Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4
Herbert M. van Wering,1
Tjalling Bosse,2
Anna Musters,2
Evelien de Jong,1
Naomi de Jong,2
Caroline E. Hogen Esch,3
Francois Boudreau,4
Gary P. Swain,5
Lauren N. Dowling,6
Robert K. Montgomery,6,7
Richard J. Grand,6,7 and
Stephen D. Krasinski6,7,8
1Department of Medicine, Free University of Amsterdam, Amsterdam 1081HV; 2Department of Medicine, University of Amsterdam, Amsterdam 1100DD; and 3Department of Medicine, Leiden University, Leiden, The Netherlands 2300RC; 4Departement d'Anatomie et Biologie Cellulaire, Faculte de Medecine, Universite de Sherbrooke, Quebec J1H 5N4, Canada; 5Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; 6Division of Gastroenterology and Nutrition, Department of Medicine, Children's Hospital, Boston 02115; 7Department of Pediatrics, Harvard Medical School, Boston 02115; and 8Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University, Boston, Massachusetts, 02111
Submitted 5 April 2004
; accepted in final form 28 May 2004
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ABSTRACT
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Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1
to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1
, requiring physical association between GATA-4 and HNF-1
and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1
, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1
. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1
and/or a GATA-specific pathway independent of HNF-1
.
lactase-phlorizin hydrolase; intestinal differentiation; GATA-4; hepatocyte nuclear factor-1
THE ABSORPTIVE ENTEROCYTE, comprising >95% of the cells on the villus epithelium, is a highly differentiated columnar cell that expresses specialized proteins required for intestinal function, including digestive enzymes, membrane receptors and transporters, and cytoplasmic carriers (17, 62). These proteins also display complex topographic patterns resulting in distinct functions along the proximal-distal (horizontal) axis of the small intestine. Lactase-phlorizin hydrolase (LPH), the enzyme responsible for the digestion of milk lactose into the absorbable monosaccharides, glucose and galactose, is an excellent model for the study of intestine-specific gene expression and intestinal differentiation, because in adult mammals, LPH is expressed only in the small intestine (40) and is confined to absorptive enterocytes on villi, not proliferating cells of the crypt compartment (52, 53). LPH demonstrates "positional" regulation, as exhibited by a tightly controlled pattern of expression along the proximal-distal axis of rodents (6, 15, 23, 25, 32, 53, 63) and humans (47), with high levels in the jejunum and proximal ileum and reduced levels in the duodenum and distal ileum. The pattern of LPH gene expression is regulated mainly by gene transcription (23). In transgenic mice, 1 kb of the pig LPH 5'-flanking sequence (63) and 2 kb of rat LPH 5'-flanking sequence (25, 32) are sufficient for tissue-, cell type-, and differentiation-specific transgene expression. The LPH 5'-flanking sequence of mice, rats, and humans contains conserved GATA, hepatocyte nuclear factor (HNF)-1, and Cdx binding sites within 100 bp of the transcription initiation site (Fig. 1), suggesting that the factors that bind these sites may be important for LPH gene expression and act in concert. Recent studies have focused on the role of the GATA-4, -5, and -6 subfamily of transcription factors in the regulation of LPH gene expression (12, 14, 26, 66).

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Fig. 1. The lactase-phlorizin hydrolase (LPH) gene contains conserved transcription factor binding sites in the 5'-flanking sequence. The 100 to 25 bp regions of the human (2), rat (68), mouse (Celera mouse genomic DNA database; courtesy of Dr. M. Fleming, Dept. of Pathology, Children's Hospital, Boston, MA) and pig (65) LPH 5'-flanking sequence are shown. GATA, hepatocyte nuclear factor (HNF)-1, and Cdx binding sites are underlined, and the TATA box is in bold. Previous studies (26) have shown that there are 2 functional GATA sites in the human LPH promoter but only 1 in the rat and mouse LPH promoters.
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The GATA-4, -5, and -6 subfamily of GATA factors, like the GATA-1, -2, and -3 subfamily, contains a pair of zinc fingers of the four-cysteine type and an adjacent basic region that together mediate binding to the consensus DNA sequence, WGATAR. GATA-1, -2, and -3 are expressed in developing blood cells and are critical for hematopoiesis (49), whereas GATA-4, -5, and -6 are expressed in more diverse patterns that include cardiac tissue, small intestine, stomach, liver, lung, spleen, ovary, testis, and bladder (1, 19, 30, 42, 43, 60), where they are thought to regulate tissue-specific gene expression (37). Gene knockout studies indicate that GATA-4 and GATA-6 are critical for embryonic development, as GATA-4 null mice die during early cardiogenesis (29, 38) and GATA-6 null mice fail at the time of gastrulation (22, 46). GATA-5 knockout mice display minor genitourinary abnormalities in females but normal intestinal structure (39); intestinal gene expression has not been reported. In postweaning and adult mice, GATA-4, -5, and -6 mRNAs are expressed throughout the small intestine (1, 11), although only GATA-4 and GATA-6 proteins have thus far been detected (5, 9, 11). GATA-4, -5, and -6 subfamily members have been shown to bind and activate the promoters of several intestinal genes including LPH (12, 14, 26, 66), sucrase-isomaltase (SI) (4, 26), intestinal fatty acid binding protein (ifabp) (16), liver fatty acid binding protein (lfabp) (8), sodium-hydrogen exchanger isoform 3 (NHE3) (20), adenosine deaminase (ADA) (11), and trehalase (treh) (48). GATA factors may interact with HNF-1
, a homeodomain protein, to synergistically activate intestinal gene promoters (4, 9, 26, 66). We have found that GATA-5 physically associates with HNF-1
to synergistically activate the LPH promoter by an evolutionarily conserved mechanism (66) but is incapable of activating the LPH promoter independently of HNF-1
(26). Together, these studies support an important role for GATA factors in the regulation of intestinal gene expression.
