GATA-6 stimulates a cell line-specific activation element in
the human lactase promoter
Kevin
Fitzgerald1,
Leonard
Bazar1, and
Mark I.
Avigan1,2
Departments of 1 Pathology and
2 Medicine, Georgetown
University School of Medicine, Washington, District of Columbia 20007
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ABSTRACT |
Lactase-phlorizin
hydrolase (LPH) synthesis is restricted to differentiated small
intestinal enterocytes and is highly regulated during development.
Analysis of expression of LPH promoter segments fused with luciferase
transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA,
mapped an 18-base pair (bp) segment 100 bp upstream of the
transcription start site that is required for transactivation.
Remarkably, the LPH upstream element (LUE) has no stimulatory activity
in both human intestinal and nonintestinal lines in which LPH mRNA is
absent. Electrophoretic analysis of sequence-specific DNA-nuclear
protein complexes demonstrated the presence of a Caco-2 cell-specific
protein(s) (CCP), which is uniformly absent in LPH nonproducer cell
lines. Mutational analysis of the LUE demonstrated that bases contained
within a GATA consensus motif are critical for both CCP binding and
transcription from the LPH promoter. Caco-2 cells express high levels
of GATA-6 mRNA in a cell line- specific manner, suggesting that GATA-6
is a CCP that complexes with the LUE. When expressed by a plasmid,
GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a
flanking downstream element. These studies are consistent with an
important role of an intestinal GATA binding protein in cell
type-specific transactivation of the LPH promoter.
lactase-phlorizin hydrolase; transcription; intestine; Caco-2
cells; GATA binding proteins
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INTRODUCTION |
THE SMALL INTESTINAL epithelium is composed of a
self-renewing monolayer of cells. As cells migrate from the crypts to
the villus tips, they cease to proliferate and terminally differentiate before apoptotically dying and exfoliating. A single stem cell in each
crypt gives rise to four distinct epithelial lineages, which include
Paneth cells, goblet cells, enteroendocrine cells, and absorptive
enterocytes (11). Each lineage of cells expresses a distinct repertoire
of molecules that are required for physiological functions of the
intestine.
To assimilate lactose, differentiated absorptive enterocytes express
lactase-phlorizin hydrolase (LPH), an enzyme that hydrolyzes the
-1,4-galactoside bond of the disaccharide into its monosaccharide components. Similar to other glycohydrolases, whose expression is
restricted to absorptive enterocytes, LPH transcription is regulated
during development (29). LPH expression on the surface of apical
membrane microvilli, which commences at the crypt-villus junction, is
programmed in enterocytes both during maturation and development and is
unaffected by the presence of substrate in the lumen (20). In the human
LPH promoter, DNA sequences upstream of a single TATA box possess
binding sites for transcription factors that are expressed in a variety
of cell lineages, including Sp1, SRF AP-2, CTF/nuclear factor-1, cyclic
adenosine monophosphate response element binding protein, and Oct1/Oct2
(4). In addition, there is a GATA motif, ~60 bp upstream
of the TATA box. This motif is conserved in the LPH promoters of all
the mammalian species that have so far been analyzed (4, 5, 27).
Members of the family of GATA binding proteins all contain a segment
with two conserved zinc finger DNA binding domains. These target the
consensus sequence (A/T)GATA(A/G). GATA-1, GATA-2, and GATA-3 play a
critical role in the regulation of hematopoiesis and are also expressed
in nonhematopoietic tissues, such as Sertoli cells (GATA-1),
endothelial cells (GATA-2), and nerve tissue (GATA-2 and GATA-3) (30).
