1 Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics, The Floating Hospital for Children, New England Medical Center, and 2 Tufts University School of Medicine and the Center for Gastroenterology Research on Absorptive and Secretory Processes, Boston 02111; 3 Tufts University School of Nutrition Science and Policy, Medford, Massachusetts 02155; 4 Free University of Amsterdam School of Medicine, 1081 HV Amsterdam; and 5 University of Amsterdam School of Medicine, 1105 AZ Amsterdam, The Netherlands
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ABSTRACT |
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The effects of
GATA-4, -5, and -6, hepatocyte nuclear factor-1 (HNF-1
) and -
,
and Cdx-2 on the rat and human lactase-phlorizin hydrolase (LPH) and
human sucrase-isomaltase (SI) promoters were studied using transient
cotransfection assays in Caco-2 cells. GATA factors and HNF-1
were
strong activators of the LPH promoters, whereas HNF-1
and Cdx-2 were
strong activators of the SI promoter, although GATA factors were also
necessary for maximal activation of the SI gene. Cotransfection of
GATA-5 and HNF-1
together resulted in a higher activation of all
three promoters than the sum of the activation by either factor alone,
demonstrating functional cooperativity. In the human LPH promoter, an
intact HNF-1 binding site was required for functional synergy. This
study is the first to demonstrate 1) differential activation
of the LPH and SI promoters by multiple transcription factors
cotransfected singly and in combination and 2) that GATA and
HNF-1 transcription factors cooperatively activate intestinal gene
promoters. Synergistic activation is a mechanism by which higher levels
of tissue-specific expression might be attained by overlapping
expression of specific transcription factors.
lactase-phlorizin hydrolase; sucrase-isomaltase; Cdx-2; hepatocyte nuclear factor-1
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INTRODUCTION |
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ABSORPTIVE ENTEROCYTES ORIGINATE from undifferentiated, multipotent stem cells located near the base of small intestinal crypts (20). Through a continuous process of cell division, migration, differentiation, apoptosis, and exfoliation, these cells are renewed every 3-5 days. Absorptive enterocytes comprise over 95% of mucosal cells on small intestinal villi and are responsible for the terminal digestion and absorption of nutrients. To carry out these specialized functions, absorptive enterocytes acquire a differentiated phenotype characterized by the expression of functionally relevant proteins such as digestive enzymes, receptors, transporters, and cytoplasmic carriers. Differences in the panel of genes expressed along the proximal-to-distal (horizontal) axis result in regional differences in intestinal function. The mechanisms that underlie the process of intestinal differentiation are poorly understood.
The effects of specific transcription factors in the regulation of absorptive enterocyte-specific differentiation can be studied by the use of marker genes whose expression parallels that of key events in the differentiation process. Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are intestinal disaccharidases that are expressed in villus enterocytes and display a differentiation-specific pattern of expression. The LPH and SI genes are similarly expressed in jejunum and proximal ileum but demonstrate different patterns during development. LPH gene expression is highest in newborn mammals and declines during weaning, whereas SI gene expression is low or undetectable at birth and increases to adult levels during weaning. In most humans, LPH expression declines, similar to that in other mammals, but in a subpopulation of humans, LPH expression remains high throughout adult life. Previous work in our laboratory has shown that the developmental pattern of lactase activity in rats (8) and the genetic pattern in humans (14) are correlated with the abundance of its mRNA and also that the horizontal and developmental patterns of LPH expression in rats are transcriptionally regulated (32). It is now widely accepted that LPH expression is controlled mainly by the rate of gene transcription.
Studies (33, 40, 41, 64, 68) in transgenic mice indicate
that the 5'-flanking regions of both the LPH and SI genes direct
transgene expression to villus epithelium. Analysis of the published
sequence reveals that the LPH (6, 71) and SI genes
(9, 63, 73) contain GATA, hepatocyte nuclear factor-1 (HNF-1), and Cdx-2 consensus binding sites in their 5'-flanking regions
(see Fig. 1). These transcription factor families have all been shown
to influence the regulation of cellular differentiation. Because GATA,
HNF-1, and Cdx-2 factors are expressed contiguously only in intestinal
epithelium and their cognate binding sites in the LPH and SI genes are
adjacent to the TATA box and to each other, we hypothesized that these
transcription factors together are important regulators of LPH and SI
gene expression.