Because there is growing evidence that GATA-4 is the principal GATA regulator of intestinal genes (5, 9, 11), the goal of the present study was to define the importance and underlying mechanism of action of GATA-4 in the regulation of the LPH gene. Our data show that GATA-4 is the principal intestinal GATA factor that binds to the GATA binding site on the mouse LPH promoter, and its expression is highly correlated with that of LPH in the jejunum and ileum. GATA-4 activates the LPH promoter synergistically with HNF-1
, similar to GATA-5, as well as independently of HNF-1
, distinguishing it from GATA-5. Structure-function studies reveal that the zinc finger and basic regions mediate differential functions between GATA-4 and GATA-5, but the underlying mechanism is not due to differences in affinity for binding to the GATA sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with HNF-1
and/or a GATA-specific pathway that is independent of HNF-1
. These data reveal for the first time independent functions among individual intestinal GATA factors.
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MATERIALS AND METHODS
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Plasmids.
Previously characterized expression vectors for mouse GATA-4 (29), GATA-5 (43), and GATA-6 (42), (gifts of M. Parmacek, Univ. of Pennsylvania) and HNF-1
(28) (gift of G. Crabtree, Stanford Univ.) were obtained for these studies. Because the original HNF-1
expression vector replicates inefficiently during bacterial amplification, the HNF-1
coding region was subcloned into pcDNA1, as previously described (66).
For transfection studies, the human LPH promoter containing 118 bp of 5'-flanking region was fused 5' to the human growth hormone reporter (h118wt) (26). This promoter region contains two GATA binding sites, an HNF-1 site and a Cdx site, as previously described (26, 67) (Fig. 1). In specified experiments, previously constructed plasmids (26) were used in which mutations were introduced into the two GATA sites (h118mG1G2), the HNF-1 site (h118mH), or in all three sites together (h118mG1HG2). Mutations introduced into these sites have been previously shown to disrupt specific protein-DNA interactions (26). pRC-CMV (Invitrogen, Carlsbad, CA) served as a negative control expression vector for all cotransfection experiments.
To construct plasmid templates for the synthesis of RNA probes for RNase protection assays, mouse cDNA sequence (LPH, bp 285 to 535; GATA-4, bp 456 to 736; HNF-1
, bp 2936 to 3174) was amplified and subcloned into pBluescript II KS(+). Templates for antisense probes were linearized and transcribed using [
-32P]UTP (800 Ci/mmol; PerkinElmer Life Sciences, Boston, MA): LPH antisense, XbaI and SP6 polymerase; GATA-4 antisense, EcoRI and T3 polymerase; and HNF-1
antisense, KpnI and T7 polymerase. A mouse
-actin probe (25) was used as a control for tissue RNA.
All plasmids used in these studies were amplified in Escherichia coli DH5
under ampicillin selection and isolated under endotoxin-free conditions using the High Purity Plasmid Maxiprep System (Marligen Biosciences, Ijamsville, MD).
Mice.
Approval was obtained from the Institutional Animal Care and Use Committee for all experiments involving mice. Mice were housed under standard conditions in the Animal Research at Children's Hospital animal facility and provided food and water ad libitum. All study animals were 816 wk of age, and all tissue was collected between 1300 and 1600 to avoid any fluctuation in gene expression due to circadium cycles (51).
Isolation of nuclear extracts from intestinal epithelium.
Nuclear extracts were isolated from the intestinal epithelium of adult mouse intestine as previously described (50). Mice were anesthetized with avertin anesthesia (240 mg/kg), the abdomens were opened longitudinally, and the intestines were removed and transferred to a glass plate on a bed of ice. The samples used for experimentation included 8-cm segments centered at the geometric center of the small intestine (jejunum) or pooled samples from the first centimeter adjacent to the pylorus (segment 1), 2-cm segments centered at the first and second quarter junctions (segment 2), 2-cm segments centered at the geometric center (segment 3), 2-cm segments centered at the third and fourth quarter junctions (segment 4), and 3-cm segments adjacent to the ileocecal junction (segment 5). Segments were isolated; rinsed with ice-cold PBS containing fresh 2 mM PMSF, 1 mM benzamidine, and 60 IU/µl aprotinin; opened longitudinally; and cut into 5-mm cross-sectional segments. The segments were transferred to a 50-ml tube containing 5 ml of BD Cell Recovery Solution (BD Bioscience, Bedford, MA), 2 mM PMSF, 2 mM benzamidine, and 120 IU/µl aprotinin and incubated at 4°C for 1820 h. After gentle manual inversion for 1 min, the mucosal pieces were removed and a sample was visualized under Trypan blue staining to confirm the dissociation of epithelial cells while the remaining cells were collected by centrifugation and washed with ice-cold PBS. Nuclear extracts were then isolated as previously described (59), and the quality was determined by expected GATA and HNF-1 complex formation in EMSAs and by detection of GATA-4 and HNF-1
by use of Western blot analysis.
In vitro transcription and translation.
