Department of Pediatrics, Stanford University Medical Center, Stanford, California 94305
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ABSTRACT |
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The GATA family of transcription
factors regulate tissue-specific patterns of gene expression during
development. We have characterized the interaction between GATA
proteins and the lactase gene promoter. Nuclear protein bound to the
lactase gene GATA region cis element (97 to
73) was
analyzed by electrophoretic mobility shift assays (EMSA) and supershift
assays with GATA antibodies. Lactase promoter activities were assayed
in Caco-2 cells transfected with wild-type and mutated luciferase
promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA
with the GATA region probe yields a specific DNA-protein complex that
requires the GATA factor binding site WGATAR. The complex is recognized
by GATA-4- and GATA-6-specific antibodies. GATA-4/5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is
mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to
the lactase cis element correlates with functional promoter
activation. We conclude that each of the GATA family zinc finger
proteins expressed in the intestine, GATA-4, -5, and -6, can interact
with the lactase promoter GATA element and can function to activate the
promoter in Caco-2 cells.
GATA-4; GATA-5; GATA-6; enterocyte
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INTRODUCTION |
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THE INTESTINAL EPITHELIUM is comprised of four principal cell types that are derived from a proliferating stem cell population (see Ref. 10 for review). The stem cells, located in crypts near the intestinal villus base, undergo terminal differentiation as they migrate from the crypt to the villus tip, where they ultimately die and are sloughed off into the gut lumen. The four principal intestinal epithelial cell types have highly specialized functions. The absorptive enterocytes are the predominant intestinal cell type, and they function to digest and absorb luminal nutrients across the apical brush-border membrane. Goblet cells secrete mucus that provides a protective barrier lining the gut. The enteroendocrine cells secrete intestinal hormones involved in signaling gut motility. Paneth cells terminally differentiate during migration toward the base of the crypt and are lysozyme- and defensin-producing cells. The molecular mechanisms regulating terminal differentiation of the intestinal cell types have not been defined.
The GATA family of zinc finger transcription factors are important regulators of cell lineage differentiation during vertebrate development. Six GATA family members have been identified in vertebrate species. Each vertebrate GATA factor contains two conserved zinc fingers that bind to a consensus sequence (WGATAR) that is present in the transcriptional regulatory region of multiple lineage-specific genes (13, 17). The GATA-1, -2, and -3 subfamily proteins control critical steps in erythroid and lymphoid development (8, 22, 23, 29). GATA-4, -5, and -6 subfamily proteins are expressed in overlapping patterns in the developing heart and endoderm-derived organs of the gastrointestinal tract including the stomach, intestine, liver, and pancreas (1, 5, 16, 19, 20). The functional role of GATA-4, -5, and -6 proteins in regulating heart and gut development is largely unknown. Homozygous disruption of GATA-4 expression during embryogenesis results in the lack of a primitive heart tube and foregut in mice (15). Targeted mutagenesis of the GATA-4 gene in embryonic stem cells disrupts visceral endoderm differentiation (25). GATA binding sites have been identified in transcriptional regulatory elements of several cardiac-specific target genes. Overexpression of GATA-4 protein has been shown to transactivate these cardiac-specific cis elements in cell culture (11, 12, 18).
With respect to a role in regulating enterocyte differentiation, distinct patterns of expression for GATA-4, -5, and -6 were recently described by Gao et al. (9). The expression patterns of the GATA factors differ along the proximal-distal villus axis in the chicken intestine and during differentiation in culture. In situ transcript levels for GATA-6 are highest in the proliferating cell region of the crypts, whereas levels for GATA-4 and GATA-5 increase toward the villus tip. On stimulation to differentiate, intestinal HT-29 cells begin to express GATA-5 whereas GATA-6 transcript levels decline. Few intestinal target genes, however, have been identified for the GATA-4/5/6 factor subfamily. In the same report by Gao et al. (9), the gene encoding Xenopus intestinal fatty acid binding protein (IFABP) was identified as an in vitro target for the GATA-4/5/6 factors in intestinal cell culture. In addition, Fitzgerald et al. (7) recently reported that GATA-6 stimulates promoter activity of the human lactase gene promoter. We report here that GATA-4 and GATA-5, in addition to GATA-6, can activate the rat lactase promoter in intestinal cell culture. These studies provide compelling evidence for the potential of each member of the GATA-4/5/6 factor subfamily to regulate expression of multiple enterocyte-specific genes.