Recently, the cDNAs of three new GATA binding proteins have been cloned
(1, 17, 21). The mRNAs of GATA-4, GATA-5, and GATA-6 are expressed
primarily in the heart and gut. Each of these transcripts encodes amino
acid sequences in the zinc finger domains, which are conserved across
species (1, 17, 26, 33). Both GATA-4 and GATA-6 are expressed in
mammalian enterocytes. Although overlapping, the developmental and
tissue-specific patterns of their expression differ significantly. In
vitro, studies have indicated that GATA-4 contributes to the regulation
of genes crucial for myocardial function and for extracellular matrix
formation in the yolk sac (3, 13, 23). Targeted disruption of GATA-4 in
mouse embryonic stem cells resulted in the loss of visceral endoderm
formation (23). The rat homologue of GATA-6 has been shown to bind to
and activate the promoters of both the
- and
-subunit genes of
the
H+-K+-adenosinetriphosphatase
enzyme in the gastric parietal cells of rats and humans (26). These
findings, along with the presence of conserved GATA elements in the
promoters of intestinal fatty acid-binding protein and
sucrase-isomaltase, suggest that GATA binding proteins may play a
significant role in the regulation of some gut-specific genes. In this
report, we show evidence that GATA-6 may play an important role in
stimulating cell type-specific activation of the human LPH promoter.
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MATERIALS AND METHODS |
Cell lines, plasmids, RNA purification, and DNA sequencing.
Caco-2 and HT-29 human colon adenocarcinoma cells, SW-13 human adrenal
carcinoma cells, and cervical carcinoma HeLa cells (purchased from the
American Type Culture Collection) and human jejunal HIE-7 cells (24)
were cultured in Dulbecco's modified Eagle's medium with 10%
heat-inactivated fetal bovine serum. A 5.5-kb segment of the human LPH
gene that contains 3.5 kb of the promoter region, exon 1 and part of
intron 1, was excised from
LPH 7 (4) (generously provided by N. Mantei, Swiss Federal Institute of Technology, Zurich, Switzerland),
using Sac I and Sal I, and cloned into pUC 19, to
produce p19/5.5.
Plasmid DNA was purified using columns from QIAGEN (Chatsworth, CA) and
quantitated spectrophometrically and by electrophoretic analysis. RNA
isolation was performed as described (8). Polymerase chain reaction
(PCR) amplifications were performed using
Taq DNA polymerase, 25 pmol primers,
and 100 ng of LPH promoter containing plasmid templates.
Oligonucleotide primers with linker sites were used to amplify
increasingly long upstream segments of the LPH promoter whose 3'
end corresponded to the previously designated
1 position
(relative to the LPH translation start site) (4). For this purpose, the 3' primer LPH (3')
(5'-TAGCTAAGCTTGTCGACTTTCTAGGAACTGTTAGGAGG-3') was paired
with 5' primers that included LPH-315
(5'-CTACAGGCGCATGCCACGATGCCTG GCTAA-3'), LPH-95
(5'GCAGGATCCTTAAATATTAAGTCTTAATTA-3'), LPH-85 (5'CAAGCTCGAGTCTTAATTATCACTTAG-3'), and LPH-46
(5'-CAAGCTCGAGTTATAAAGTAAGGGTTCC-3'). PCR
segments with 5-bp-long blocks of mutations in a segment
between
112 and
93 were produced with the following
5' primers (mutated sequence shown in bold lowercase letters):
LPH-mutA
(5'-GCAGGATCCtcgacTAGATAACCCAGTTAAA-3'), LPH-mutB
(5'-GCAGGATCCGATCAgctcgAACCCAGTTAAA-3'),
LPH-mutC (5'-GCAGGATCCGATCATAGATccaaaAGTTA-
AA-3'), and LPH-mutD (5'-GCAGGATCCG
ATCATAGATAACCCctggcAA-3'). For
electrophoretic mobility shift assays (EMSAs), the same mutations were
introduced into annealed 22-bp double-stranded oligonucleotide probes,
whose corresponding wild-type upper strand sequence was
5'-GATCATAGATA ACCCAGTTAAA-3', to yield LPH upstream
element (LUE)/mutA, LUE/mutB, LUE/mutC, and LUE/mutD. The PCR products
listed above containing the wild-type and mutant sequences were cloned
into the polylinker of pXP1 (Pharmacia Biotech) to produce
LXP/
315, LKP/
95, LXP/
85, LXP/
46, LXP/mutA,
LXP/mutB, LXP/mutC, and LXP/mutD, respectively. An LPH promoter
restriction fragment encompassing LPH bases
3500 and
1
was also cloned into pXP1 to produce LXP/
3500. By restriction of
a Bam I site in the polylinker of
LXP/
315 and of unique sites in the LPH promoters at either
200 (BstX I) or
112 (Bcl I), LXP/
200
and LXP/
112 were produced. LXP/
315
, a plasmid with an
internal deletion between bases
112 to
46, was produced
by ligation of a plasmid fragment encompassing bases
315 to
112 to the linearized 5' end of the LPH segment of
LXP/
46 (site of the TATA box). DNA sequences of the LPH promoter
segments cloned into pXP1 were analyzed with the Sequenase 2.0 kit
(U.S.
