Hepatocyte nuclear factor-4 regulates intestinal expression of the guanylin/heat-stable toxin receptor

E. Scott Swenson, Elizabeth A. Mann, M. Lynn Jump, and Ralph A. Giannella

Division of Digestive Diseases, Veterans Affairs Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have investigated the regulation of gene transcription in the intestine using the guanylyl cyclase C (GCC) gene as a model. GCC is expressed in crypts and villi in the small intestine and in crypts and surface epithelium of the colon. DNase I footprint, electrophoretic mobility shift assay (EMSA), transient transfection assays, and mutagenesis experiments demonstrated that GCC transcription is regulated by a critical hepatocyte nuclear factor-4 (HNF-4) binding site between bp -46 and -29 and that bp -38 to -36 were essential for binding. Binding of HNF-4 to the GCC promoter was confirmed by competition EMSA and by supershift EMSA. In Caco-2 and T84 cells, which express both GCC and HNF-4, the activity of GCC promoter and/or luciferase reporter plasmids containing 128 or 1973 bp of 5'-flanking sequence was dependent on the HNF-4 binding site in the proximal promoter. In COLO-DM cells, which express neither GCC nor HNF-4, cotransfection of GCC promoter/luciferase reporter plasmids with an HNF-4 expression vector resulted in 23-fold stimulation of the GCC promoter. Mutation of the HNF-4 binding site abolished this transactivation. Transfection of COLO-DM cells with the HNF-4 expression vector stimulated transcription of the endogenous GCC gene as well. These results indicate that HNF-4 is a key regulator of GCC expression in the intestine.

Escherichia coli heat-stable enterotoxin; gene transcription


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE GUANYLYL CYCLASE C (GCC) gene encodes the receptor for Escherichia coli heat-stable enterotoxin (STa) (31) and for the endogenous peptides guanylin and uroguanylin (7, 12). In most adult mammals, GCC is expressed primarily in the gastrointestinal tract. In response to ligand binding, the intracellular domain of GCC catalyzes formation of cGMP, resulting in inhibition of Na+ and H2O absorption and stimulation of cystic fibrosis transmembrane conductance regulator-dependent Cl- secretion (10). Activation of GCC by STa can cause severe secretory diarrhea, a major cause of infant mortality in developing countries (11). The pathophysiological role of GCC is well established, and, although it seems likely that the physiological function of GCC is to regulate electrolyte balance in the gut, mice lacking GCC do not exhibit overt alteration of intestinal function (23, 32).

We have previously determined that GCC is expressed at high levels throughout the epithelium of the mouse intestine, from duodenum to colon (36). GCC mRNA is abundant in both crypts and villi of the small intestine, as well as in the crypts and surface epithelium of the colon. A similar crypt-villus distribution was seen by in situ hybridization of GCC mRNA in human small intestine and colon (unpublished observations) and by in situ 125I-labeled STa receptor autoradiography (16). Low levels of GCC mRNA were detected in neonatal and weanling mouse livers (36) and in fetal, neonatal, and regenerating rat livers (18, 19). The function of hepatic GCC expression is unknown.

The complex developmental and tissue-restricted pattern of GCC expression suggests that GCC could serve as a valuable model for the study of intestine-specific gene expression. It differs from the more extensively studied sucrase-isomaltase and intestinal and liver fatty acid binding protein genes, which are expressed primarily in differentiated villus enterocytes of the small intestine (20, 30). The GCC gene may contain distinct, potentially novel, regulatory elements that direct expression to the small intestinal and colonic crypts, as well as to villus or surface epithelium.

Our previous studies demonstrated intestinal cell line-specific transcriptional activity within the proximal 128 bp of human GCC 5'-flanking sequence; a minimum of two additional positive-acting elements reside farther upstream (22). Subsequent cloning (24) and sequencing of the 5' end of the mouse GCC gene revealed significant identity with the human GCC promoter. The similarity of the expression pattern between mouse and human and the high degree of sequence conservation in the promoter suggest that essential cis-regulatory sequences may reside in this region. The sequence of the conserved human promoter suggests possible binding sites for a number of transcription factors, including hepatocyte nuclear factor-4 (HNF-4).