Unlabeled and labeled wild-type and mutated GATA-4, GATA-5, and HNF-1
proteins were synthesized as previously described (66) by use of the TNT transciption/translation system (Promega, Madison, WI). Labeled proteins were synthesized using 35S-labeled methionine (Redivue; Amersham-Pharmacia Biotech, Piscataway, NJ).
EMSAs.
To define protein-DNA interactions, EMSAs were carried out as previously described (26) using oligonucleotides shown in Fig. 2A as probes and/or competitors. For competition or supershift EMSAs, competitors or antibodies (0.1 µg/µl), respectively, were preincubated with the nuclear extract or TNT protein for 20 min before the addition of the probe. The antibodies used in EMSAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) as the concentrated gel shift stock (GATA-4, sc-1237X; GATA-5, sc-7280X; GATA-6, sc-7244X; and HNF-1
, sc-6547X). In certain experiments, band densities from autoradiography were quantified using the Chemi-Doc gel document system and Quantity One software (Bio-Rad, Hercules, CA). All experiments were conducted on at least three different animals.

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Fig. 2. Oligonucleotides used in this study. Top: EMSA oligonucleotides. The forward strand is shown. GATA binding sites are indicated in bold. hG1 corresponds to the 5'-GATA site, and hG2 depicts the 3'-GATA site in the human LPH gene; mG1, the only functional GATA binding site in the proximal mouse promoter, is conserved in position with that of hG1. Bottom: mutagenic oligonucleotides. The forward strand sequence is shown. Incorporation of new codons is indicated in bold, and new restriction sites are shown in underlined italics.
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RNase protection assays.
The quantitative pattern of LPH, GATA-4, and HNF-1
mRNA expression was defined by RNase protection assays using a previously described protocol (23, 25). For proximal-distal analysis, five 1-cm segments were taken from regions as described in Isolation of nuclear extracts from intestinal epithelium Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA), quantified by optical density at A260 nm, and checked for degradation on an agarose gel. 32P-labeled probes were hybridized at 68°C in 50% formamide overnight and digested with RNase A and T1, and the protected fragments were separated on 6% denaturing polyacrylamide gels and revealed by autoradiography. All experiments were conducted on at least three different animals.
Western blot analysis.
To quantify GATA-4 and HNF-1
proteins along the proximal-distal axis, Western blot analysis was conducted. Nuclear extracts of mouse intestine (20 µg) were electrophoresed in 10% SDS-polyacrylamide gels and transferred onto Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA) by electroblotting. After transfer, gels were stained with Coomassie blue to check for transfer efficiency. The membranes were blocked at room temperature for 1 h or overnight at 4°C in 5% nonfat dried milk in PBS. The antibodies used in Western blot analysis were purchased from Santa Cruz Biotechnology and included goat anti-GATA-4 (sc-1237, 1:2,000 dilution) and goat anti-HNF-1
(sc-6547, 1:3,000 dilution). GATA-4 and HNF-1
were visualized with horseradish peroxidase-linked secondary antibodies and the Supersignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL), following the manufacturer's instructions. To verify equal loading of samples, the blots were stripped in 62.5 mM Tris·HCl (pH 6.7), 2% SDS, and 100 mM
-mercaptoethanol at room temperature for 1 h, blocked, and reprobed with mouse monoclonal anti-
-actin (Sigma-Aldrich, St. Louis, MO) at 1:3,000. Experiments with nonimmune serum were used as negative controls. Positive controls were assays conducted using TNT proteins. All experiments were conducted on at least three different animals.
Immunohistochemistry.
To define coexpression of GATA-4 and HNF-1
and to verify their proximal-distal expression patterns, immunohistochemistry was performed on serial sections of adult mouse jejunum (segment 2) and ileum (segment 5). Intestinal segments were isolated, rinsed in ice-cold PBS, fixed in 4% paraformaldehyde in PBS, embedded in paraffin as longitudinal segments, and sectioned (6 µm) onto Superfrost Plus (Fischer) slides. After heating for 30 min at 37°C and deparaffinization, sections were boiled in a microwave for 6 min in 10 mM sodium citrate (pH 6.0), rinsed, blocked with protein blocking agent (Coulter-Immunotech, Miami, FL) for GATA-4 immunostaining or TENG-T (10 mM Tris, pH 7.5, 5 mM EDTA, 0.15 mM sodium chloride, 0.25% gelatin, and 0.05% Tween 20) for HNF-1
immunostaining, and incubated overnight at 4°C with affinity-purified primary antibodies from Santa Cruz Biotechnology. Goat anti-GATA-4 (sc-1237) and goat anti-HNF-1
(sc-6547) were each used at a concentration of 0.2 µg/ml. The primary antibodies were visualized using a biotinylated secondary antibody and an avidin-horseradish peroxidase conjugate (Vectastain ABC kit, Vector Labs). Signal development was achieved using 3,3'-diamino benzidine (Sigma) for 46 min. The tissue was lightly counterstained with methyl green. Sections were imaged under bright-field microscopy by use of SPOT image capture software (Diagnostic Instruments, Sterling Heights, MI). All experiments were conducted on at least three different animals.
Cell culture and transient cotransfection assays.
