1 may thus influence the developmental expression of Sp1-regulated genes, such as gastrin.

Thus far, two Sp1 binding sites have been identified in the gastrin promoter. Sp1 binding to a proximal G-rich element (-68 to -53) is an important component of epidermal growth factor-stimulated gastrin gene expression in rat pituitary GH4 cells, as described previously (25) . As the current report demonstrates, an additional upstream Sp1 binding site is strongly activating in gastrin-expressing islet cell lines. Mutations in either Sp1 site (Fig. 1, constructs 1 and 10) are associated with diminished transcriptional activity compared to the wild type. Prior studies have established that Sp1 binding at multiple elements has a synergistic effect on transcriptional activation (46) . One proposed mechanism for such long range interactions involves DNA looping mediated by tetramers of Sp1 binding to proximal and distal enhancers simultaneously (47) . Such cooperative interactions between Sp1 elements may similarly play a role in activating gastrin transcription during islet cell differentiation. Inhibiting binding at just one of these Sp1 sites by an islet cell factor binding to the adjacent gAT site may be a critical determinant in switching off gastrin transcription. Such an interaction may explain why repression of gastrin expression coincides with full differentiation of the pancreatic islets, when many pancreas-specific transcription factors are maximally expressed. Future studies will need to focus on identifying the relative levels of these different factors at different times in pancreatic development. In addition to elucidating the molecular mechanisms controlling pancreatic development, these studies may provide insight into the reactivation of gastrin expression with the neoplastic transformation of pancreatic islets.


Figure 1: Linker scanning mutagenesis of the human gastrin 5`-flanking region. 11 linker scanning mutant gas200 pXP2-luciferase constructs as described under ``Materials and Methods'' were transfected with DEAE-dextran into 38A islet cells and luciferase activity was assayed at 48 h. The XX in the promoter map represents the 10-bp block substitution. Luciferase activity is expressed as % activity of the wild-type gas200 pXP2 construct (WT) and represents the mean ± S.D. of 4-6 samples. Each transfection experiment was repeated at least 3 times.




FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK07191, DK42147, and DK01410. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an American Gastroenterological Association/Merck Senior Fellowship Research Award. To whom correspondence should be addressed: Gastrointestinal Unit, Jackson 7, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-3766; Fax: 617-726-3673.

Present address: Myogenics, 38 Sidney St., Cambridge, MA 02139.

**
Supported by an American Gastroenterological Association/Pharmacia research scholarship award. Present address: Div. of Digestive Diseases, University of North Carolina, Chapel Hill, NC 27599.

The abbreviations used are: bp, base pair(s); WT, wild type; gAT, gastrin-AT; RIN, rat insulinoma; GNE, gastrin negative element.


ACKNOWLEDGEMENTS

We thank Drs. Timothy C. Wang and David A. Brenner for critical review of the manuscript and helpful advice, Philip J. Davis for technical assistance, and Dr. Anil K. Rustgi for antisera. Oligonucleotides were provided by the Center for the Study of Inflammatory Bowel Disease at the Massachusetts General Hospital.


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