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The GATA family of zinc finger proteins, defined by a highly conserved DNA-binding domain that interacts specifically with DNA elements containing a consensus WGATAR sequence (W = A or T; R = A or G), has been implicated in cell lineage differentiation during vertebrate development. GATA-1, -2, and -3 are expressed in developing blood cells during hematopoiesis, whereas GATA-4, -5, and -6 are expressed together only in cardiac tissue and small intestine, but individually (or in overlapping patterns) in the stomach, liver, lungs, spleen, ovary, testis, and bladder (3, 29, 36, 50, 51). In both mouse (3) and chicken intestine (18), GATA-4 transcripts are distributed all along the villus, with increasing levels accumulating toward the villus tips. In chicken intestine (18), GATA-5 mRNA is localized to differentiated cells of the villi, whereas GATA-6 transcripts are localized to cells of the upper crypt and lower villus. Mice homozygous for the GATA-4 (35, 48) or GATA-6 (31, 52) null allele die in utero and therefore provide little information regarding maintenance of intestinal differentiation in adults. GATA-5 knockout mice survive and reproduce despite pronounced genitourinary abnormalities in females (49) but show no gross abnormalities in intestinal structure or histology (intestinal gene expression was not reported). Recent studies (15, 17, 18) have suggested that GATA-4, -5, and -6 may modulate the expression of intestinal genes. Gao et al. (18) showed that GATA-4, -5, and -6 can directly stimulate the activity of the promoter for the Xenopus intestinal fatty acid binding protein (IFABP) gene. Fitzgerald et al. (17) demonstrated a 15-fold induction of a human LPH promoter-reporter construct using a chicken GATA-6 expression vector. Fang et al. (15) showed that all three members of this GATA subfamily are capable of activating the rat LPH promoter. Two potential GATA-binding sites are present in the rat and human LPH genes (see Fig. 1), although the 3' site in the rat LPH gene contains only 5 bp of the 6-bp consensus. The human SI gene contains a single putative GATA motif within 100 bp 5' to the transcriptional start site, the characterization of which has not yet been reported.
HNF-1 transcription factors contain a novel DNA-binding homeodomain
distantly related to the POU domain and bind as dimers to the consensus
sequence GTTAATNATTAAC (42, 67). Two members of this
family, HNF-1 and HNF-1
, have been described (43, 55) and share highly homologous DNA binding domains but
divergent activation domains. Originally thought to be liver specific,
these two proteins are also expressed in kidney, stomach, and small and
large intestine (5, 34, 43). HNF-1
is also expressed in
lung and ovary (43). In mouse small intestine, HNF-1
and HNF-1
mRNAs are expressed at high levels in crypt cells, and expression is gradually reduced until there are low levels at the
villus tips (58). Reports (1, 38, 54) of mice
lacking HNF-1
gave variable results, including sterility, liver
enlargement, renal dysfunction, non-insulin-dependent diabetes
mellitus, and reduction in the expression of certain liver genes such
as albumin,
1-antitrypsin, phenylalanine hydroxylase,
and liver FABP. The effect of HNF-1
gene disruption on
gastrointestinal function has not been described. HNF-1
knockout
mice have a disorganized visceral endoderm and die at embryonic
day 7.5 (4, 11). In addition to regulating the
expression of liver genes, HNF-1 factors also modulate genes that are
expressed only in small and/or large intestine, including LPH
(59), SI (72), and guanylin
(23). In the pig LPH gene, mutational analysis in
cotransfection experiments localized an HNF-1
-mediated response to
an HNF-1 binding site within 227 bp of the transcriptional start site
(called CE-LPH2c) (59), which is conserved in position in
the human (
90 to
70 bp) and rat LPH genes (
86 to
66 bp) (see
Fig. 1). The human SI promoter has two HNF-1 binding sites, SIF2 (
90
to
70 bp) and SIF3 (
176 to
159 bp), that interact with both
HNF-1
and HNF-1
(72). These two family members,
which can form homodimers as well as heterodimers, differentially
activate both the LPH and SI promoters (59, 72),
suggesting that the relative abundance of HNF-1 isoforms may be
important for the regulation of genes with HNF-1 binding sites.
Cdx-2, a homeodomain-containing transcription factor that binds to the consensus sequence TTTAY (Y = A or C), is highly expressed in adult mammalian small intestine and colon and is also detectable in pancreas (19, 25, 26, 60). In mouse small intestine, Cdx-2 is expressed in all epithelial cells in both crypts and villi, with the exception of Paneth cells at the base of crypts (24). Mice heterozygous for a Cdx-2 null mutation develop adenocarcinoma of the small intestine and colon, whereas homozygote null mutant embryos die between 3.5 and 5.5 days postcoitum (10). Cdx-2 has been shown (61) to induce differentiation in IEC-6 cells, suggesting a role in cellular differentiation. Cdx-2 has also been shown to bind and activate the LPH (7, 16, 22, 53, 65, 66) and SI genes (60) via conserved cis elements called CE-LPH1a and SIF1, respectively, both of which are adjacent to their TATA boxes (Fig. 1). CE-LPH1a contains a single binding site, whereas SIF1 contains two adjacent sites on opposite strands. Additional Cdx-2 binding sites, CE-LPH1b (59) and CE-LPH-1c (47), have been identified in the pig LPH gene.
At present, the relative importance of GATA-4, -5, and -6, HNF-1 and
, and Cdx-2 on LPH and SI gene expression and possible species
differences between rat and human LPH gene expression have not been
characterized together under the same experimental conditions.
Furthermore, possible interactions among these transcription factors
have only recently been examined (47). Mitchelmore et al.
(47) have demonstrated that HNF-1
and Cdx-2 physically interact and cooperatively activate the pig LPH promoter. In the present study, we demonstrate for the first time that GATA, HNF-1, and
Cdx-2 transcription factors regulate the LPH and SI promoters differently and that GATA-5 and HNF-1
synergistically activate these promoters.