HNF-4, a member of the nuclear hormone receptor superfamily of zinc-finger transcription factors, has been shown to regulate several genes that are expressed in the liver and intestine (34). The studies reported here indicate that the GCC promoter contains a critical HNF-4 binding site, between bp -46 and -29, that is necessary for maximal expression of GCC in intestinal cell lines. Electrophoretic mobility shift assays (EMSAs) and transient transfection assays using GCC promoter and/or luciferase reporter plasmids demonstrate that HNF-4 binds this element and regulates GCC transcription. In addition, transient transfection of COLO-DM cells (which express neither HNF-4 nor GCC) with an HNF-4 expression vector stimulates transcription of the endogenous GCC gene. These results strongly support our hypothesis that HNF-4 is an important regulator of GCC expression in the intestine.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and sequencing of the human and mouse GCC 5' nontranscribed region. The cloning, sequencing, and preliminary transcriptional analysis of the human and mouse GCC 5' region have been previously described (22, 24). Nucleotide sequences have been submitted to the GenBank and European Molecular Biology Laboratories database with accession numbers U20230 (human) and AF027301 (mouse). The human GCC promoter constructions previously described (22) were used, with one modification. Promoter fragments were cloned into the pGL3-basic luciferase vector (Promega, Madison, WI) to increase the assay sensitivity over the previously used pGL2-basic vector. To accomplish this transfer, promoter fragments were excised from GCC promoter/pGL2-basic constructs at the Hind III and Sst sites, gel purified, and ligated into the Hind III-Sst I sites in the polylinker of pGL3-basic. Correct transfer of the promoter fragment into pGL3-basic was confirmed by restriction digest and partial sequencing. Two GCC promoter fragments were used in these studies: -128/+120 and -1973/+124. Cloning of these fragments into pGL3-basic resulted in plasmids (-128/+120)Luc and (-1973/+124)Luc.

Mutagenesis of the HNF-4 site in the GCC promoter. A 3-bp mutation (TTG to GCA) was introduced into the putative HNF-4 binding site (at -38 to -36) by a PCR-based strategy, using primers that amplified nt -288 to +162 (sense primer 5'-TAAGGATTTCGTTTCAATGTGC-3', antisense primer 5'-AACCATACTCCTTGTGCC-3'), a phosphorylated mutagenic oligonucleotide (mutant 1, Table 1), and a previously cloned and sequenced 2.8-kb genomic Xba I fragment (22) as template.

                              
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Table 1.   Wild-type and mutant GCC sequences

Construction of plasmids. The PCR product described above was cloned into the EcoR V site of pBluescript IIKS (Stratagene, La Jolla, CA), resulting in plasmid (-288/+162/MutHNF4)KS. This plasmid was digested with Nsi I and Sty I to release fragment -128/+120/MutHNF4. The 248-bp fragment was gel purified, blunt ended, and cloned into the EcoR V site of pBluescript IIKS. The -128/+120/MutHNF4 promoter fragment was excised from plasmid (-128/+120/MutHNF4)KS at flanking Hind III and Sst I sites, gel purified, and ligated into the polylinker of pGL3-basic at the Hind III and Sst I sites, generating plasmid (-128/MutHNF4)Luc containing a TTG-to-GCA mutation at nt -38 to -36.

Plasmid (-288/+162/MutHNF4)KS was cut with Afl II and Sty I, releasing fragment (-257/+120/MutHNF4). This fragment was then cloned into the plasmid (-1973/+124)Luc, replacing nt -257/+120 with -257/+120/MutHNF4. This cassette replacement strategy resulted in a TTG-to-GCA mutation at nt -38 to -36 in the context of the (-1973/+124)Luc reporter construct.

Correct introduction of the HNF-4 binding site mutation at bp -38 to -36 and the absence of secondary (unintended) mutations were verified by PCR cycle sequencing (University of Cincinnati, DNA Core Facility).

Cell lines. Caco-2 cells were a gift from Dr. Alain Zweibaum, Institut National de la Recherche Medicale, Villejuif Cedex, France. Hep G2 cells were a gift from Dr. Judith Harmony, University of Cincinnati. COLO-DM, NIH/3T3, and T84 cells were obtained from American Type Culture Collection (Rockville, MD).

Cell culture and DNA transfections. Caco-2 and COLO-DM cells (both derived from human colon carcinomas) were grown in DMEM supplemented with 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 atmosphere. NIH/3T3 cells were grown in DMEM supplemented with 10% FCS, with penicillin and streptomycin as above. T84 cells (derived from human colon carcinoma) were grown in DMEM-Ham's F-12 medium with 10% FCS plus penicillin and streptomycin. Hep G2 cells were grown in minimum essential medium supplemented with 10% FCS plus penicillin and streptomycin. For reporter assays, 3 × 105 Caco-2 cells, 106 T84 cells, or 106 COLO-DM cells were plated per well in six-well plates 24 h before transfection. Cell density was ~40-50% of confluence at the time of transfection. All transfections were performed by calcium phosphate coprecipitation (4). COLO-DM and Hep G2 cells were harvested 48 h after transfection. Caco-2 and T84 cells were harvested 72 h after transfection.