Biochem).
Transfections.
Transient transfections of Caco-2 cells were performed by
electroporation, using a Bio-Rad gene pulser system and capacitance extender set at 250 V and 960 µF. Transfections were performed with 2 × 107 cells, 40 µg of a
selected LXP reporter construct, and 2 µg of pOBCAT0 (2). Cells grown
to 80% confluence were trypsinized, thoroughly suspended in 0.5 ml of
cell culture media, chilled to 4°C, and aliquoted in 0.4-cm
electroporation cuvettes (Bio-Rad). After electroporation, the cells
were cultured for 42 h in petri dishes 100 mm in diameter. To prepare
cellular extracts from transfected Caco-2 cells, the adherent and
nonadherent cells were harvested together. At the time of harvest for
analysis of reporter expression, the adherent cells were 60-80%
confluent and growing in clusters of 10-20 cells. In some
experiments, 10 µg of an RSV/chicken GATA-6 expression vector (9)
(generously provided by J. B. E. Burch, Fox Chase Cancer Center,
Philadelphia, PA) was cotransfected with 35 µg of an LXP construct
and 2 µg of pOBCAT0 into cells. Identical amounts of DNA were also
transiently transfected into Cos-1 cells, by calcium phosphate
transfection. Each culture of transfected cells was lysed in 100 µl
of buffer containing 1% Triton X-100 and 0.1 M potassium phosphate (pH
7.8) and stored at
70°C. To measure chloramphenicol
acetyltransferase (CAT) expression, we mixed 10 µl of the cleared
supernatants in scintillation vials with 200 µl CAT reaction buffer
containing 10 µl
[3H]acetyl-CoA (Dupont
NEN) diluted 1:35 with 2.5 mM acetyl-CoA, 50 µl 5 mM chloramphenicol,
and 140 µl 0.1 M tris(hydroxymethyl)aminomethane (Tris) · Cl (pH 7.7). The assay mix was gently
overlaid with 2 ml of Econofluor scintillation fluid and immediately
placed in a 1209 Rack Beta liquid scintillation counter. The samples
were then counted at 1-h intervals. Sample readings of reaction rates were used to establish transfection efficiencies. Based on transfection efficiencies, standardized volumes of each extract (range 4-20 µl) were then analyzed for luciferase content, using a Monolight 2010 luminometer (Analytic Bioluminescence Laboratories). For each cell line
tested, differences in the volumes of extracts that were tested were
less than twofold.
Ribonuclease protection, reverse transcription-PCR, and Northern
blot analysis.
To detect LPH mRNA from human enterocytes and Caco-2 cells
32P radiolabeled riboprobes were
made from a 578-bp LPH segment (bases
198 to +380) inserted into
Bluescript SK and transcribed from the T3 and T7 promoters to produce
sense and antisense probes, respectively. To detect reporter mRNA from
Caco-2 cells transiently transfected with LXP plasmids, riboprobes were
made from a Bluescript SK plasmid containing a 208-bp segment between
bases
112 of LPH and +95 of the luciferase reporter. After
linearization of the plasmid, the probes were transcribed from the T7
and T3 promoters to produce sense and anti-sense probes, respectively.