Intestinal lactase-phlorizin hydrolase (LPH, lactase) is the absorptive enterocyte membrane glycoprotein essential for digestive hydrolysis of lactose in milk. Lactase is present predominantly along the brush-border membrane of differentiated enterocytes lining the villi of the small intestine. Expression of the lactase gene is spatially restricted along both vertical and longitudinal axes in the gut (24). Along the vertical axis, immature enterocytes derived from stem cells in the crypts migrate from the crypt to the villus tip, differentiate, and begin to express the enzymatic activity of lactase as well as other digestive hydrolases. Along the longitudinal axis, the lactase gene is expressed maximally in the proximal small intestine and declines significantly in the distal segments of the intestine. Lactase gene expression is also temporally restricted in the gut during intestinal maturation. Enzyme activity is maximal in the small intestine of preweaned mammals and declines markedly during maturation (3, 14, 21). The mechanisms regulating the spatial restriction and maturational decline in lactase activity have not been fully defined. In vitro binding studies showed that the lactase gene promoter interacts with specific nuclear proteins from intestinal cells (28). The homeodomain protein Cdx-2 binds to a distinct cis element, CE-LPH1, of the lactase 5'-flanking region and is capable of activating transcription of the lactase promoter (6, 27). Regulation of intestine-specific differentiation is likely to involve additional transcription factors that control expression of terminal differentiation genes such as LPH. Here we identify a GATA binding site element in the promoter region of the lactase gene, identify the GATA proteins interacting with the element, and characterize transcriptional activation mediated by GATA-4, -5, and -6 proteins in intestinal Caco-2 cell culture.
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MATERIALS AND METHODS |
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Materials and reagents. Restriction endonucleases and modifying enzymes were purchased from New England Biolabs (Beverly, MA). Radioisotopes were purchased from DuPont NEN. Oligonucleotides were synthesized in the Protein and Nucleic Acid facility of the Stanford University Beckman Center.
Subcloning of deletion and mutant lactase promoter-reporter
constructs.
The lactase promoter deletion and mutant reporter constructs were
generated from pgLac (6), a previously described plasmid in which a 200-bp 5'-flanking region of the rat lactase gene (nt 200
to +13) was cloned upstream of the firefly luciferase reporter gene in
the vector pGL3-Basic (Promega). Deletion promoter-reporter constructs
were generated by initial PCR amplification of pgLac using forward
oligonucleotides corresponding to nt
100 to
80, nt
73 to
53, or
nt
58 to
38 (each with an added 5' Bgl II site) and GL
primer 2 (Promega), the reverse oligonucleotide proximal to the
luciferase gene in pGL3-Basic. The Bgl II-Hind
III fragments for each of the PCR products were then cloned into
pGL3-Basic to generate the corresponding
100,
73, and
58 bp
deletion promoter-reporter constructs.
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Electrophoretic mobility shift assay and supershift assay.
Nuclear extracts were prepared from Caco-2 or QT6 cells according to a
modification of a previously described procedure (2). Cells were harvested by scraping and resuspended in 5 vol of
phosphate-buffered saline followed by centrifugation (400 g
for 10 min at 4°C). The cells were resuspended in five packed cell
volumes of cold buffer A [in mM: 10 HEPES (pH 7.9), 10 KCl,
1.5 MgCl2, 0.5 dithiothreitol, and 1 phenylmethylsulfonyl
fluoride (PMSF)] and incubated for 10 min on ice followed by
centrifugation as above. Cells were resuspended in two packed cell
volumes of buffer A, lysed by Dounce homogenization, and
then centrifuged (400 g for 20 min at 4°C). The nuclei
pellet was resuspended in 3 ml of cold buffer B [20 mM
HEPES (pH 7.9), 10% glycerol, 0.42 M NaCl, 1.5 mM MgCl2,
0.5 mM dithiothreitol, 0.2 mM EDTA, and 1 mM PMSF] followed by Dounce homogenization and stirred gently for 30 min at 4°C. After
centrifugation (100,000 g for 20 min), the supernatant was
precipitated with ammonium sulfate (final concentration 0.33 g/ml) and
then centrifuged (25,000 g for 20 min). The pellet was
resuspended in buffer C [20 mM HEPES (pH 7.9), 10%
glycerol, 0.5 mM dithiothreitol, 0.2 mM EDTA, and 1 mM PMSF], dialyzed
against buffer C for 12-18 h at 4°C, and stored at
80°C.