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MATERIALS AND METHODS |
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Chemicals and reagents. All chemicals were purchased from Sigma Chemical (St. Louis, MO), GIBCO-BRL (Gaithersburg, MD), or Fisher Scientific (Fair Lawn, NJ) unless indicated otherwise. Enzymes were purchased from GIBCO-BRL, Promega Biotech (Madison, WI), or Pharmacia Biotech (Piscataway, NJ) unless indicated otherwise. Radioactive nucleotides and [14C]chloramphenicol were purchased from DuPont-New England Nuclear (Boston, MA). [35S]methionine (Redivue) was purchased from Amersham Life Science (Arlington Heights, IL).
Plasmids.
The human growth hormone (hGH) gene was used as a reporter in
transfection assays. As described previously (33), the hGH gene was subcloned into pBluescript II KS+ (Stratagene, La Jolla, CA).
This construct, referred to as Bluescript growth hormone (BSGH), served
as a promoterless control for background and the basic construct into
which all promoter sequences were subcloned. Sequence from the rat LPH
(rLPH) gene (2038 to +15 bp) was isolated (71) and
subcloned into BSGH 5' to the hGH reporter, as previously described
(33). A segment of the human LPH (hLPH) gene (
1025 to
+11 bp) was amplified by PCR from human DNA isolated from discarded jejunum of patients undergoing elective gastric bypass surgery. The 5'
(5'-ATGGGTACCAAGATACTTATTATAGGAAGAGGA-3') and 3'
primers (5'-ATCTAAGCTTTTTCTAGGAACTGTTAGGAGG-3'), designed
from the published sequence (6), contain Kpn I
and Hind III sites, respectively (underlined), to
facilitate subcloning into BSGH. A segment of the human SI (hSI) gene
(
183 to +54 bp, gift of Dr. P. Traber, University of Pennsylvania)
(73) was also amplified
(5'-ATGGGTACCTGACAGTACAATTACTAATTAAC-3 and
5'-ACGTAAGCTTAGCCTGTTCTCTTTGCTATG-3) and subcloned into
BSGH. Deletions and mutations of all other rat LPH, human LPH, and
human SI promoter-reporter plasmids were constructed by PCR using the same Kpn I-Hind III strategy. Mutations were
introduced into primer and template oligonucleotides. All constructs
were confirmed by sequencing.
Cell culture and transfections. The Caco-2 cell line was the principal cell line used in the present study. Originally derived from a human colon adenocarcinoma, Caco-2 cells differentiate on confluence, exhibiting characteristics of small intestinal absorptive enterocytes, including a microvillus membrane and expression of small intestinal genes. The Caco-2 cells used in the present study have been previously characterized (69) with respect to LPH and SI expression.
All cells were grown in DMEM (BioWhittaker, Walkersville, MD) supplemented with 5 µg/ml penicillin-streptomycin and containing 10% FCS. Cells at 80-95% confluence were collected by trypsinization, and aliquots were transfected using 10 µg of the promoter-reporter constructs and when indicated, 8 µg of expression vector and 1 µg of RSVCAT as a control for transfection efficiency. pBluescript II KS+ (Stratagene) was added as a carrier (total DNA/transfection = 35 µg). Transfections were carried out by electroporation (Pulse Controller II, Bio-Rad, Hercules, CA) at 300 V and 950 µF in Cytomix buffer (240 mM KCl, 0.3 mM CaCl2, 20 mM HPO4/KH2PO4, pH 7.6, 50 mM HEPES, pH 7.6, 4 mM EGTA, pH 7.6, and 10 mM MgCl2) supplemented with fresh glutathione (3.08 mg/ml). After transfection, cells were plated at 75-90% confluence in six-well plates. Medium was replaced after 24 h, and cells were harvested after 48 h (i.e., 24 h after the last medium change). All plates were confluent at the time of harvest. The amount of hGH secreted into the medium over a 24-h period was used as an indicator of transcriptional activity. The concentration of hGH was measured using an 125I immunoassay kit (Allegro hGH, Nichols Institute, San Juan Capistrano, CA). To analyze CAT activity, cell lysates were prepared by freeze-thaw cycling, and the protein concentration of the supernatant was determined by a Coomassie protein assay (Pierce, Rockford, IL). CAT activity was measured in cell lysates as described previously (56). Transcriptional activity was expressed as total hGH secreted into the medium over 24 h relative to total CAT activity in cell lysates (expressed as mg hGH/U CAT activity). The transcriptional activity of BSGH was subtracted from all other constructs to correct for background.Nuclear extracts.
Nuclear extracts were isolated from confluent cells grown on 10-cm
plates directly (untransfected) or after transfection of specific
expression vectors. For transfected cells, 8 µg of expression vector
were transfected as described above, and two transfection reactions
were combined on 10-cm dishes. Nuclear extracts were prepared by
modifying previously published methods (2). Cells were
harvested by scraping, and resuspended in ice-cold buffer A
[10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)], incubated on ice for 10 min, and lysed by vortexing for 10 s. Nuclei were pelleted in a microcentrifuge for 10 s at
full speed and extracted in 100 µl of buffer C (20 mM
HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.5 mM
PMSF) for 20 min on ice. The resulting lysate was cleared of cellular
debris by centrifugation for 15 min and stored as a nuclear extract at 80°C. The protein concentration was determined as described
previously by Kalb and Bernlohr (28).