For reporter assays in Caco-2 and T84 cells, 4.5 µg (or the molar equivalent) of GCC promoter/luciferase plasmid plus 1 µg of RSV110 beta -galactosidase (to serve as a control for transfection efficiency) were used, with pBluescript II as necessary to bring the total DNA to 5.5 µg/well. For cotransfection experiments in COLO-DM cells, 3 µg of reporter (or its molar equivalent), 1 µg of pMT2-HNF4 (gift of Dr. Frances Sladek, University of California at Riverside) or a molar equivalent of pMT2, 1 µg of RSV110 beta -galactosidase to correct for transfection efficiency, and an appropriate amount of pBluescript II to bring the total amount of DNA to 5.5 µg/well were used. Cells were harvested by lysis in reporter lysis buffer (Promega) and centrifuged at 14,000 g, and luciferase activity was assayed (luciferase assay system, Promega) from the supernatant. beta -Galactosidase activity was determined (GalactoLight Plus, Tropix, Bedford, MA) to normalize for variation in transfection efficiency. For harvest of RNA from COLO-DM, 107 cells were plated in 100-mm dishes and transfected with 10 or 25 µg pMT2-HNF4 or the molar equivalent of empty vector, pMT2. RNA was isolated from nontransfected Caco-2 and Hep G2 cells harvested at confluence.

RNA isolation and RT-PCR. Total RNA was prepared by the acid guanidinium phenol/chloroform method (6). For RT-PCR analysis of GCC expression in HNF-4-transfected COLO-DM cells, 2 µg of total RNA were reversed transcribed (SuperScript II reverse transcriptase, Life Technologies) using an antisense primer corresponding to nt 3199-3180 (5'-CCTTGTCTGTGGTATTCAGC-3') and then amplified by PCR for 30 cycles (sense primer: nt 2782-2801, 5'-CTGGAGTTGTGGGAATCAAG-3') in a Perkin-Elmer Cetus thermocycler. The PCR conditions were as follows: denaturation for 1 min at 95°C, annealing for 1 min at 62°C, and extension for 1 min at 72°C, with 5 units AmpliTaq Gold polymerase (Perkin-Elmer). Amplified products were then digested with Hinc II (which cleaves the GCC cDNA at nt 2839) and separated on a 1.8% agarose gel.

Nuclear extract preparation. Nuclear extracts were prepared from NIH/3T3, COLO-DM, or postconfluent Caco-2 cells using a modified (1) mini-extract procedure (29). Cells were rinsed with PBS, scraped, transferred to microcentrifuge tubes, and pelleted by centrifugation at 3000 g. Cells were resuspended in lysis buffer [composed of 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% NP-40, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. Nuclei were pelleted by centrifugation, rinsed with lysis buffer without NP-40, repelleted, and then resuspended in extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF). After 10 min on ice, the extracted proteins in the supernatant were separated from insoluble nuclear debris by centrifugation at 14,000 g. After extraction, nuclear protein was dialyzed against 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, and 0.5 mM PMSF and stored at -70°C. Protein concentration was determined by a modified Lowry method (Bio-Rad DC, Bio-Rad, Hercules, CA).

Footprint assay. An oligonucleotide corresponding to bases +162 to +145 (5'-AACCATACTCCTTGTGCC-3') was labeled by T4 polynucleotide kinase and [gamma -32P]ATP (NEN, Boston, MA). This oligomer was then incorporated into a 334-bp PCR product (sense primer 5'-GATGTATTGCCCTCTTTCTC-3') encompassing bp +162 to -172, labeled on the 5' end of the noncoding strand. The PCR conditions were as follows: denaturation for 1 min at 95°C, annealing for 1 min at 60°C, and extension for 1 min at 72°C, with 5 units AmpliTaq Gold polymerase (Perkin-Elmer), for 35 cycles. Unincorporated nucleotides were removed by a BioSpin 6 minicolumn (Bio-Rad). Crude nuclear protein (20 µg) was incubated with probe (20,000 counts/min) using 0.5 µg of poly(dI/dC) as nonspecific competitor. After digestion with 0.01 or 0.05 units of DNase I (Pharmacia, Piscataway, NJ), protein was digested with proteinase K, and the DNA was extracted with phenol/chloroform and precipitated in ethanol with tRNA as carrier. After centrifugation and resuspension in formamide-loading buffer, digested products were separated on an 8 M urea-6% polyacrylamide sequencing gel. Sequencing reactions (Sequenase, US Biochemicals, Cleveland, OH) were performed using the +162 to +145 primer and alpha -35S-labeled dATP (NEN). The template for both the probe synthesis and the sequencing reactions was a previously sequenced cloned genomic Xba I fragment. The gel was dried onto Whatman paper and autoradiographed (Biomax MS) for 3 days at -70°C.

