Division of Digestive Diseases, Veterans Affairs Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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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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.
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 RSV110RNA 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
[-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
-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
[
-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.
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RESULTS |
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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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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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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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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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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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DISCUSSION |
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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-1 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-1
in the promoter constructs we
used for transfection, the endogenous GCC gene may contain HNF-1
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
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-4 and HNF-4
) and that splice variants exist for each
(8). Although both are expressed in the intestine, only HNF-4
mRNA
is found in liver. Homozygous deletion of HNF-4
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-4
expression on GCC promoter activity. It is not known whether the
HNF-4
antiserum we used recognizes HNF-4
(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/EBP 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/EBP
(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.
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ACKNOWLEDGEMENTS |
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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
1-antitrypsin and hepatocyte growth factor-like protein genes and Elizabeth Yee for technical assistance.
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FOOTNOTES |
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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.
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