In vitro transcription-translation.
Labeled ([35S]methionine) and unlabeled proteins were
synthesized using a reticulocyte lysate transcription-translation
system (TNT, Promega) according to the manufacturer's instructions.
Because all expression vectors contain a T7 DNA-dependent RNA
polymerase site between the viral promoter and the cDNA insert, a
T7-based TNT system was employed. However, because in vitro
transcription of the GATA-5 expression vector using a T7 polymerase
transcription-translation system was inefficient and PCR could not
amplify the GATA-5 coding region, we subcloned a 1.5-kb Sma
I-Eco RV fragment containing the GATA-5 coding region
into the Sma I site of pGEM-7Zf() and isolated (screened
by restriction digests, confirmed by sequencing) plasmids whose
orientation enabled correct transcription from the SP6 polymerase site.
GATA-5 was efficiently synthesized from this plasmid using an SP6 TNT system.
EMSAs.
Oligonucleotides used as probes and competitors in EMSAs are shown in
Fig. 2. Probes were made by annealing a
10-fold molar excess of 10- or 11-base reverse-strand oligonucleotide
to the forward strand by boiling for 2 min in annealing buffer (100 mM NaCl, 10 mM Tris, pH 8.0, and 1 mM EDTA), then slow cooling for 2 h (<32°C). The annealed oligonucleotides were extended with the
large fragment of DNA polymerase I (Klenow) in the presence of
[32P]dATP in extension buffer (50 mM Tris, pH 8.0, 10 mM
MgCl2, and 1 mM DTT) for 1 h at 37°C. After removing
an aliquot for specific activity determinations by TCA precipitation,
we separated the labeled probe from the short antisense
oligonucleotides using a spin column with a 12-base cutoff (STE
Select-D G-25, 5 Prime3 Prime, Boulder, CO). Purified probes were
stored at
20°C. The specific activity of all probes exceeded
107 cpm/pmol. Competitors were synthesized by annealing
equimolar amounts of full-length sense and antisense oligonucleotides
as described above.
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RT-PCR. RNA was isolated from Caco-2 cells grown on six-well plates using TRIzol reagent (GIBCO-BRL) according to the manufacturer's instructions. To synthesize first-strand cDNA, purified RNA was annealed to oligo(dT) and extended with Superscript II (GIBCO-BRL) according to the manufacturer's suggested protocol. PCR was carried out on the first-strand cDNA using the following oligonucleotides: human LPH (5'-GTCCCACTACCGTTTTTCCA-3', 5'-CGTCTGTGGTAGGTCCCAGT-3'), human SI (5'-CTCTCCATCGGTCTTTCCAA-3', 5'-AGAAGGCTCTGGGAGGTGTT-3'), human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5'-GGGTCATCATCTCTGCCCCCTCTG-3', 5'-CCATCCACAGTCTTCTGGTGGCA-3'), and mouse GATA-5 (5'-GCCTCTTCTCCCACTCTCCT-3', 5'-GTAGGACCCCACTGAGACCA-3'). After an initial melting step at 95°C for 2 min, the oligonucleotides were annealed to the cDNA template at 55°C for 40 s, extended at 72°C for 60 s, and melted at 95°C for 20 s. After 30 cycles, the reaction was extended for 10 min at 72°C, then cooled to 4°C. The amplification products were analyzed on 1.8% agarose gels stained with ethidium bromide.
Statistics. The t-test or one-way ANOVA was employed in all statistical analyses using InStat software (Graphpad Software, San Diego, CA). The Dunnett's or Tukey multiple comparison test was employed for all statistically significant ANOVA analyses.
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RESULTS |
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Characterization of individual effects of transcription factors on
LPH and SI promoters.
The individual effects of multiple transcription factors on the LPH and
SI promoters have not been compared under the same experimental
conditions. Thus transient cotransfection assays in Caco-2 cells were
carried out using individual expression vectors for each of the
transcription factors under study. As shown in Fig.
3A, GATA factors are capable
of activating the LPH and SI promoters. Transcriptional activities of
promoter-reporter constructs were increased when cotransfected with
expression vectors for GATA-4, GATA-5, or GATA-6 compared with pRC-CMV,
a plasmid containing only the CMV promoter. GATA-stimulated
transcriptional activities were higher for constructs that contain
consensus GATA binding motifs in their promoters (i.e., rLPH108,
rLPH2038, hLPH118, hLPH1025, and hSI183). Activation was generally
strongest with the GATA-5 expression vector, demonstrating
statistically significant increases from pRC-CMV controls for rLPH108,
rLPH2038, hLPH118, and hSI183. Transcriptional activities of the basal
promoter constructs rLPH37 and hLPH37 and the negative control
promoter-reporter construct RSVdE cotransfected with the GATA
expression vectors revealed minimal responses, although all were
significantly increased over the pRC-CMV controls with GATA-5
cotransfection (P < 0.01 for each). These data
demonstrate that GATA family transcription factors activate the LPH and
SI promoters, consistent with previous reports for LPH (15,
17). Activation of the SI promoter by GATA-5 has not been
previously reported.