After hybridization, the mRNA-riboprobe duplexes were treated with
ribonucleases (RNases) A and T1, denatured, and separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were then
dried and autoradiographed. To determine the presence of LPH mRNA in
cell lines, RNA samples were reverse transcribed with random hexamer
primers and reverse transcriptase (RT). The RT products were then
amplified with LPH primers to generate a 225-bp segment between +15 and
+237 (5' primer: 5'-GAGCTGTCTTGGCATGTAGT-3'; 3'
primer: 5'-GGAGACTGCTGAAGTACTCTGGC-3'), which was detected
by agarose gel electrophoresis. For Northern blot analysis of GATA-6
mRNA, electrophoretically separated total RNA was transferred to a
nylon membrane. GATA-4 mRNA was detected with a 44-base oligonucleotide
probe (AGGCTGTGCAGGACCGGGCTGTCGAAGGGGCCGGCG- GAGGCGGC), which is
complementary to both human and rodent GATA-4 forms, but not to other
GATA-binding protein family members (1, 21, 26, 33), after
32P end-labeling with T4
polynucleotide kinase. A human GATA-6 cDNA probe (kindly provided by T. Evans, Albert Einstein College of Medicine, Yeshiva University, Bronx,
NY), consisting of a 1.5-kb EcoR I
fragment not overlapping with the conserved zinc finger DNA binding
domain, was 32P radiolabeled with
random hexamers and the Klenow fragment of Escherichia
coli DNA polymerase, and hybridized to
membrane-bound mRNA. After washing, the membranes were dried and
autoradiographed.
Nuclear extract preparation and analysis of protein-DNA binding.
Nuclear extracts of cell lines were prepared according to a modified
protocol of Dignam et al. (10), and extracts were stored at
70°C. Briefly, after being rinsed and scraped in cold
phosphate-buffered saline (PBS), the cells were centrifuged. The cell
pellet was then resuspended in an equal volume of cold 2×
buffer
A [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.25 mM
dithiothreitol (DTT), 100 µg/ml phenylmethylsulfonyl fluoride (PMSF),
1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml
aprotinin]. After the cells were swollen on ice and disrupted by
passage through a 26-gauge needle, the mixture was layered over a
sucrose cushion (0.8 M sucrose, 10 mM Tris · Cl, pH
7.5, 80 mM KCl, 5 mM MgCl2, 0.25 mM DTT, and the protease inhibitors listed above) and centrifuged at
2,000 revolutions/min (rpm) for 15 min. The nuclear pellet was
resuspended in an equal volume of 2×
buffer
C (20 mM HEPES, pH 7.9, 420 mM NaCl,
1.5 mM MgCl2, 200 mM EDTA, 0.25 mM
DTT, and the protease inhibitors listed above in 20% glycerol). The
mixture was then centrifuged at 37,500 rpm for 30 min, and the
supernatant was stored at
70°C. Protein quantitation was
determined by the Bradford protein assay (Bio-Rad) (7). EMSAs were
performed using plasmid restriction endonuclease fragments or
double-stranded DNA oligonucleotides that were
32P end-labeled with T4
polynucleotide kinase. The radiolabeled double-stranded
oligonucleotides or restriction fragment probes (0.2-0.4 ng) were
added to 8 µl of EMSA binding buffer [25 mM Tris · Cl, 200 mM glycine, 1 mM EDTA, 10% glycerol,
1 mM 2-mercaptoethanol, 50 mM NaCl, 0.025% bromophenol blue, and 1 µg poly(dI-dC)] before the addition of 1 µl nuclear extract
(5 µg/sample). The mixtures were incubated at room
temperature for 30 min and then loaded onto 5% polyacrylamide gels.