To characterize the function of GATA factors on the LPH promoter, transient cotransfection assays were conducted. HeLa cells were used because these cells do not synthesize endogenous HNF-1
(26) and are therefore a model for both HNF-1
-independent and HNF-1
-dependent (cotransfected) activation. Cells were transfected using Effectene reagent (Qiagen) according to the manufacturer's instructions. Optimal conditions were 1 µg total DNA (0.4 µg of promoter-reporter construct, 0.2 µg of expression vector when included, and pBluescript as carrier) and 4 µl of Effectene reagent for transfection in six-well plates. Media were replaced after 24 h and collected for human growth hormone analysis after an additional 24 h. Human growth hormone (hGH) was quantified using an 125I-labeled radioimmunoassay kit (Allegro hGH; Nichols Institute Diagnostics, San Clemente, CA). All plates were confluent at the time of harvest. To control for transfection efficiency, all transcriptional activities were expressed relative to pXGH5, as documented previously (66). All HeLa cells used in the present experiment were mycoplasma free, as determined using the Mycoplasma detection kit (American Type Culture Collection, Manassas, VA).
Site-directed mutagenesis.
To delete or disrupt specific structures within GATA-4, site-directed mutagenesis as originally described by Kunkel (27) and later modified (66) was carried out on the GATA-4 (G4) and GATA-5 (G5) expression vectors. Mutagenic oligonucleotides are outlined in Fig. 2B as the forward strand for clarity, although the reverse strand was used for all uracil templates. G4(AD) resulted in the insertion of a BamHI site and a new start codon. The activation domains, which are flanked by BamHI sites after mutagenesis, were removed (AD) by a BamHI digest followed by an intramolecular ligation. G4(C290S) resulted in a cysteine-to-serine substitution in the COOH-terminal zinc finger at amino acid position 290. G4(AD/Nhe), G4(CTD/Nhe), G5(AD/Nhe), and G5(CTD/Nhe) incorporated NheI sites between the activation domains (AD) and zinc fingers (Zn) or the zinc fingers and COOH-terminal domains (CTD) of both GATA-4 and GATA-5 for domain-swapping experiments. The zinc finger and basic regions were removed by digestion with NheI, interchanged, religated, and checked for proper orientation and codon integrity by sequencing.
Glutathione S-transferase pull-down assays.
To partially map the structures necessary for protein-protein interactions, glutathione S-transferase (GST) pull-down assays were carried out as previously described (4, 66). GST-HNF-1
protein synthesis was induced in E. coli DH5
with the use of 0.1 µM isopropyl-1-thio-D-galatcopyranoside for 3 h (57) and purified using glutathione-Sepharose beads (Amersham) according to the manufacturer's instructions. For pull-down assays, GST-HNF-1
was incubated with labeled wild-type or mutated GATA-4 and glutathione-coated beads in binding buffer (66) for 2 h at 4°C. The beads were washed five times in PBS, and bound proteins were released by boiling in SDS sample buffer and resolved by SDS-PAGE.
Statistics.
Means were compared by one-way ANOVA. For statistically significant ANOVA, specific differences among groups were determined by multiple comparison analysis. The Dunnett's multiple comparison test was used when data were compared with a specified control. Otherwise, the Tukey-Kramer multiple comparison test was employed. All analyses were conducted using InStat software (GraphPad Software, San Diego, CA).
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RESULTS
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GATA-4 is emerging as an important activator of intestinal genes (4, 9, 11), but little is known about its importance and underlying mechanism in the activation of the LPH gene. As a first step, we sought to define the GATA factors from intestinal epithelium that bind the LPH promoter. With the use of nuclear extracts from epithelial cells of mouse jejunum and the mouse GATA binding site (mG1) as a probe in EMSAs (Fig. 3A), a specific protein-DNA complex was formed that supershifted with the addition of a GATA-4 antibody. Faster mobility complexes, which we believe are products of GATA-4, also disappeared with the addition of the GATA-4 antibody. Quantitative analysis from jejunal extracts of four different mice using scanning densitometry revealed that 75% (range 6092%, P < 0.05) of the complex that formed in the absence of the GATA-4 antibody supershifted when the GATA-4 antibody was added. No supershift was formed using GATA-5 or GATA-6 antibodies or with nonimmune serum. All antibodies used in these assays recognized their respective antigens, as shown by supershift analysis of nuclear extracts from transfected Caco-2 cells (Fig. 3B). These data indicate that GATA-4 in nuclear extracts from epithelial cells of the adult mouse jejunum is the primary GATA protein that binds to the mouse LPH promoter, implicating GATA-4 as the principal intestinal GATA regulator of LPH gene expression in adults.

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Fig. 3. GATA-4 isolated from mouse epithelium binds the GATA site on the mouse LPH gene. A: supershift EMSAs were carried out with the use of nuclear extracts (20 µg) isolated from the epithelial cells of the jejunum of adult mice and the mG1 site (conserved 5'-GATA site at 89 bp) as a probe. A GATA complex (GC) was formed that supershifted (supershift complex; SC) with the anti-GATA-4 antibody (G4) but not with the anti-GATA-5 (G5) or anti-GATA-6 antibodies (G6) or nonimmune (NI) serum. B: antibodies used in this study recognize their specific antigens. Supershift EMSAs were carried out with the use of nuclear extracts (10 µg) isolated from Caco-2 cells untransfected (lane 2) or transfected with expression vectors for GATA-4 (lanes 3 and 4), GATA-5 (lanes 5 and 6), or GATA-6 (lanes 7 and 8).