Transient transfection assays. Caco-2 cells or QT6 cells were cultured in DMEM with 10% fetal bovine serum. Forty-eight to sixty hours before transfection, the cells were split and 35-mm dishes were seeded with 2 × 105 cells. For each reporter construct, a DNA transfection mixture was prepared consisting of 0.6 µM of the construct, 0.5 µg of pRL-CMV (Promega) as an internal control, and pBluescript KS+ II to adjust to 3.5 µg of total DNA. The individual DNA mixtures were transfected into cells (50-80% confluent) with Lipofectamine reagent (BRL) according to the protocol of the manufacturer. For cotransfection experiments, 0.3 pmol of pxGATA-4, -5, or -6 expression constructs (9), in which Xenopus GATA cDNAs were cloned downstream of a cytomegalovirus (CMV) promoter in pCDNA3, was transfected along with 0.6 pmol of the luciferase reporter constructs. Cells were harvested 48 h after transfection (70-90% confluent), and luciferase activity was measured by the Dual-Luciferase reporter assay system (Promega) as described by the manufacturer, in a Monolight 3010 luminometer. Transfection with the dual reporters (firefly luciferase for the lactase promoter-reporter plasmids and renilla luciferase for the pRL-CMV control) allowed for simultaneous expression and measurement of both reporter enzymes. The lactase promoter-reporter activity correlated with the effect of the promoter sequence or GATA expression, and the activity of the cotransfected pRL-CMV provided an internal control. Experimental lactase promoter-reporter activities were normalized to the activity of the pRL-CMV internal control and expressed as relative luciferase activity (means ± SD, n = 3), thereby minimizing experimental variability caused by differences in cell viability or transfection efficiency.
RNA analysis by RT-PCR. Total RNA was extracted from proliferating Caco-2 cells using Tri reagent (Molecular Research Center) according to the protocol of the manufacturer. RNA concentrations were determined by optical densitometry at 260 nm, and the absence of RNA degradation was confirmed by agarose gel electrophoresis. For RT-PCR, cDNA was initially synthesized from 1.0 µg of Caco-2 cell total RNA using avian myeloblastosis virus RT and the Advantage RT-for-PCR kit (Clontech) according to the protocol of the supplier and brought to a final volume of 100 µl. PCR reactions were then carried out with synthetic oligonucleotide primers corresponding to exon sequences for the human GATA-4 (Ref. 30; GenBank accession no. D78260), GATA-6 (Ref. 26; accession no. NM005257), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (internal standard; Ref. 4; accession no. J04038) genes: GATA-4F 5'-CATCAAGACGGAGCCTGGCC-3' and GATA-4R 5'-TGACTGTCGGCCAAGACCAG-3' (218-bp product); GATA-6F 5'-CCATGACTCCAACTTCCACC-3' and GATA-6R 5'-ACGGAGGACGTGACTTCGGC-3' (213-bp product); and GAPDH-F 5'-GGGTCATCATCTCTGCCCCCTCTG-3' and GAPDH-R 5'-CCATCCACAGTCTTCTGGTGGCA-3' (208-bp product). To prevent nonspecific amplification hot-start PCR reactions (50 µl) were carried out with 5 µl of the reverse-transcribed cDNA, 0.4 µM primers, 0.20 mM dNTPs, 20 mM Tris · HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, and 2.5 units of Taq polymerase (GIBCO BRL) preincubated with 1:28 with TaqStart Antibody (Clontech). Control amplifications confirmed that the reactions were entirely dependent on the RT reaction. Amplification conditions were 94°C for 45 s, 55°C for 45 s, and 75°C for 45 s performed for 15-40 cycles to optimize for PCR products in the linear range of exponential amplification. Simultaneous PCR reactions were performed with first-strand cDNA generated from RNA of different human tissues (MTC; Clontech). PCR products were analyzed after electrophoresis on 2% agarose gels using a Molecular Analyst densitometer (Bio-Rad) and sequenced to confirm their identity.