After separation of the protein-DNA complexes, the gels were dried and
autoradiographed.
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RESULTS |
The LPH promoter is only activated in Caco-2 cells.
To identify a human cell line that endogenously expresses LPH mRNA, PCR
amplification was performed on the RT products of total RNA isolated
from intestinal and nonintestinal cell lines using primers from LPH
exon 1. Of the five cell lines tested, only Caco-2 cells exhibited
expression of the LPH transcript (Fig. 1A). This human
colon carcinoma cell line forms a monolayer of polarized cells, after
growing to confluence, and expresses a number of proteins associated
with the brush borders of small intestinal enterocytes (14, 15). LPH
mRNA expression is low in preconfluent Caco-2 cells and increases
significantly in postconfluent culture, as the cells become more
differentiated (Fig. 1B). Although the electrophoretically separated RT-PCR product of preconfluent cell
LPH mRNA was not detected by ethidium bromide staining, after Southern
blotting it was detected by hybridization with a radiolabeled LPH cDNA
probe (data not shown). A series of LPH promoter segments of increasing
length were fused with luciferase (Fig. 2). To determine whether LPH promoter activity is restricted in a cell line-specific manner, the LXP fusion constructs were transiently transfected into
Caco-2 cells as well as the other four cell lines tested above (Table
1). Luciferase expression was then analyzed after standardization to CAT levels generated by a cotransfected plasmid containing the CAT gene linked to the SV40 early promoter. All the cell
lines tested were efficiently transfected with this CAT reporter
plasmid and exhibited high levels of CAT expression. In sharp contrast,
transcription from the LPH promoter was only measurable in Caco-2
cells. In these LPH-producing cells LPH promoter activity of the LXP
constructs was high, whereas the promoterless construct was
transcriptionally silent (Table 1). Initial promoter mapping, using the
LXP constructs with increasingly extended LPH promoter segments,
indicated that the region required for transcriptional activation in
Caco-2 cells is between the LPH TATA box (LXP/
46) and a site 66 bp further upstream (LXP/
112). Consistent with this finding, a
construct with an internal deletion between the TATA box and
112
(LXP/
315
) was transcriptionally inactive. These experiments
revealed the unique ability of Caco-2 cells to activate transcription
from the LPH promoter.

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Fig. 1.
A: lactase-phlorizin hydrolase (LPH)
mRNA expression is detectable in postconfluent Caco-2 cells but not in
other intestinal and nonintestinal cell lines. Reverse
transcription-polymerase chain reaction (RT-PCR) was performed on total
RNA isolated from each cell line. Primers encompassing a region in the
first exon of LPH from +15 to +237 were used to amplify a 225-bp
segment (arrow indicates PCR product). Control lanes include primers
without reverse-transcribed products (Primers only) and Caco-2 cell
reverse-transcribed mRNA without primers (Template only). With control
primers encompassing a segment in the
2-microglobulin cDNA, PCR of
the RT products from each tested cell line produced equivalent levels
of electrophoretically detectable products (not shown).
B: LPH mRNA expression is induced
after confluence in Caco-2 cell cultures; d, day;
2-microglob,
2-microglobulin control
primers.
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Fig. 2.
Schematic representation of LPH promoter segments of increasing length
fused with luciferase (LXP). The 5' end of each promoter segment
is numbered relative to the LPH translation start site. TATA represents
the LPH TATA box, which is retained in all fusion constructs. ATG
indicates the translation start site in the luciferase gene, and the
filled arrowhead indicates the promoter transcription start site.
Experimental results involving the use of these constructs are seen in
Table 1 and Figs. 4 and 5.
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Analysis of transcription start sites of the endogenous and
transfected LPH promoters.