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To delineate the function of GATA-4 in the regulation of LPH gene expression in vivo, the relationship between GATA-4 protein and LPH mRNA in adult mouse intestine was determined. Because GATA-4 demonstrates cooperativity with HNF-1
(9, 66), characterization of HNF-1
was also undertaken. The regulated LPH gene expression along the proximal-distal axis was chosen as a model (Fig. 4A) (6, 15, 23, 25, 32, 53, 63). GATA-4 mRNA abundance was highest in the proximal and middle segments of intestine and reduced in distal segments, coincident with a reduction in LPH mRNA abundance in this region. This pattern was similar to that previously described for GATA-4, using Northern blot analysis (11). Neither LPH nor GATA-4 mRNA was expressed in colon. GATA-5 and GATA-6 mRNAs were expressed in all segments, with GATA-5 being lowest in the proximal intestine and highest in the distal intestine and GATA-6 being evenly expressed throughout the small intestine (Krasinski, unpublished observation), as previously reported (11). HNF-1
mRNA abundance was similar throughout the small intestine and colon. GATA-4 and HNF-1
, as determined by quantitative Western blot analysis (Fig. 4B), were correlated with their respective mRNAs: GATA-4 was highest in proximal segments and reduced in distal segments, and HNF-1
was relatively evenly expressed throughout the mouse small intestine. Immunohistochemistry on serial sections (Fig. 4C) revealed that in jejunum, GATA-4 and HNF-1
were coexpressed in the nuclei of villus enterocytes, in agreement with previous studies (5, 9), although we also noted for the first time crypt enterocyte staining for both of these proteins. Serial dilutions of both antibodies (not shown) revealed an absence of background at higher concentrations (up to 0.8 µg/ml) and an equal disappearance of crypt and villus staining at lower concentrations (
0.02 µg/ml), verifying the crypt staining. The reason for the differences in published data is unknown. GATA-4 immunostaining was not detected in the ileum, whereas that of HNF-1
was present in all villus enterocytes, in agreement with quantitative mRNA (Fig. 4A) and protein (Fig. 4B) analyses. Together, these data reveal for the first time detailed qualitative and quantitative analyses of GATA-4 and HNF-1
along the entire length of the adult mouse small intestine and support a role for GATA-4 in defining the pattern of LPH gene expression in jejunum and ileum.
GATA-4 has already been shown to activate the LPH promoter in the Caco-2 intestinal cell line (12, 26), but the underlying mechanism has not yet been defined. Thus transient cotransfection assays using expression vectors for GATA-4, GATA-5, and HNF-1
were conducted in HeLa cells, which do not synthesize endogenous HNF-1
(26) and are therefore a model for both HNF-1
-independent and cooperative activation. A previously described plasmid containing 118 bp of the human LPH promoter, including the conserved GATA and HNF-1 binding sites (Fig. 1), fused to the human growth hormone reporter (h118wt) (26, 66) was used in these studies. As shown in Fig. 5, GATA-5 did not activate the human LPH promoter significantly, consistent with previous data in HeLa cells (26), but GATA-4 independently activated this promoter over basal expression (>25-fold, P < 0.05). Both GATA-4 and GATA-5 synergistically activated the human LPH promoter with HNF-1
, as previously shown (66). GATA-4 and GATA-5 were synthesized in similar amounts as indicated by band densities in EMSAs using the XGATA probe, which binds both proteins equally (16, 26, 66), and nuclear extracts from transfected HeLa cells (not shown). These data indicate that GATA-4 activates the human LPH promoter by dual mechanisms, one that is independent of HNF-1
(GATA-4-specific activation) and one that demonstrates cooperativity with HNF-1
(GATA/HNF-1
-cooperative activation).
To define the underlying mechanisms of each pathway, the requirement of particular structures in GATA-4 necessary for GATA-4-specific and GATA/HNF-1
-cooperative activation was determined by the introduction of mutations into GATA-4 that delete or disrupt these structures (Fig. 6). GATA-4 is comprised of a pair of NH2-terminal activation domains, a pair of zinc fingers, and a basic region that mediate protein-DNA (44) and protein-protein (10, 31, 34, 54, 61) interactions and a COOH-terminal domain that also may bind proteins (7). Deletion of the activation domains resulted in a 60% reduction in transcriptional activity in the absence of HNF-1
and a 50% reduction in the presence of HNF-1
, although cooperative activation in the presence of HNF-1
remained above the summation (dotted) line, indicating synergy (Fig. 6A). A mutation in a critical cysteine residue (C290S) that disrupts the structure of the COOH-terminal zinc finger resulted in the elimination of both GATA-4-specific and GATA/HNF-1
-cooperative activation (P < 0.05 for each, Fig. 6A). EMSAs (Fig. 6B) and GST pull-down assays (Fig. 6C) indicated that G4(AD) is capable of binding to DNA and physically associating with HNF-1
, respectively, whereas G4(C290S) is incapable of binding to DNA (Fig. 6B), as previously shown (44), or physically associating with HNF-1
(Fig. 6C). These data indicate that the COOH-terminal zinc finger of GATA-4 is necessary for GATA-4-specific and GATA/HNF-1
-cooperative activation by conferring on GATA-4 the ability to bind DNA and physically associate with HNF-1
, respectively.
To define the importance of GATA and HNF-1 binding sites on the LPH promoter for GATA-4-specific and GATA/HNF-1
-cooperative activation, transient cotransfection assays were carried out in HeLa cells using h118 LPH-hGH constructs in which the GATA and/or HNF-1 binding sites were mutated (Fig. 7). In a promoter construct in which both GATA sites were mutated, GATA-4-specific activation was significantly reduced to <20% of that of the wild-type promoter (P < 0.05), whereas GATA/HNF-1
-cooperative activation was reduced to
50% of that of the wild-type promoter but remained above the summation (dotted) line, indicating synergy. In a promoter construct in which only the HNF-1 binding site was mutated, GATA-4 activation was not compromised, whereas GATA/HNF-1
-cooperative activation was reduced to
25% of that of the wild-type promoter and fell below the summation line, indicating loss of synergy. In a construct where all three binding sites were mutated, both GATA-4-specific and GATA/HNF-1
-cooperative activation were significantly reduced (P < 0.05 for each). These findings demonstrate that the GATA binding sites are necessary for GATA-4-specific activation, and the HNF-1 binding site is required for GATA/HNF-1
-cooperative activation.