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Combined effects of transcription factors on LPH and SI expression.
To characterize the combined effects of multiple transcription factors
and to identify possible functional interactions among transcription
factor families, cotransfection experiments were carried out using all
possible combinations of GATA-5, HNF-1, and Cdx-2. These
transcription factors were chosen because they were either strong
activators (GATA-5 and HNF-1
) or the only member (Cdx-2) of the
transcription factor families under study. Furthermore, all are
expressed on villi coincident with LPH and SI expression. As shown in
Fig. 5A, the individual
effects of HNF-1
and Cdx-2 produced significantly greater
transcriptional activities for hSI183 than for rLPH108 and hLPH118. The
combination of GATA-5 plus HNF-1
resulted in mean transcriptional
activities that were similar for all three promoter constructs. The
combinations of GATA-5 plus Cdx-2, HNF-1
plus Cdx-2, and all three
transcription factors together resulted in mean transcriptional
activities that were significantly greater for hSI183 than for either
of the LPH promoter-reporter constructs (P < 0.05 for
each). GATA-5 plus HNF-1
produced the highest activation of all
combinations for rLPH108 and hLPH118, whereas all three expression
vectors together produced the greatest response for hSI183.
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Cell-specific activation of human LPH promoter by GATA-5.
To determine whether the GATA-5 response is cell specific,
cotransfection experiments with the GATA-5 expression vector and the
hLPH118 construct were carried out in different cell lines. Table
2 shows a wide range of transcriptional
responses (fold change over baseline) in both intestinal (T84, Caco-2,
HT-29, COLO 320DM) and nonintestinal cell lines [HepG2, Madin-Darby
canine kidney (MDCK), HeLa, 3T3]). The highest responses were found in T84, Caco-2, (both colonic adenocarcinoma), HepG2 (hepatocellular carcinoma), and MDCK (kidney epithelial) cells, whereas the lowest responses were observed in HeLa (cervical carcinoma), COLO 320DM (adenocarcinoma of the colon), and 3T3 (fibroblast) cells. Parallel results were found for rLPH108 and hSI183 (not shown). EMSAs using a
probe specific for HNF-1 or Cdx-2 binding (Fig.
7) revealed a close correlation between
the presence of HNF-1 binding activity and GATA-5 activation of hLPH118
(Table 2). Those cell lines that had abundant HNF-1 binding activity
(Fig. 7A) also displayed high levels of GATA-5 activation
responses (Table 2), whereas those cell lines with no evidence of HNF-1
binding activity demonstrated limited GATA-5 responses. There was no
correlation between the presence of Cdx-2 binding activity (Fig.
7B) and GATA-5 activation.
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Mutational analysis of GATA and HNF-1 binding sites in human LPH
promoter.
To characterize the effects of disruptions in protein-DNA interactions
during individual and combined GATA-5 and HNF-1 cotransfections, mutations were introduced into the GATA and HNF-1 binding sites of the
human LPH promoter. To first demonstrate that the introduced mutations
disrupt specific protein-DNA interactions, EMSAs were carried out using
nuclear extracts from Caco-2 cells transfected with the GATA-5
expression vector and a probe that spans the 5' GATA and the HNF-1 site
of the human LPH gene (hG1H). As shown in Fig.
9, two main complexes were identified.
The faster mobility complex was competed with an oligonucleotide
containing the wild-type GATA site (hG1; Fig. 9, lane
3), but not with an oligonucleotide containing the specific
mutation in the GATA motif (Fig. 9, lane 4). This complex
also supershifted with a GATA-5 antibody (Fig. 9, lane 5)
and was therefore indicated as GATA-5 complex. The slower mobility
complex competed specifically with an oligonucleotide containing an
intact human HNF-1 site (hH; Fig. 9, lane 6), but not with
an oligonucleotide containing a mutated site (Fig. 9, lane
7), and supershifted specifically with an HNF-1
antibody (Fig.
9, lane 8). This complex was indicated as HNF-1 complex. Noteworthy is that specific competition with hH revealed partial competition of the GATA-5 complex. An oligonucleotide probe containing the 3' GATA site (hG2) also binds GATA factors, but a
mutation introduced into this site does not compete (not shown). These data demonstrate that mutations introduced into the GATA and HNF-1 sites of the human LPH promoter disrupt specific protein-DNA
interactions at these sites.
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DISCUSSION |
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This is the first study to extensively compare, under the same
experimental conditions, the effect of specific transcription factors
on multiple intestinal gene promoters and to identify functional
cooperativity between two major classes of transcription factors, the
zinc finger GATA proteins and the homeobox HNF-1 family. Studies to
date have generally examined regulation of individual intestinal gene
promoters, and recently, the effect of multiple transcription factors
(46, 47, 72), but only on single promoters. In the present
study, we found that GATA, HNF-1, and Cdx-2 transcription factors
regulate the rat LPH, human LPH, and human SI promoters differently and
that GATA-5 and HNF-1 cooperatively activate these promoters.