Electrophoretic mobility shift assay. Oligonucleotides containing bases from -50 to -20 of the GCC promoter (as wild type or with specific mutations, see Table 1) in both orientations were synthesized, annealed, and labeled by T4 polynucleotide kinase and [gamma -32P]ATP (NEN). After removal of unincorporated label (BioSpin 6 minicolumn, Bio-Rad), 0.5 µg of nuclear protein was allowed to bind the labeled probe (50,000 counts/min, ~35 fmol) in the presence of 0.25 µg of poly(dI/dC) as nonspecific competitor, in a 20-µl reaction at room temperature for 20 min. For supershift reactions, polyclonal rabbit anti-rat HNF-4 antiserum (provided by Dr. Frances Sladek, University of California at Riverside) was diluted 1:5 in PBS-3% BSA; 1 µl of diluted antiserum was added to the binding reaction halfway through the 20-min incubation. Bound and unbound probe were separated on 5% polyacrylamide gels at 4°C, in 0.25× Tris-borate-EDTA. The gels were dried onto Whatman paper and autoradiographed overnight at -70°C.

Oligonucleotides containing known HNF-4 binding sites from the alpha 1-antitrypsin (13) or hepatocyte growth factor-like protein gene (38) were generously provided by Dr. Susan Waltz (Children's Hospital Medical Center, Cincinnati, OH). Oligomers containing binding sites for SP1 (2) or GATA-4 (14) were synthesized based on published sequences.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human and mouse GCC promoters are highly conserved. We have previously isolated partial genomic clones of both human (22) and mouse (24) GCC. Sequence determination of the mouse promoter revealed that the proximal 290 bp are 78% identical to the human promoter (Fig. 1). Transfection experiments using human GCC reporter constructs (22) demonstrated equivalent activity with -257GCC/Luc and -129GCC/Luc, suggesting that only the proximal 129 bp are required for GCC promoter activity in vitro. A number of consensus binding sites for known transcription factors are conserved between the two promoters in this region and are thus potential regulators of GCC transcription.


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Fig. 1.   Alignment of the 5' nontranscribed domain of the human and mouse guanylyl cyclase C (GCC) genes. Transcription start site (+1) for human GCC was previously determined (14). Alignment of human and mouse promoters is inferred on the basis of sequence homology. Nonidentical bases in the mouse GCC promoter are indicated by lowercase letters. Gaps introduced to optimize the alignment are indicated by dashes. Potential transcription factor binding sites are boxed. C/EBP, CCAAT/enhancer binding proteins; cdx-2, homeodomain protein related to caudal; HNF-4, hepatocyte nuclear factor-4; GATA, GATA-binding factors.

DNase I footprint and EMSA suggest HNF-4 binding to the GCC promoter. DNase I footprint analysis was performed to assess the position of cis-regulatory elements in the conserved GCC proximal promoter. With the use of nuclear extract prepared from the human Caco-2 intestinal cell line that expresses GCC, the assay (Fig. 2) indicated three regions of protection corresponding to bases at site A (-9 to -21), site B (-29 to -46), and site C (-65 to -81). Protected bases at site A include a nonconsensus TATA sequence (CATAAC) at -22 to -17 (Fig. 1), suggesting that site A reflects protection by TATA-binding protein and basal transcription apparatus. Sequence analysis of site B (-44 to -31, 5'-TGAACTTTGGTTTA) indicated homology with HNF-4 binding sites (5'-TGAACTTTGAACTT). This potential HNF-4 binding site is highly conserved in the corresponding region of the mouse GCC promoter. A nonconserved GATA factor binding site (-28 to -33) is also present within the site B footprint. Site C includes a potential cdx-2 binding site at -81 to -75 and also shows partial sequence identity (at bp -73 to -56) to a heptad repeat sequence in the liver fatty acid binding protein gene, which plays a role in colon-specific transgene expression (33).