To determine if the 5' end of LPH mRNA in Caco-2 cells is
consistent with the transcription start site in human primary
enterocytes, RNase protection analysis was performed. We hybridized 10 µg of total RNA from both human enterocytes and Caco-2 cells to a
578-bp riboprobe made from the LPH bases
198 to +380. Nuclease
digestion resulted in a 399-bp protected fragment from both samples,
suggesting that identical LPH transcription start sites are utilized 15 bp upstream of the translation start site (Fig. 3). This
site is within a 5-bp segment that was previously found to encompass
the transcription start site (4). To determine whether the appropriate LPH promoter sequence was utilized by the transfected LXP constructs, mRNA from Caco-2 cells transiently transfected with LXP/
315 was analyzed by RNase protection. For this purpose, a 207-bp riboprobe, which consisted of 112 bp of the LPH promoter segment fused with a
95-bp segment of the reporter plasmid, was used. RNase protection analysis of mRNA from transfected cells demonstrated a 114-bp protected
fragment, which was absent when mRNA from nontransfected cells was
analyzed (Fig. 4). The size of the protected fragment is
consistent with appropriate usage of the LPH transcription start site
in the transfected LPH-luciferase gene.

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Fig. 3.
RNase protection analysis demonstrates identical transcription start
sites of the LPH gene in human enterocytes and Caco-2 cells. Arrow
indicates a 399-bp protected fragment after nuclease digestion of LPH
mRNA-probe hybrid products from human enterocytes and Caco-2 cells.
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Fig. 4.
RNase protection analysis shows identity of the transcription start
sites in a transfected LXP plasmid and the endogenous LPH gene. Arrow
indicates a 115-bp protected fragment after nuclease digestion of the
LXP/ 315 mRNA-probe hybrid product from transfected Caco-2 cells
that is absent in nontransfected cells. The size of this product
corresponds to the predicted transcription start site of the LPH
gene.
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Identification of an 18-bp LPH upstream segment that is required for
LPH transcription and that binds a Caco-2 cell line-specific nuclear
protein(s).
To locate a regulatory element within the 66-bp region required for the
activation of the LPH promoter in Caco-2 cells, LXP constructs were
made that contained segments of the LPH promoter 39 and 49 bp upstream
of the TATA box (LXP/
85 and LXP/
95, respectively). When
transiently transfected into Caco-2 cells, these constructs exhibited
no significantly greater activity than the TATA box construct
LXP/
46 (Fig. 5). This result indicated that the
crucial area for LPH transcription activation is in an 18-bp segment
that is located between
95 and
112 bp upstream of the LPH
translation start site. This segment has been designated the LUE.

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Fig. 5.
Mapping of an LPH upstream element (LUE) that is required for promoter
transcription activation in Caco-2 cells. Transfections and
measurements were performed as in Table 1. The values of luciferase
expression represent fold increases over pXP1 background activity.
Error bars indicate the standard errors of the mean (SEM).
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It was of interest to determine if Caco-2 cells produce a protein(s),
absent in the other tested cell lines, that binds the LUE in a
sequence-specific manner. Analysis of proteins in Caco-2 cell nuclei
that bind in the vicinity of the LUE was performed using a
32P radiolabeled 22-bp
double-stranded oligonucleotide encompassing the 18-bp LUE (Fig.
6A). Binding to this
probe was also tested by EMSA with nuclear extracts from other cell
lines in which the LPH promoter is not transactivated. As shown in Fig.