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RESULTS |
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Regulatory regions of lactase gene 5'-flanking DNA mapped by
deletional analysis of reporter constructs.
Promoter activity was previously mapped to within 200 bp upstream
from the transcription start site of the rat lactase gene (6). To identify regions of the lactase gene capable of
mediating regulation of gene transcription, deletion fragments of this
promoter region were cloned upstream of the firefly luciferase cDNA in the reporter plasmid pGL3-Basic and transfected into Caco-2 cells. The
100,
73, and
58 bp lactase reporter constructs, shown in Fig.
2A, were transiently
transfected into Caco-2 cells, a human adenocarcinoma-derived cell line
that mimics a small intestinal enterocyte phenotype with respect to
expression of several digestive hydrolases including lactase and
sucrase-isomaltase. Caco-2 cell extracts were assayed for relative
luciferase activity 48 h after transfection.
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Specific nuclear protein(s) from Caco-2 cells interacts with a GATA
region element.
Inspection of the sequence for the 73 to
100 bp positive
cis element region of the lactase promoter reveals a
consensus binding site for the GATA family of zinc finger proteins. To
identify interactions between this GATA region cis element
and nuclear proteins in Caco-2 cells, we used the electrophoretic
mobility shift assay (EMSA) or the gel shift assay. The wild-type GATA region oligonucleotide was radiolabeled, incubated in the presence of
Caco-2 cell nuclear extract, and then migrated through a 6% nondenaturing acrylamide gel. The autoradiograph in Fig.
3 reveals the position of the rapidly
migrating unbound probe at the base of the gel and a DNA-protein
complex of slower mobility that is formed after incubation with Caco-2
nuclear extract. The DNA-protein complex is not competed away
by 100-fold excess unrelated unlabeled SP1 oligonucleotide but is
competed for by 100-fold excess unlabeled wild-type GATA region
oligonucleotide. A slightly faster migrating nonspecific band is not
displaced with either competitor. The nuclear protein bound to the GATA
region probe in Caco-2 cells therefore represents a specific
DNA-protein interaction.
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GATA family proteins interact with the lactase promoter element.
Members of the GATA family of transcription factors (GATA-4, -5, and
-6) are expressed in intestine and are capable of binding to the GATA
cis element in the IFABP promoter (9). To
determine whether GATA family proteins in Caco-2 cells bind to the
lactase gene-positive cis element identified above, we
incubated the radiolabeled GATA region probe and nuclear extract from
Caco-2 cells in the absence or presence of a polyclonal GATA-4 and
GATA-6 antibody (Fig. 4). GATA-5 binding
could not be assayed because human-reactive GATA-5-specific antibody
was not available. The complexes were analyzed by electrophoresis on a
nondenaturing gel and analyzed for specific supershift of the
previously identified complex. Both the GATA-4 and GATA-6 antibodies
recognized the nuclear protein bound to the GATA region probe,
resulting in a complex of reduced gel mobility or a supershift (Fig.
4). There is residual bound DNA-protein complex that is not
supershifted by the GATA-4 or GATA-6 antibody in each reaction,
consistent with the presence of both proteins interacting with the GATA
region probe. Although GATA-5 binding could not be assayed directly,
residual complex remained even in reactions incubated in the presence
of both GATA-4 and-6 antibodies (Fig. 4). As expected, no supershift
was observed for the binding reaction carried out in the presence of
nonimmune serum (Fig. 4).
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GATA-4 and GATA-6 mRNA is present in proliferating Caco-2 cells.
To confirm GATA-4 and GATA-6 gene expression in Caco-2 cells, the
presence of GATA-specific mRNA was assayed (Fig.