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Fig. 2.   DNase I footprint analysis of the proximal GCC promoter. Probe contained bases +162 to -172, labeled on the noncoding strand. Dideoxy sequencing reaction (left) using a +162 to +145 primer allows identification of bases protected by Caco-2 nuclear extract (right), in comparison to nonspecific protection by BSA (middle). A hypersensitive site is visible just upstream of site B at -50.

EMSA suggests HNF-4 binding to the GCC promoter. The binding of nuclear proteins to site B was further examined by EMSA, comparing results with Caco-2 cell nuclear extracts to nuclear extracts from COLO-DM and NIH/3T3 cells, which do not express GCC. An oligonucleotide probe containing bp from -50 to -20 of human GCC formed a unique specific complex with crude nuclear extract from Caco-2 (Fig. 3A, lanes 2-4) but not COLO-DM (lanes 5-7) or NIH/3T3 (lanes 8-10). Formation of the Caco-2-specific complex could be blocked by addition of 20-fold molar excess of unlabeled -50/-20 GCC (lane 3) but not by 20-fold excess of nonspecific competitor oligomer containing an SP1 binding site (lane 4). In addition to the Caco-2-specific complex, there were two more rapidly migrating complexes seen with all three nuclear extracts.


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Fig. 3.   A: Caco-2-specific complex is seen by electrophoretic mobility shift assay (EMSA) with the -50 to -20 region of GCC. Arrow indicates a specific DNA-protein complex seen only with Caco-2 nuclear extract (lanes 2-4). Faster migrating complexes are also seen that are present with COLO-DM and NIH/3T3 extracts (lanes 5-10). All complexes are competed by unlabeled self oligomer (S) but not by an unrelated oligomer (NS) containing an SP1 binding site. Competitors are present at 20-fold molar excess. B: an intact HNF-4 site is required to compete Caco-2-specific complex formation. Competition of -50/-20 GCC-Caco-2 complex by wild-type -50/-20 self oligomer (S, lane 3), -50/-20 GCC with HNF-4 site mutation (m1, lane 4), GATA-4 binding site from the troponin C gene (G, lane 5), -50/-20 GCC with GATA site mutation (m2, lane 6), and SP1 binding site oligomer (NS, lane 7) is shown. Lane 1 is control. The complex is not seen with extract from COLO-DM cells (lane 8), which do not express GCC. Competitors are at 20-fold excess. C: GCC HNF-4 site competes for binding to the alpha 1-antitrypsin gene HNF-4 binding site. Competition of alpha 1-antitrypsin gene HNF-4 binding site complex (lane 2) with self competitor (AT, lane 3), -50/-20 GCC (GC, lane 4), and SP1 binding site oligomer (NS, lane 5) is shown. Lane 1 is control. Competitors are at 30-fold molar excess. D: supershift of Caco-2-specific complex by HNF-4 antiserum. Arrows indicate the positions of the -50/-20 GCC-specific complex before (lane 2) and after (lane 3) addition of HNF-4 antiserum, illustrating the decrease in complex mobility (supershift). Lane 1 is control.

The -50/-20 GCC probe contains potential binding sites for HNF-4 and GATA factors so mutant oligomers were designed to disrupt each potential binding site independently (Table 1 and Fig. 3B). Mutant 1 contains a 3-bp mutation in the putative HNF-4 site at -38 to -36. Mutant 2 contains a 2-bp mutation in the potential GATA factor binding site at -30 to -29. Formation of the Caco-2-specific complex (Fig. 3B, lane 2) could be abolished by addition of 20-fold molar excess unlabeled wild-type GCC (lane 3) or mutant 2 competitor (lane 6), which has a mutant GATA site and a wild-type HNF-4 site. Formation of the Caco-2-specific complex was poorly competed by mutant 1 (lane 4), which contains a mutation within the putative HNF-4 binding site (Table 1) and was not competed by an oligomer containing an SP1 binding site (lane 7). A GATA-4 binding site from the cardiac troponin C gene (15) (lane 5) was not able to compete for Caco-2-specific complex formation. Furthermore, with the use of Caco-2 nuclear extract, complex formation with the radiolabeled mutant 1 (HNF-4 site mutant) probe was severely impaired, whereas labeled mutant 2 (GATA site mutant) formed a complex of the same mobility and intensity as that seen with the wild-type probe (data not shown). On the basis of these results, the Caco-2-specific complex most likely represents binding of HNF-4 (or a closely related factor) rather than GATA factor(s) to the -50/-20 GCC region.