Together, these data suggest that the specific expression of intestinal
genes is modulated by complex interactions involving multiple
transcription factors.
Our data are in agreement with previous reports demonstrating
individually that members of the GATA-4, -5, and -6 subfamily are
capable of activating the human (17) and rat LPH promoters (15), HNF-1 is capable of activating the pig LPH
(59) and human SI (72) promoters, and Cdx-2
is capable of activating the pig (65) and rat LPH
(16) and human SI promoters (60). Our data
also parallel a recent report by Mitchelmore et. al. (47),
who showed that HNF-1
and Cdx-2 cooperatively activate the pig LPH
promoter, a finding we have also demonstrated for the human LPH
promoter. Thus our data are in general agreement with previous reports.
GATA and HNF-1 transcription factors together are key activators of LPH
gene expression, whereas Cdx-2 has a comparatively weak effect on the
LPH promoters. Although GATA-4, -5, and -6, HNF-1, and Cdx-2 are all
capable of activating the LPH promoters individually in cotransfection
assays, comparative analyses reveal that the rat and human LPH
promoters generally demonstrate greater transcriptional responses to
the GATA factors and HNF-1
than to Cdx-2 (Table 1). Similarly,
activation of endogenous LPH transcription in Caco-2 cells occurred
with overexpression of GATA-5 or HNF-1
but not with Cdx-2 (Fig. 6).
In contrast to our data, in which Cdx-2 showed no effect on LPH mRNA
abundance, Fang et al. (16) reported a modest twofold
increase in LPH mRNA abundance in Cdx-2-transfected Caco-2 cells, which
could be due to a more sensitive quantification technique (densitometry
of the negative image of ethidium bromide-stained bands in agarose
gels) employed in their study. Our results depict the comparative
effects of single and multiple transcription factors, and thus relative
to GATA-5 or HNF-1
cotransfection, Cdx-2 appears to be a
comparatively weak activator of endogenous LPH gene transcription in
Caco-2 cells. Finally, the combination of GATA-5 and HNF-1
produces
the strongest activation of the LPH promoters, demonstrating functional
cooperativity between these two transcription factor families
(discussed below). Together, these data suggest that members of the
GATA and HNF-1 transcription factor families are significant activators
of LPH gene expression, whereas Cdx-2, although perhaps necessary for
LPH expression in vivo, has a comparatively lesser role in modulating
LPH gene expression.
Although some experiments argue that HNF-1 and Cdx-2 have a greater
role than GATA factors in the modulation of the SI promoter, other
experiments suggest that all three transcription factor families are
important in the activation of SI gene expression. As shown in Table 1,
transcriptional responses for the human SI promoter were strongest for
HNF-1 and Cdx-2 compared with those for the GATA factors.
Furthermore, greater basal expression of the human SI promoter compared
with the LPH promoters could be due to the relative abundance of
endogenous transcription factors synthesized in Caco-2 cells. Based on
the amount of extract used, the intensity of complexes on
autoradiographic film, and the length of exposure time, data from EMSAs
suggest that Caco-2 cells synthesize abundant amounts of HNF-1 and
Cdx-2 proteins but limited amounts of GATA proteins (not shown). Thus
it could be argued that HNF-1 and Cdx-2 have a greater role than GATA
factors in the activation of the SI promoter (in contrast to the LPH
promoters), resulting in a greater basal (unstimulated) expression.
HNF-1 and Cdx-2 have greater affinities for SIF3 and SIF1,
respectively, compared with their cis element counterparts
in the LPH genes (Fig. 4), which could explain the greater basal
transcriptional activities of the SI vs. the LPH promoters. Noteworthy
is that hSIF1 contains two Cdx-2 motifs, whereas rC and hC (i.e.,
CE-LPH1a) each have a single binding motif, which likely accounts
for the differences in affinity between the two elements.
Cotransfection experiments using combinations of expression vectors,
however, suggest that all three transcription factor families
cooperatively activate the human SI promoter. For hSI183, cotransfection of the combination of GATA-5, HNF-1, and Cdx-2 expression vectors produced the highest transcriptional activity of any
combination of transcription factors (Fig. 5A) and
demonstrated a strong synergistic interaction response (Fig.
5B). Furthermore, all three transcription factors were also
important for endogenous SI gene expression, because an amplified
product corresponding to human SI mRNA was detected only in the RNA
isolated from Caco-2 cells transfected with the GATA-5, HNF-1
, and
Cdx-2 expression vectors (Fig. 6). Activation of SI individually by
HNF-1 and Cdx-2 transcription factors has previously been demonstrated
(60, 72), but the present study is the first to show
activation of the SI promoter by the GATA family and cooperative
activation of the SI promoter by members of the three different
transcription factor families.
The rat and human LPH promoters, with some exceptions, demonstrated
similar responses to the transcription factors under study. Both
promoters were significantly activated by GATA, HNF-1, and Cdx-2
transcription factors (Fig. 3), and their patterns with cotransfection
of multiple expression vectors were similar to each other (Fig.