5). Total RNA isolated from proliferating
Caco-2 cells was reverse-transcribed and then PCR-amplified with GATA-4
and GATA-6 gene-specific primers. A single PCR product of the exact
predicted size was amplified for both the GATA-4 and GATA-6 primer sets
in Caco-2 cells. There was no detectable amplification of the RT
reaction performed in the absence of RT as a control for genomic DNA
contamination. In addition, GATA-4 and GATA-6 mRNA was detected in
adult human small intestine and liver. GATA-6 mRNA was also detected in
human spleen. Expression of the GAPDH mRNA was detected as a positive control in all of the samples. GATA-4 mRNA was undetectable in control
HeLa cells (data not shown).
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GATA-4, -5, and -6 can activate lactase gene transcription.
To determine whether the intestinal GATA proteins are capable of either
activating or repressing lactase gene transcription, Caco-2 and QT6
cells were cotransfected with wild-type or mutated promoter-reporter
constructs and with the expression constructs for GATA-4, -5, and -6. In cloning the constructs, Xenopus GATA cDNA inserts were
ligated downstream of a recombinant CMV promoter in the expression
vector pCDNA3. Cotransfection with each of the GATA proteins results in
a three- to fourfold transcriptional activation of the wild-type
lactase promoter-reporter construct pgLac (Fig.
6A, compare pxGATA-4, -5, and
-6 vs. pCDNA3). Gao et al. (9) reported a similar
activation of the Xenopus IFABP gene promoter using the
identical GATA-4/5/6 expression constructs. To determine whether GATA
factor overexpression was capable of activating the lactase promoter in
nonintestinal cells, we assayed QT6 cells cotransfected with the GATA
expression constructs. Overexpression of the GATA proteins in the
nonintestinal QT6 cells resulted in comparable transactivation of the
lactase promoter-reporter construct (Fig. 6B).
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GATA factor binding to DNA correlates with functional promoter
activation.
To correlate binding of the GATA factors to the lactase cis
element DNA with the functional promoter activation data, we
performed gel shift and supershift analysis with nuclear extract
isolated from QT6 cells transfected with the GATA-4/5/6 expression
constructs as described above. A GATA-specific complex, a slower
migrating complex A, a faster migrating prominent
complex B, and a nonspecific complex are detected in QT6
cells cotransfected with the GATA-4, -5, and -6 constructs (Fig.
8A). The
relative migration of the GATA-specific complex is slightly different
for each factor, with the GATA-6 reactions consistently resulting in a
less abundant complex. The GATA-specific complex is not detected in the
QT6 cells cotransfected with the empty pcDNA3 expression vectors. The
GATA-specific complex is competed by 100-fold excess unlabeled probe
but not by the MutB probe, in which the GATA sequence is mutated. In
addition, the GATA-4-specific complex is supershifted with the GATA-4
antibody (Fig. 8B). The faint GATA-6-specific complex is
similarly supershifted with GATA-6 antibody (data not shown), and the
GATA-5-specific complex could not be assayed because human-reactive
GATA-5 antibody was not available. These results indicate that the
lactase promoter construct is inactive in QT6 cells (Fig.
2B) but that overexpression of GATA factors can result in
factor binding to the lactase GATA region cis element and
can function to stimulate promoter activity.
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DISCUSSION |
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The GATA-4, -5, and -6 subfamily of zinc finger proteins are
expressed in overlapping patterns in the developing heart and endoderm-derived organs of the gastrointestinal tract including the
stomach, intestine, liver, and pancreas (1, 5, 16, 19,
20). GATA binding sites have been identified in transcriptional regulatory elements of several cardiac-specific target genes. However,
few intestinal target genes have been identified for the GATA-4/5/6
factor subfamily. Fitzgerald et al. (7) demonstrated that
GATA-6 can activate the human lactase promoter in cell culture. Subsequently, the gene encoding IFABP was identified by Gao et al.