EMSA was used to determine whether the -50/-20 GCC oligomer could compete for HNF-4 binding to known HNF-4 binding sites. When an oligomer containing the alpha 1-antitrypsin gene HNF-4 binding site (13) was used as radiolabeled probe (Fig. 3C), addition of either unlabeled alpha 1-antitrypsin (lane 3) or -50/-20 GCC oligomer (lane 4) resulted in competition of the Caco-2-specific complex. Similar results (not shown) were seen with a probe containing the HNF-4 binding site from the hepatocyte growth factor-beta -like protein gene (38). Direct evidence of HNF-4 binding to -50/-20 GCC is shown in Fig. 3D. Addition of HNF-4 antiserum caused a supershift of the Caco-2-specific complex (lane 3 vs. lane 2) without changing the mobility of the smaller complexes.

HNF-4 binding site is critical for GCC promoter activity in Caco-2, T84, and Hep G2 cells. Caco-2 and T84 are human intestinal cell lines that express both GCC and HNF-4. Both (-128/+120)Luc and (-1973/+124)Luc reporter plasmids were highly active in Caco-2 and T84 cells (Fig. 4). The larger promoter construct had greater transcriptional activity, confirming the presence of additional positive regulatory sequences between -128 and -1973, which we have previously reported (22). Introduction of the 3-bp mutation into the HNF-4 binding site at -38 to -36 in either promoter construct (-128/MutHNF4)Luc or (-1973/MutHNF4)Luc dramatically reduced GCC promoter activity in both cell lines. This result suggests that upstream positive regulatory element(s) requires a functional HNF-4 binding site in the proximal GCC promoter. In the Hep G2 liver cell line, which expresses HNF-4 but not GCC, detectable transcriptional activity was seen with both constructs. This activity was attenuated by mutation of the HNF-4 site. In contrast to the intestinal cell lines, both the small and large promoter constructs were equally active in Hep G2 cells. This suggests that, in Hep G2 cells, HNF-4 drives transcription from both promoters to a limited extent and that additional transacting factors required for maximal expression are present in intestinal cells but not Hep G2 cells. Alternatively, Hep G2 cells may contain factors that repress GCC transcription, even in the presence of HNF-4.


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Fig. 4.   Effect of HNF-4 binding site mutagenesis on transcriptional activity of GCC/Luc reporter plasmids in Caco-2, T84, and Hep G2 cells. Caco-2 and T84 cell lines express endogenous GCC and HNF-4. Hep G2 cells express HNF-4 but not GCC. Cells were transfected with plasmids containing luciferase driven by no promoter (LUC BASIC), SV40 promoter (SV40), or a GCC promoter element, (-128/+120)Luc or (-1973/+124)Luc. -128/MutHNF4 and -1973/MutHNF4 contain a 3-bp mutation (m1, TTG to GCA) at -38 to -36. Data represent the luciferase-to-beta -galactosidase ratio and are expressed as "fold" activation relative to promoterless luciferase. n = Number of independent transfections for each construct.

GCC promoter is activated by HNF-4 in COLO-DM cells. The COLO-DM cell line, derived from a human colon adenocarcinoma, expresses markers characteristic of enteroendocrine cells, such as norepinephrine, serotonin, and neuropeptide Y (28). Despite their intestinal origin, COLO-DM cells express neither GCC (see Fig. 6) nor HNF-4 (Northern blot, data not shown). To assess the ability of HNF-4 to activate the GCC promoter in a nonexpressing intestinal cell line, GCC reporter constructs were cotransfected with HNF-4 expression vector (pMT2HNF4) in COLO-DM cells (Fig. 5). GCC promoter constructs (-128/+120)Luc and (-1973/+124)Luc were activated ~23-fold by HNF-4, whereas promoterless luciferase (basic) and SV40-luciferase were not, indicating a specific effect of HNF-4 on GCC promoter activity. Incorporation of the 3-bp mutation at bp -38 to -36 (Table 1) into either reporter construct (-128/MutHNF4)Luc or (-1973/MutHNF4)Luc abolished the transactivation effect of pMT2-HNF4 (Fig. 5).