5A). A significant difference between the two promoters,
however, was revealed by cotransfection of the GATA-6 expression
vector, in which the rat LPH promoter demonstrated a greater
transcriptional response than the human LPH promoter (Table 1). This
suggests a preferential activation of the rat promoter by GATA-6 and
may indicate differential affinities of the GATA factors for specific
sequence in the binding sites, although this has not been specifically
tested. A second difference was found with HNF-1 cotransfection, in
which the human LPH promoter demonstrated a significantly greater
activation response than the rat LPH promoter (Table 1), suggesting a
preferential activation of the human LPH promoter by HNF-1
(although
no difference in HNF-1 binding affinity was noted between the rat and
human HNF-1 sites; Fig. 4B). Finally, the combination of
HNF-1
plus Cdx-2 as well as the combination of all three expression
vectors demonstrated antagonistic or additive responses for rLPH108,
but strong synergistic responses for hLPH118 (Fig. 5B,
discussed further below). Together, these data suggest that the
position, configuration, and/or specific sequence within binding motifs
of the LPH promoter may influence the magnitude and direction
(activation vs. repression) of responses to specific combinations of
transcription factors. Implications for the differential regulation of
LPH gene expression between humans and other mammals are unknown at
this time.
The basal promoter constructs (i.e., rLPH37 and hLPH37), with varying
degrees of statistical significance, were activated by many of the
transcription factors tested (Fig. 3). It is possible that sequence in
the hGH reporter gene may activate reporter expression in response to
specific expression vectors. However, the activity of BSGH after
specific transcription factor stimulation was subtracted from that of
all other constructs stimulated with the same transcription factor,
thus eliminating activity due to the reporter itself. In a
preliminary report (70), we showed that Cdx-2 is capable of binding and activating the rat and human LPH promoters at their TATA
box regions. Thus Cdx-2 may directly activate these promoters at their
TATA boxes, whereas GATA and HNF-1 factors, which do not bind to the
37-bp to
1-bp region (not shown), may activate transcription of a
basal promoter indirectly (in contrast to direct protein-DNA
interactions) or by direct interactions with the transcriptional initiation complex.
In the human LPH gene, we confirm that the two GATA motifs bind GATA-5 with similar affinities (Fig. 4A) and demonstrate that both sites play a role in the activation of the human LPH promoter (Fig. 10). Mutations introduced into either one of the sites had little effect on GATA-stimulated activity, but mutations introduced into both sites resulted in a significant reduction in GATA-5-stimulated transcriptional activity (Fig. 10). In a previous report, Fitzgerald et. al. (17) demonstrated that a mutation in the 5' GATA site of the human LPH promoter resulted in a decrease in GATA-6 activation from the wild-type control. The reason for the apparent difference between our study and that of Fitzgerald et. al. (17) is unknown, but could be due to differences in the ability of chicken GATA-6 compared with mouse GATA-5 to activate the human LPH promoter. In the human LPH gene, the 5' GATA site (hG1) is on the forward strand whereas the 3' GATA site (hG2) is on the reverse strand, which could result in possible differences in the ability of different GATA proteins to activate the LPH gene through the hG2 site. We conclude from our data that GATA-5 is capable of activating the human LPH promoter through either GATA motif and that the two sites together do not activate the LPH gene any more than either of the sites individually.
Binding site affinity experiments suggest that GATA-5 prefers to bind
WGATAT. Unbiased analysis of random GATA motifs revealed that the
consensus for the GATA-1, -2, and -3 subfamily is WGATAR, where W is A
or T and R is A or G, but clear preferences in core and adjacent
sequences were noted (30, 45). A similar characterization of the GATA-4, -5, and -6 subfamily has not been reported. Comparison of the relative affinity of GATA-5 for each of the GATA motifs (Fig.
4A), as summarized in Fig.
11, reveals that GATA-5 binds equivalently if A or T (but not G) is in the 5' position and prefers T
in the 3' position, although it continues to bind with lesser affinity
if A or G are in this position. We therefore propose that the GATA-5
consensus is WGATD, where D is A, G, or T.
|
In cotransfection experiments, the combination of HNF-1 and Cdx-2
demonstrated synergistic activation of the human LPH promoter (hLPH118), but antagonistic interactions on the rat LPH promoter (Fig.
5B). Our data on the human LPH promoter are in agreement with a previous report by Mitchelmore et al. (47), who
demonstrated that HNF-1
and Cdx-2 functionally interact, resulting
in a synergistic activation of the pig LPH promoter. Although the
reason for the apparent species difference in response to HNF-1
and
Cdx-2 is not readily known, structural similarities and differences
among the rat, human, and pig LPH promoters were noted. There is
greater conservation of sequence identity in the first 100 bp of
5'-flanking region between the human and pig LPH genes (93%) than
between the human and rat LPH genes (78%). This includes the HNF-1
binding motif, in which the 3' half is completely conserved between the human and pig sequence and more closely correlates with the HNF-1 consensus than that of the rat LPH HNF-1 (rH) site. Noteworthy, however, is that rH and hH demonstrate similar affinities for HNF-1
binding in competition EMSAs (Fig. 4). Sequence differences also
include the TATA boxes, in which the human and pig LPH genes contain a
true TATA motif, whereas the rat LPH gene contains a CATA motif,
although the effect of such a difference on transcriptional regulation
is unknown. The recent demonstration (47) of a second Cdx-2 binding site (called CE-LPH1c) 5' to the HNF-1 site in the pig
LPH gene is unlikely to explain the species differences in response to
these transcription factors, because CE-LPH1c is not present in the
human LPH gene but is present in the rat LPH gene (
102 to
97 bp).