Because binding of GATA-4 to DNA is a requirement for GATA-4-specific activation (Fig. 7), and since the zinc finger and basic regions of GATA factors mediate DNA binding (37, 44, 66), we next tested the hypothesis that the zinc finger and basic regions are responsible for the differential abilities of GATA-4 and GATA-5 to activate the LPH promoter in the absence of HNF-1
. In these studies, expression vectors were constructed in which the zinc finger and basic regions of GATA-4 and GATA-5 were interchanged and tested for their ability to activate the LPH promoter in HeLa cells. G4(ZnG5), which contains the activation and COOH-terminal domains of GATA-4 but the zinc finger and basic region of GATA-5, activated the LPH promoter significantly less than that of wild-type GATA-4 but similar to that of wild-type GATA-5 (Fig. 8A). In contrast, G5(ZnG4), which contains the activation and COOH-terminal domains of GATA-5 but the zinc finger and basic region of GATA-4, activated the LPH promoter similarly to that of wild-type GATA-4. These expression vectors synthesized similar amounts of GATA protein, as shown by EMSA using nuclear extracts from transfected HeLa cells and the XGATA probe (Fig. 8B). These proteins were all capable of binding GATA sites on DNA (Fig. 8B), were recognized by epitope-specific antibodies (supershift, not shown), and synergistically activated the LPH promoter with HNF-1
(>2-fold synergy for all, not shown), indicating that the proteins were structurally intact. These findings reveal that GATA-4-specific activation of the LPH promoter is mediated by the zinc finger and basic regions.

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Fig. 8. GATA-4-specific activation is mediated by the zinc finger and basic regions. A: heterologous GATA-5 containing the GATA-4 zinc finger region demonstrates GATA-4-specific activation. HeLa cells were cotransfected with h118wt and wild-type or heterologous GATA-4 or GATA-5 containing zinc finger swaps. Data are means ± SE; n = 4. *P < 0.01 compared with wild-type GATA-4 and G5(ZnG4) by the Tukey-Kramer multiple-comparison test. B: mutated GATA factors bind GATA sites on DNA. EMSAs carried out using nuclear extracts from transfected HeLa cells (5 µg) and the XGATA probe reveal similar intensities of specific complexes.
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Because the zinc finger and basic regions of GATA factors mediate binding to DNA (37, 44, 66), we hypothesized that the differences in the ability of GATA-4 and GATA-5 to activate the LPH promoter in the absence of HNF-1
are due to a greater affinity of GATA-4 compared with GATA-5 for the GATA binding sites on the human LPH promoter. To test this hypothesis, a series of affinity EMSAs were conducted (Fig. 9). In the first experiment, competition EMSAs were carried out in which the ability of the GATA binding sites on the LPH promoter to preferentially compete GATA-4 or GATA-5 away from a standardized GATA binding site was determined (Fig. 9A). Previous studies (16, 26, 66) indicated that the GATA binding site in the Xenopus ifabp gene (i.e., XGATA) binds both GATA-4 and GATA-5 similarly and thus was used as the standard control probe. GATA-4 and GATA-5 are different in size (43) and thus can be added together and analyzed simultaneously. Supershift analysis verified that the slower mobility complex was GATA-4 (lane 3) and the faster mobility complex was GATA-5 (lane 4). XGATA competed both bands similarly (lanes 57), as anticipated. The hG1 and hG2 oligonucleotides also competed each band relatively equally, suggesting that they bind GATA-4 and GATA-5 with similar affinities. In the second experiment, affinity was tested directly as previously described (7) using constant amounts of synthetic GATA-4 or GATA-5 and limiting amounts of probe (Fig. 9B). Probe titrations revealed no difference in complex intensities between GATA-4 and GATA-5 for both hG1 and hG2. Together, these data indicate that the GATA sites on the human LPH promoter bind GATA-4 and GATA-5 with similar affinities and thus do not account for the differences in the ability of these two transcription factors to activate the LPH promoter in cotransfection experiments. Experiments to identify alternative mechanisms underlying the GATA-4/GATA-5 differences are currently in progress in our laboratory.

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Fig. 9. GATA-4 and GATA-5 have similar affinities for the GATA binding sites on the human LPH promoter. A: EMSAs showing similar competition of GATA-4 and GATA-5 complexes by the GATA binding sites in the LPH promoter. With the use of the XGATA probe and a mixture of in vitro transcribed and translated GATA-4 and GATA-5 proteins (lanes 213), competition EMSAs were carried out with titrated amounts of oligonucleotide competitors (5-, 20-, and 100-fold molar excess), including XGATA (lanes 57), hG1 (lanes 810), and hG2 (lanes 1113). SC formation confirmed that the band with the slower mobility was the GATA-4 complex (G4C, lane 3) and that with the faster mobility was the GATA-5 complex (G5C), as anticipated, based on the size of the proteins. B: EMSAs showing similar affinities of GATA-4 and GATA-5 for the binding sites on the LPH promoter. Constant amounts of synthetic GATA-4 or GATA-5 were incubated with limiting amounts of XGATA, hG1, and hG2 probes.