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Fig. 5.   Potentiation of GCC promoter activity by HNF-4 in COLO-DM cells. COLO-DM cells do not express either GCC or HNF-4. GCC/luciferase constructs are identical to those in Fig. 4. Each reporter construct was cotransfected with the HNF-4 expression vector (pMT2HNF4, solid bars) or empty vector (pMT2, open bars). Data represent the ratio of luciferase to beta -galactosidase activities and are expressed as fold activation for each construct. The number of independent transfections for each construct is indicated in parentheses.

The striking activation of the GCC promoter by HNF-4 led us to investigate whether HNF-4 could activate transcription of the endogenous GCC gene in COLO-DM cells. Total RNA was isolated from COLO-DM cells transiently transfected with pMT2-HNF4 or empty pMT2 vector. Northern blot (not shown) and RT-PCR analysis (Fig. 6) demonstrated that GCC could be detected in COLO-DM cells transfected with 25 µg of pMT2-HNF-4 but not in vector-transfected COLO-DM cells or nontransfected Hep G2 cells. A diagnostic Hinc II digest verified the identity of the RT-PCR product.


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Fig. 6.   HNF-4 activates endogenous GCC transcription in COLO-DM cells. GCC mRNA was detected by RT-PCR (418-bp product) only with RNA from COLO-DM cells transfected with 25 µg of HNF-4 (lane 5) and with Caco-2 RNA (lane 8). Authenticity of this product was confirmed by digestion with Hinc II, which cleaves 57 bp from the 418-bp GCC product (lanes 6 and 9). In lanes 1, 4, 7, and 10, reverse transcriptase was omitted to show that amplification was not due to contaminating DNA. The faint band in lanes 2, 3, 11, and 12 is a nonspecific product that is not the correct 418 bp in size. Molecular weight size markers (end lanes) are as indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The experiments described herein strongly support the hypothesis that intestinal expression of GCC is regulated by HNF-4. DNase I protection and EMSA suggest that HNF-4 binds the GCC promoter between bp -46 and -29 and that bp -38 to -36 are critical for binding. Competition EMSA and antibody supershift using HNF-4 antiserum confirm binding of HNF-4. GCC promoter activity in Caco-2 and T84 cells is highly dependent on the HNF-4 binding site. Cotransfection with HNF-4 expression vector strongly activates GCC/luciferase reporters in COLO-DM cells, which express neither GCC nor HNF-4, and disruption of the HNF-4 binding site abolishes transactivation by HNF-4. Finally, transfection of COLO-DM cells with HNF-4 was sufficient to activate transcription of the normally silent endogenous GCC gene in COLO-DM cells.

HNF-4 occupies an important position in a regulatory cascade that affects the transcription of many genes (34). An HNF-4 binding site in the promoter of the HNF-1alpha transcription factor gene directly regulates its expression in liver, intestine, and other tissues. Therefore, HNF-4-mediated transactivation of a promoter may occur indirectly through induction of another transcription factor. Although there are no binding sites for HNF-1alpha in the promoter constructs we used for transfection, the endogenous GCC gene may contain HNF-1alpha binding sites in regions we have not yet examined. However, our observation that HNF-4-mediated transactivation of GCC promoter/luciferase constructs was abolished by mutation of the HNF-4 binding site suggests that HNF-4 activates GCC transcription, at least in part, through a direct effect on the HNF-4 site in the proximal promoter. Two additional potential HNF-4 binding sites are located at -1305 and -1225 in human GCC (22), but they did not contribute additional activation by HNF-4 above that seen with the proximal HNF-4 site alone (Fig. 5, -1973/+124 vs. -128/+120) nor were they able to compensate for a mutated proximal HNF-4 site (Fig. 5, -1973/+124 vs. -1973/MutHNF4).

Under the conditions used for supershift EMSA (Fig. 3D), not all of the Caco-2-specific complex was shifted. This could simply reflect a relative excess of HNF-4 vs. antibody, or it could reflect the presence of one or more comigratory complexes whose mobility was not retarded by HNF-4 antiserum. In addition, the two more rapidly migrating specific complexes, seen with all nuclear extracts tested (Fig. 3), did not change mobility. These other complexes likely represent the binding of additional regulatory proteins to the HNF-4 site, as they all are competed by an oligomer containing the alpha 1-antitrypsin gene HNF-4 binding site (Fig. 3C). Regulation of gene expression by HNF-4 involves a complex mechanism by which other, more widely distributed nuclear hormone receptor transcription factors compete for binding at HNF-4 sites. These include factors ARP-1, EAR3/COUP-TF, and EAR2, which have mostly been described as suppressors of transcription at HNF-4 sites (17, 26), although in some circumstances ARP-1 may stimulate gene transcription (21). The net effect of these nuclear hormone receptors on transcriptional activation or repression at a given promoter appears to be determined by the relative abundance of each factor, its binding affinity for a given DNA sequence, the presence or absence of other factors, and interactions with other regulatory elements.