6B, Caco-2 cells contain a protein(s)
that binds the LUE in a sequence-specific manner. In contrast, none of
the other cell lines contain this protein. To determine which bases are
necessary for binding by the Caco-2 cell line-specific protein(s)
(CCP), excess molar amounts of 22-bp double-stranded oligonucleotides
encompassing the LUE sequence with tandem blocks of 5-bp mutations
(Fig. 6A) were used as cold
competitors for CCP binding to the radiolabeled LUE. Binding of the CCP
to the LUE was effectively competed by the wild-type sequence as well
as by sequences with base pair substitutions in segments containing
bases
112 to
108 (LUE/MutA) and bases
97 to
93 (LUE/MutD) (Fig. 6B). In
contrast, mutations with base pair substitutions containing bases
107 to
103 (LUE/MutB) and
102 to
98
(LUE/MutC) reversed efficient competition of the excess oligonucleotides with the radiolabeled LUE for binding to the CCP. The
loss of CCP binding indicates the crucial role of the 10-bp site within
the LUE in forming a DNA sequence-specific complex with the nuclear
protein. In the EMSA, a series of protein-DNA complexes that migrated
more rapidly than the CCP-LUE complex were also observed. By
competition with excess wild-type and mutant LUE sequences, these were
not DNA sequence specific. To confirm that the LUE-containing LPH
promoter binds the CCP in a sequence-specific manner, a restriction
fragment containing bases
112 to
1 was added in excess
molar quantities to the binding reaction. This fragment successfully
competed with the 22-bp radiolabeled LUE probe for CCP binding. In
contrast, a nonspecific 125-bp restriction fragment was ineffective as
a competitor (Fig. 6C),
corroborating that the CCP binds to the double-stranded LPH promoter in
a sequence-specific manner.

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Fig. 6.
A Caco-2 cell line-specific protein(s) (CCP) binds the LPH upstream
element in a 10-bp stretch that contains a GATA consensus motif.
A: wild-type (WT) and mutated
sequences of a 22-bp segment containing the LUE ( 112 to
91) that were tested for binding to CCP. Bases that were mutated
in tandem 5-bp segments are shown in lowercase letters. Bases that
match the position of the GATA motif are underlined. Of the mutated LUE
sequences, LUE/mutB and LUE/mutC contain substituted bases within the
GATA consensus motif, whereas LUE/mutA and LUE/mutD contain substituted
bases that flank this site. B: gel
shift assay of the radiolabeled WT/LUE fragment bound by CCP. Where
indicated, the binding reactions were performed with a molar excess of
nonradiolabeled WT or mutated LUE competitors. Arrow indicates the site
of migration of the CCP-LUE complex.
C: formation of the CCP-LUE complex
(arrow) is competed by excess molar amounts of a 119-bp plasmid
restriction fragment encompassing LPH bases 112 to 1, but
not of a control 125-bp restriction fragment. A series of more rapidly
migrating complexes are not competed in a DNA sequence-specific
fashion.
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Analysis of the LUE DNA sequence indicated that the 10-bp segment that
is crucial for CCP binding contains a GATA consensus site (WGATAR). As
described above, the binding analysis suggested that the CCP is a GATA
binding protein. Members of this family of factors are well known for
their tissue-specific and developmental regulation of transcription.
GATA-4 and GATA-6 are expressed in chicken as well as in human and
mouse intestinal epithelial cells and may play a significant role in
the tissue-specific transcriptional activation of gastrointestinal
genes (1, 17, 21-23, 26).
LUE bases within and 3' of the GATA motif are required for
promoter activity.
If CCP binding to the LUE is necessary to stimulate the LPH promoter,
it is predicted that mutations that abolish the protein-DNA interaction
also block transactivation. To test this prediction, four LXP
constructs were made that incorporated each of the four tandem blocks
of LUE mutations (LXP/MutA, LXP/MutB, LXP/MutC, and LXP/MutD),
previously analyzed for binding in Fig.