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DISCUSSION
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GATA-4 is emerging as the principal GATA regulator of intestinal genes. GATA-4 was the primary GATA factor in intestinal epithelial cells that binds the GATA sites in the SI (4), lfabp (8), and ADA (11) promoters. In intestinal epithelial cells of chimeric mice that do not express GATA-4, lfabp expression was absent compared with wild-type cells in the same animal, providing key in vivo data demonstrating a critical importance of GATA-4 for intestinal lfabp expression and a lack of overlapping functions with other intestinal GATA factors (8). In the present study, we show that GATA-4 is the principal GATA factor that binds also to the LPH promoter (Fig. 3) and correlates well with LPH mRNA in jejunum and ileum (Fig. 4), implicating GATA-4 as the primary GATA regulator of LPH gene expression in vivo. GATA-4 synergistically activates the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1
(van Wering et al., Ref. 66), supporting the hypothesis that GATA/HNF-1
interactions are a conserved pathway among intestinal GATA factors. GATA-4 also activates the LPH promoter by a mechanism that is distinct from that of GATA-5, demonstrating for the first time independent functions among individual intestinal GATA factors. These data support the addition of LPH to the growing list of intestinal differentiation markers regulated by GATA-4 and reveal complex mechanisms not previously appreciated by which GATA-4 activates an intestinal gene promoter.
The inability to detect binding of GATA-5 or GATA-6 to intestinal gene promoters could be due to a lower affinity of GATA-5 and GATA-6 compared with GATA-4 for the binding sites under study, to modification or recruitment pathways in the adult intestine that modulate their ability to bind specific binding sites on DNA, or to a low abundance of these proteins in adult intestine. Synthetic GATA-4 and GATA-5 each bind the LPH promoter equally (Fig. 9), suggesting that differences in binding are not due to intrinsic differences in affinity. GATA-4, -5, and -6 mRNAs are all expressed in the intestine of adult mice (Krasinski, unpublished observation) (11), indicating that all three genes are transcribed. The presence of GATA-4 protein in epithelial cells of adult mouse jejunum has been demonstrated by supershift EMSAs (Fig. 3) (4, 9, 11), quantified by Western blot analysis (Fig. 4) (4), and localized by immunostaining (Fig. 4) (9, 11). GATA-5 could not be detected by immunostaining despite positive control immunostaining of GATA-5 in stomach (9). Although quantitative Western blot analysis has not been reported, these data suggest that GATA-5 protein is not highly expressed. GATA-6 in adult mouse intestine has been revealed by immunostaining (9) but not yet quantified by Western blot analysis. GATA-6 was found to bind the treh promoter in 8-day-old mice but only after glucocorticoid treatment increased its abundance (48). Taken together, these data are consistent with the hypothesis that GATA-5 and GATA-6 are present in a low abundance. These data do not, however, exclude the possibility that GATA-5 or GATA-6 undergoes modification or interacts with other proteins in the adult intestine that reduce its ability to bind to specific sites on DNA. Although it is possible that GATA-5 and GATA-6 are preferentially degraded during nuclear extract isolation, other proteins, including GATA-4, HNF-1
,
-actin (Fig. 4), and Cdx-2 (not shown), are readily detected in these extracts. GATA-5 and GATA-6 may regulate intestinal gene expression at developmental time points other than adult, but the data presented here and in other studies (4, 9, 11) implicate GATA-4 as the key GATA regulator of intestinal gene expression in adult mice in vivo.
LPH gene expression in the adult mouse small intestine is highly correlated with that of GATA-4 in the jejunum and ileum but not in the duodenum (Fig. 4). In the jejunum and proximal ileum, LPH and GATA-4 are both expressed at high levels, and in the distal ileum, LPH and GATA-4 are both expressed at reduced levels, consistent with the hypothesis that GATA-4 is an activator of LPH gene expression in these regions and defines, in part, the proximal-distal pattern of LPH gene expression. In the duodenum where LPH expression is low, GATA-4 expression is high. Reduced LPH gene expression in the duodenum could be due to a reduction or modification of a critical activator or to activation of a repressor pathway. HNF-1
, a proposed activator of the LPH promoter (26, 35, 36, 58), is expressed equally throughout the small intestine (Fig. 4), as previously shown (55), and is thus unlikely to account for differential LPH gene expression along the proximal-distal axis. Cdx-2, also a proposed activator of the LPH promoter (13, 26, 36, 64, 65, 67), is reduced in proximal vs. distal intestinal segments (5, 56) and thus may be responsible for the reduced duodenal expression of LPH. It is also possible that LPH is specifically repressed in the duodenum. Preliminary data suggest that Pdx-1, the intestinal expression of which is confined to the duodenum (18), represses LPH gene expression in this region of small intestine (69). Precedence for a repressor pathway is supported by Cux/CDP, a homeodomain-containing transcriptional repressor closely related to the cut protein in Drosophila melanogaster, which represses SI gene expression in distal ileum and colon, as shown by ectopic ileal and colonic SI expression in mice that do not express Cux/CDP (3). Together, these data support the hypothesis that the pattern of LPH gene expression along the proximal-distal axis is defined, in part, by GATA-4, but that other pathways are also involved.