(9) as an vitro target for the GATA-4/5/6 factors in
intestinal cell culture. Both genes are expressed in absorptive
enterocytes. To identify regions of the intestinal lactase promoter
involved in regulating lactase transcription, we have characterized the promoter activity of various lactase reporter-promoter constructs. Fragments of the 5'-flanking region linked to the luciferase reporter gene and transfected into Caco-2 cells were assayed for transcriptional activity. A 100 bp promoter-reporter construct possessed maximal activity whereas the
73 bp deletion construct was essentially inactive compared with the promoterless pGL3-Basic vector (Fig. 2A). As expected, the promoter constructs were inactive when
transfected into nonintestinal QT6 cells, which do not express the
lactase gene (Fig. 2B). Close inspection of the sequence in
this positive element mapped between nucleotides
73 and
100 of the
lactase promoter revealed a consensus binding sequence for the GATA
family of zinc finger transcription factors. Fitzgerald et al.
(7) mapped a positive GATA cis element to a
similar region in the human lactase gene. The 28-bp GATA region
sequence is highly conserved in the rat, pig, and human lactase genes,
providing support for an important functional role in regulating
intestinal lactase transcription.
The nuclear proteins interacting with the GATA region element were
assayed using nuclear extract prepared from Caco-2 cells. The
predominant DNA-protein complex seen on gel shift analysis was
determined to be a specific interaction that was competed for by excess
GATA region probe but not by an unrelated oligonucleotide (Fig. 3). To
further define specificity of binding, we assayed for the ability of
four mutant GATA region oligonucleotides to compete for nuclear protein
binding to the probe. Only Mut-B, in which the wild-type sequence of
GATA at nucleotides 90 to
87 was replaced with
CGGT, was unable to compete for binding to the wild-type
probe. The inability of the Mut-B oligonucleotide to compete for
binding suggests that the GATA sequence is essential for
nuclear protein binding. The sequences surrounding the GATA region
comprise the consensus binding site, WGATAR, for the GATA family of
zinc finger transcription factors. Fitzgerald et al. (7)
reported a similar interaction between Caco-2 cell nuclear proteins and
the upstream GATA cis element of the human lactase promoter.
We have identified the proteins interacting with the GATA region
element. Specific antibodies against GATA-4 and GATA-6 recognized the
nuclear protein bound to the GATA region probe, resulting in a
supershifted EMSA complex (Fig. 4). Both GATA-4 and GATA-6 proteins
therefore are expressed in Caco-2 cells and appear to interact with the
GATA element of the lactase promoter. GATA-5 binding could not be
directly assayed because human-reactive GATA-5 antibody was not
available. However, we speculate that the residual EMSA complex not
supershifted in the presence of both GATA-4 and -6 antibodies may
represent GATA-5 binding.
GATA-4/5/6 are each expressed in developing gastrointestinal and gut-derived tissues (5). Fitzgerald et al. (7) demonstrated that GATA-6 RNA is present in Caco-2 cells but were unable to detect GATA-4 protein or RNA in Caco-2 cells by gel supershift or Northern blot hybridization. To assay for the presence of GATA-4 and -6 mRNA, we performed RT-PCR amplification using total RNA isolated from Caco-2 cells (Fig. 5). Specific RT-PCR products corresponding to both GATA-4 and GATA-6 mRNA species were detected. GATA-4 mRNA was undetectable in control HeLa cells (data not shown). These gel shift and RT-PCR results indicate that GATA-4, in addition to GATA-6, interacts with the GATA binding element (Fig. 4) and that both mRNA species are expressed in Caco-2 cells (Fig. 5). The discrepancies may be attributable to differences in GATA-specific antibody affinities and the enhanced sensitivity of RT-PCR for mRNA detection. Variations or shifts in the phenotypic expression pattern of Caco-2 cells cultured by different investigators over time may also account for the discrepancies. In addition, as expected (26), mRNA for both GATA-4 and GATA-6 was detected in both liver and small intestine, but only GATA-6 mRNA was detected in spleen, and no expression was detected in thymus (Fig. 5).