In the small intestine, HNF-4 is expressed in both crypts and villi (26), similar to the expression pattern of GCC. HNF-4 is also expressed in the colon (8), although the crypt-surface distribution has not been reported. Both HNF-4 (9) and GCC (36) mRNAs are also present in developing mouse intestine. Although HNF-4 is abundant in the liver and kidney, GCC is expressed only transiently in neonatal mouse liver (36) and is undetectable (by Northern blot) in adult mouse liver or kidney (36). We have shown that GCC mRNA was not detectable in the HNF-4-expressing liver cell line Hep G2 (Fig. 6). Possible models include the presence of tissue-specific transcriptional suppressors that preclude the action of HNF-4 (such as ARP-1, EAR3/COUP-TF, or EAR2) or that HNF-4 acts in combination with one or more other tissue-restricted transcription factors (absent from liver and kidney) in regulating intestinal transcription of GCC.

It is now known that there are two distinct human genes that encode HNF-4 (HNF-4alpha and HNF-4gamma ) and that splice variants exist for each (8). Although both are expressed in the intestine, only HNF-4alpha mRNA is found in liver. Homozygous deletion of HNF-4alpha results in early embryonic lethality in mice (5), demonstrating functional disparity between the two genes. The relative roles of the two genes in intestinal transcription have not been determined. Our in vitro experiments in COLO-DM cells measured the effect of only HNF-4alpha expression on GCC promoter activity. It is not known whether the HNF-4alpha antiserum we used recognizes HNF-4gamma (F. Sladek, personal communication).

CCAAT/enhancer binding proteins (C/EBP) make up a family of bZIP transcription factors, some of which are expressed in the intestine (3). Synergistic interactions between HNF-4 and C/EBPalpha have been reported (25) for the apolipoprotein B promoter where binding sites overlap. Similarly, the liver-specific activity of the ornithine transcarbamoylase enhancer depends on the combination of HNF-4 and C/EBPbeta (27). The potential C/EBP binding site in the GCC promoter at -104 to -108 is therefore a candidate for further evaluation of the mechanism of transcriptional activation for the GCC gene.

A potential role for cdx-2, a homeodomain transcription factor related to the Drosophila protein caudal, is suggested by the results of the COLO-DM experiments. It is intriguing that in COLO-DM, a non-GCC-expressing intestinal cell line, forced expression of HNF-4 was sufficient to activate endogenous GCC. COLO-DM cells, but not the Hep G2 liver cell line, express cdx-2, which has been shown to be important for the intestine-specific transcription of sucrase-isomaltase as well as other intestinal genes (35, 37). Like GCC, cdx-2 is expressed from the duodenum to the colon and is expressed in both crypt and villus or surface epithelium (15). It is possible that both HNF-4 and cdx-2 are required for intestinal expression of GCC. There is a potential cdx-2 binding site at nt -81 to -75 (within the site C footprint) present in both the -128/+120 and the -1973/+124 reporter constructs. Although it is an imperfect match (6/7) to the consensus cdx-2 binding site (35), it is fully conserved in the same position in the mouse promoter (Fig. 1).

This paper demonstrates that HNF-4 transcriptional regulation is necessary for the intestinal expression of GCC. However, it is clear that other transcriptional factors also contribute to the regulation of GCC. Further investigation of candidate transcription factors and their binding sites in the GCC promoter may reveal crucial interactions with HNF-4 important for intestine-specific gene expression.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Frances Sladek, University of California at Riverside, for providing the pMT2-HNF4 expression vector and HNF-4 antiserum. We also thank Dr. Susan Waltz, Children's Hospital Medical Center, Cincinnati, OH, for providing oligonucleotides containing HNF-4 binding sites from the alpha 1-antitrypsin and hepatocyte growth factor-like protein genes and Elizabeth Yee for technical assistance.


    FOOTNOTES

This work was supported by Veterans Affairs Grant 5393108.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. A. Giannella, Division of Digestive Diseases, Box 670595, Univ. of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0595 (E-mail: Ralph.Giannella{at}uc.edu).

Received 25 August 1998; accepted in final form 24 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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