Because rLPH108 does not demonstrate synergistic activation by HNF-1
and Cdx-2, it is likely that the presence or absence of this site does
not play a direct role in the cooperative activation by these two
transcription factors. Finally, with regard to binding site position,
the distance between the 5' base of the HNF-1 motif and that of the
Cdx-2 binding site at CE-LPH1a is 33 bp for the human and pig promoters
and 28 bp for the rat LPH promoter, representing a difference of
approximately one-half turn of the helix, which could affect the
ability of HNF-1
and Cdx-2 to interact. At present, the process
underlying the cooperative interactions between HNF-1
and Cdx-2 is
unknown, but the findings in the present study imply that the mechanism
is more complex than simple protein-protein interactions and the
presence of HNF-1 and Cdx-2 binding sites.
In the present report, we demonstrate for the first time functional
cooperativity between GATA-5 and HNF-1. This cooperativity was
strongest for the human LPH promoter but was present for the rat LPH
and human SI promoters (Fig. 5B) as well. Functional
cooperativity between zinc finger GATA proteins and homeobox
transcription factors has been previously demonstrated (13, 37,
57) for GATA-4 and Nkx-2.5, a cardiac-restricted homeobox
protein. GATA-4 was shown to physically associate with Nkx-2.5 and
cooperatively activate cardiac-specific promoters. Furthermore,
mutational analysis of each transcription factor indicated that
physical interaction was required for functional synergy. Deletional
analysis in GST pull-down assays revealed that the COOH-terminal zinc
finger of GATA-4 was required to interact with Nkx-2.5, and helix III
of the homeodomain of Nkx-2.5 was necessary to associate with GATA-4. Both regions are known to bind their cognate DNA binding sites, demonstrating a close association between protein-protein and protein-DNA interactions. Indeed, the homeodomain of HNF-1
was shown
to be necessary for interactions with Cdx-2 (47). Based on
these models, we hypothesize that the COOH-terminal zinc finger of
GATA-5 and the homeodomain of HNF-1
physically interact and that
this interaction is necessary for synergistic activation of the human
LPH promoter.
Based on the findings in the present study, we hypothesize that the patterns of expression of certain intestinal genes are due, at least in part, to the simultaneous presence of GATA, HNF-1, and Cdx-2 transcription factors. Members of all three transcription factor families are present in the upper crypt and lower villus, which correspond to regions of LPH and SI transcriptional activation during intestinal differentiation. However, the net effect on intestinal gene expression during differentiation is far more complex than the simple presence of these transcription factors and their cognate binding sites. Data in the present study suggest that the number of binding sites and their relative positions on the promoter are important. Furthermore, as shown by transcription from longer promoter constructs (Fig. 3) or from the endogenous genes (Fig. 6), which do not always parallel expression of shorter promoter constructs, other interactions involving sequence further upstream may also influence transcriptional activation. Finally, interaction with other transcription factors, such as HOXC11 (46), as well as with specific cofactors, including friend of GATA-2 (FOG-2) (39, 62), dimerization cofactor for HNF-1 (DCoH) (44), and p300 (27) must be considered.
We conclude that the complex hierarchies of control that govern gene expression during intestinal differentiation and development are determined by the stoichiometry of transcription factors and cofactors within intestinal cells at any given time and the type, number, and arrangement of cis-acting elements in the regulatory regions of intestinal genes. LPH and SI, markers of intestine-specific gene expression and intestinal differentiation, are regulated by complex interactions involving multiple transcription factors. Synergistic activation by combinations of transcriptions factors is likely a mechanism by which specific patterns of tissue-specific expression might be attained by the overlapping expression of specific transcription factors. Characterizing specific interactions will provide insight into the regulatory mechanisms that control the pathways of intestinal differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Leah Moyer for excellent technical assistance and Todd Evans, Menno Verhave, and Robert K. Montgomery for helpful insight and suggestions. We are also grateful for the support from the Silvio O. Conte Digestive Disease Core Centers: Douglas Jefferson of the Cell Culture Core, Anne Kane of the Microbiology Core, and Andrew Leiter of the Molecular Biology Core.
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FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-32658-19, Silvio O. Conte Digestive Disease Core Center Grant P30-DK-34928, and grants from the Charles H. Hood Foundation, March of Dimes Birth Defects Foundation, and the New England Medical Center (S. D. Krasinski), and the Nutricia Research Foundation (H. M. Wering).
Address for reprint requests and other correspondence: S. D. Krasinski, Division of Pediatric Gastroenterology and Nutrition, New England Medical Center, Box 383, 750 Washington St., Boston, MA 02111.
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 28 September 2000; accepted in final form 5 March 2001.
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