GATA transcription factor function has been implicated in the regulation of intestinal epithelial cell differentiation (9). To determine whether GATA family proteins are capable of either activating or repressing lactase gene transcription, Caco-2 cells were transfected with wild-type or mutated promoter-reporter constructs and with GATA-4, -5, and -6 expression constructs. Each of the GATA expression constructs was capable of activating the luciferase reporter driven by the wild-type lactase promoter in Caco-2 cells (Fig. 6A). Fitzgerald et al. (7) initially reported a similar transactivation function for GATA-6. Our results indicate that the GATA-4 and GATA-5 proteins, in addition to GATA-6, are capable of functioning to activate lactase promoter activity in intestinal cells. In addition, there was transactivation of the lactase promoter-reporter construct by the GATA-4/5/6 expression constructs in cotransfected QT6 fibroblasts (Fig. 6B). GATA nuclear protein overexpression is therefore sufficient to activate the lactase promoter in a nonintestinal cell type that does not express lactase.
The mutated pMutB lactase promoter-reporter construct, in which the WGATAR binding site was disrupted, was not significantly transactivated by GATA factor overexpression (Fig. 7). As predicted from the results of the gel shift analysis, the GATA proteins cannot bind to the mutant lactase promoter in pMutB and therefore cannot activate transcription. GATA protein activation of the pMutD construct was also disrupted, suggesting that bases mutated just downstream of the GATA element are also involved in lactase transcriptional activation. The nuclear proteins interacting with such downstream sequence would not have resulted in an additional gel shift complex if the entire recognition sequence was not contained within the GATA region probe. It is not unexpected that multiple transcription factors may interact with the sequence throughout the 100-bp lactase promoter region. The cotransfection results, combined with the gel shift analysis, suggest that the GATA region of the lactase promoter is essential for GATA protein binding and that when bound, GATA-4, -5, and -6 are capable of activating transcription of the lactase promoter.
To correlate binding of the GATA factors to lactase cis element DNA with the functional promoter activation data, we performed gel shift and supershift analyses with nuclear extract isolated from the cotransfected QT6 cells and the GATA region probe. QT6 cells cotransfected with the GATA expression constructs form a GATA-specific protein-DNA complex that is supershifted with GATA-specific antibody (Fig. 8). GATA factor binding to the GATA region cis element of the lactase promoter therefore is correlated with functional activation of the lactase promoter. Of note, a faster-migrating prominent complex B band was formed in gel shifts with QT6 cell nuclear extract and the GATA region probe and was not supershifted with GATA antibody. Whether other factors, possibly those comprising complex B, are involved in repressing lactase transcription in the nonintestinal QT6 cells is yet to be determined.
Fitzgerald et al. (7) demonstrated that GATA-6 can stimulate activation of the lactase promoter. We have demonstrated that each of the members of the zinc finger GATA binding protein family expressed in intestine (GATA-4 and GATA-5 in addition to GATA-6) can recognize a GATA consensus binding sequence within a positive cis element of the lactase promoter and can activate transcription. Gao et al. (9) demonstrated that the GATA-4/5/6 proteins are also capable of binding to a GATA cis element in the promoter of the IFABP gene and can activate transcription of the IFABP promoter in enterocyte cell culture. These findings for two intestine-specific genes (lactase and IFABP) support a role for the GATA family of transcription factors in regulating intestinal gene expression. Gao et al. (9) described differences in relative abundance of specific GATA proteins along the small intestine crypt-villus axis and in differentiating intestinal cells in culture. Relative levels of each GATA factor within enterocytes may therefore regulate transcription during cell differentiation. Other gene regulatory nuclear proteins may also interact with the GATA proteins to mediate their ability to activate or repress transcription during enterocyte differentiation. It will be of interest to determine whether the complex interactions between multiple GATA transcription factors function to regulate gut differentiation and development.
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ACKNOWLEDGEMENTS |
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We thank Dr. Todd Evans for providing the pxGATA-4, -5, and -6 expression vectors. We thank Drs. D. H. Alpers, G. M. Gray, and M. P. Scott for helpful discussions and suggestions. We thank Libra White for expert secretarial assistance.
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FOOTNOTES |
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E. Sibley was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02552.
Address for reprint requests and other correspondence: Eric Sibley, Dept. of Pediatrics, Stanford Univ. Medical Center, 750 Welch Rd., Suite 116, Palo Alto, CA 94304 (E-mail: erc{at}leland.stanford.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.
Received 27 December 1999; accepted in final form 12 August 2000.
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