GATA-4 may activate the LPH promoter in concert with HNF-1
through an evolutionarily conserved mechanism. Serial sectioning experiments (Fig. 4) demonstrate that GATA-4 and HNF-1
are coexpressed in the same absorptive enterocytes on villi, providing the topographic basis for molecular interactions. Importantly, LPH expression in adult mice always occurs in regions that coexpress GATA-4 and HNF-1
, although LPH is not always expressed where GATA-4 and HNF-1
are coexpressed, such as in crypts and duodenum, suggesting that GATA-4 and HNF-1
are necessary but not sufficient for LPH gene expression. Preliminary data in our laboratory using germline hnf-1
null mice demonstrate
90% reduction in LPH gene expression in adult jejunum (24), revealing that HNF-1
is an important component of LPH gene expression in vivo. In cell culture models, GATA-4 (Figs. 5 and 6) and GATA-5 (66) physically associate with HNF-1
to synergistically activate the LPH promoter, and all three GATA factors interact with HNF-1
to synergistically activate the lfabp promoter (8). For both GATA-4 and GATA-5, physical association with HNF-1
is mediated by their COOH-terminal zinc fingers and basic regions (66) (Fig. 6). Binding sites on the LPH promoter for HNF-1
, but not for GATA, are necessary for both GATA-4/HNF-1
and GATA-5/HNF-1
synergy (66) (Fig. 7). The parallel requirements for both GATA-4/HNF-1
and GATA-5/HNF-1
synergy suggest that cooperative interaction with HNF-1
is a conserved pathway among intestinal GATA factors for the activation of intestinal genes.
GATA-4 and GATA-5 also demonstrate differential regulation of the LPH promoter in that in the absence of HNF-1
, GATA-4 is capable of activating the LPH promoter alone but GATA-5 is not (Fig. 5). Using these differences as a model in structure-function studies involving a domain swapping strategy, we determined that GATA-4-specific activation of the LPH promoter is mediated by the zinc finger and basic regions (Fig. 8). However, although the zinc finger/basic regions mediate DNA binding, GATA-4 and GATA-5 surprisingly demonstrate virtually identical affinities for the binding sites on the LPH promoter (Fig. 9), suggesting that the underlying differences in the ability of GATA-4 and GATA-5 to activate the LPH promoter are not due to differences in affinity for binding to DNA. Differences in the ability of individual members of the GATA-4, -5, and -6 subfamily to activate target genes have been previously reported for nonintestinal genes. In contrast to the present study, which showed that differential regulation was not due to differential binding affinity, Charron et al. (7) showed that preferential regulation of the
-myosin heavy chain (MHC) gene by GATA-4 was due in part to a greater affinity of GATA-4 compared with GATA-6 for the GATA site on the
-MHC promoter. Also in contrast to the present study, which mapped differential regulation to the zinc fingers, Morrisey et al. (45) found that differential regulation of the Dab2 promoter by GATA-4 and GATA-6 was not due to the zinc fingers and therefore unlikely to be due to differences in DNA binding affinity. Thus GATA-4-specific activation of the LPH gene may represent a novel intestinal pathway of GATA regulation that is distinct from that which occurs in the regulation of cardiac-specific genes.
Several issues remain a challenge for future studies. First, the contribution of GATA/HNF-1
cooperativity vs. GATA-4-specific activation of LPH gene expression in vivo remains to be determined. Although GATA-4 and HNF-1
are coexpressed in the same cells (Fig. 4) and synergistically activate intestinal gene promoters in overexpression experiments in cell culture assays (8, 26, 66), the importance of GATA-4/HNF-1
cooperativity at stoichiometric levels in vivo for intestinal gene expression remains unknown. Second, the mechanisms underlying the complex topographic patterns of intestinal gene expression are yet to be defined. Although LPH expression occurs where GATA-4 and HNF-1
are highly coexpressed, LPH expression is low or absent in duodenum and in crypts (Fig. 4), respectively, where GATA-4 and HNF-1
are also highly coexpressed, implicating the involvement of other pathways. Finally, the mechanisms underlying the differential functions of GATA-4 and GATA-5 remain to be determined. We mapped the functional components to the zinc finger/basic regions of GATA-4 and GATA-5 (Fig. 8) but showed that the differential function was not due to differences in binding affinity to the LPH promoter (Fig. 9). Alternative mechanisms for all of these issues include the participation of other transcription factors and/or other proteins that may act as coactivators or corepressors, as well as modification pathways that could alter protein function. Indeed, GATA-4 may be directly modulated by phosphorylation (21, 33, 41) or acetylation (70), which may influence both DNA binding affinity and transactivation potential. The future challenge will be to delineate the combinatorial roles of GATA-4 and HNF-1
and their interaction with other factors and cofactors as well as their possible modification in the spatial and temporal regulation of intestinal differentiation.
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-061382 (to S. D. Krasinski) and R37-DK-32658 (to R. J. Grand), the Harvard Digestive Disease Center (grant no. 5P30-DK-34854), and grants from the Nutricia Research Foundation (to H. M. van Wering and T. Bosse).
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ACKNOWLEDGMENTS
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We thank Christina M. Piaseckyj and John J. Fialkovich for expert technical assistance. We also thank Suzanne White of the Histology Core of the Harvard Digestive Disease Center for help in the preparation of tissue sections. Finally, we thank Dr. M. Parmacek (Univ. of Pennsylvania) for the mouse GATA-4, GATA-5, and GATA-6 expression vectors and Dr. G. Crabtree (Stanford Univ.) for the HNF-1
expression vector.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. D. Krasinski, GI/Cell Biology, EN 1220, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115 (E-mail: stephen.krasinski{at}childrens.harvard.edu)
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. Section 1734 solely to indicate this fact.
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