6B. Expression by each of these
altered LPH promoter-luciferase constructs in Caco-2 cells was compared
with expression by the wild-type LUE reporter construct
(LXP/
112) (Fig. 7). Although none of the constructs containing
mutated sequences demonstrated transcriptional activity as high as the
wild-type control (LXP/
112), the construct with mutations in the
segment encompassing bases
112 to
107 (LXP/MutA) reduced
transcriptional activity by only 1.5-fold (Fig. 7). In contrast, mutations that block CCP binding (LXP/MutB and LXP/MutC) reduced transcriptional activity more dramatically, consistent with a
critical role for binding of the CCP in LPH promoter activation. Unexpectedly, LXP/MutD, which contains mutated bases located downstream of the GATA element that do not disrupt CCP binding, was also transcriptionally silent. Interestingly, the mutated bases in LXP/MutD
are part of a DNA sequence in which 11 of 13 bases are identical with
the reported consensus sequence for hepatocyte nuclear factor-1 (HNF-1)
(28, 31). Although contiguous, the GATA element is not
overlapping with this site.

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Fig. 7.
Mutations that eliminate binding of the CCP to the LUE block
transcriptional activation of the LPH promoter in Caco-2 cells.
Transfections and measurements were performed as in Table 1.
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GATA-6 stimulates the LPH promoter.
GATA-4 and GATA-6 mRNA are both expressed in small intestinal
epithelial cells. Compared with the rat heart mRNA, none of the cell
lines tested by Northern analysis, including Caco-2 cells, expressed
significant levels of GATA-4 mRNA (Fig.
8A and data not
shown). In contrast, Caco-2 cells produce high levels of
GATA-6 mRNA. Expression of GATA-6 transcripts at equivalent levels by the LPH nonproducer cell lines was not observed. The cell line-specific expression of high amounts of GATA-6 mRNA parallels the restricted expression of CCP detected by EMSA analysis. These data suggest that
GATA-6 is a CCP that binds the LUE in vivo. Because of the difference
between GATA-4 and GATA-6 mRNA levels, it is unlikely that GATA-4 is as
abundant as GATA-6 in the CCP-LUE complex. This is supported by the
observation that mobility of the CCP-LUE bound product, detected by
EMSA analysis, is not affected by GATA-4 "supershifting"
antibodies (data not shown). Unlike LPH expression, GATA-6 mRNA
synthesis in Caco-2 cells is constitutive and is not influenced by
confluence-induced differentiation (Fig.
8B).

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Fig. 8.
Caco-2 cells express constitutively high levels of GATA-6 mRNA in a
cell line-specific manner. A:
postconfluent Caco-2 cells express negligible levels of GATA-4 mRNA but
high levels of GATA-6 mRNA. Northern blot analysis of total RNA was
performed with a GATA-4 probe complementary to both human and rodent
species and a human GATA-6 probe. Rat heart mRNA served as a positive
control for detection of GATA-4. Also shown is the ethidium bromide
(EtBr) staining of 28S and 18S ribosomal RNA used to normalize the
loading of samples. B: GATA-6 mRNA
levels are the same in preconfluent and 7- and 10-day postconfluent
Caco-2 cells.
|
|
To test whether GATA-6 stimulates the LPH promoter, LXP wild-type and
mutant reporter constructs were cotransfected into Caco-2 cells with a
GATA-6 expression plasmid (pRSV/GATA-6). Consistent with the results
shown in Fig. 7, both the wild-type (LXP/
112) and LXP/MutA
promoters demonstrated low levels of basal activity in the absence of
cotransfected pRSV/GATA-6 (5- and 6-fold mean increases in activity,
respectively, relative to the activity of a promoterless reporter;
n = 3; data not shown). Cotransfection of pRSV/GATA-6 significantly stimulated LPH promoter activity of both
reporter constructs (Fig.
9A). Not
surprisingly, the constructs with mutations overlapping the GATA motif
(LXP/MutB and LXP/MutC) did not respond to the stimulatory effects of
the product of the GATA-6 expression vector. The mutated LPH promoter
with base pair substitutions on the 3' flank of the GATA motif
(LXP/mutD) was also not susceptible to transcriptional activation by
GATA-6.

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Fig. 9.
Mutation of the GATA binding site eliminates LPH promoter stimulation
by GATA-6. LXP plasmids were transiently transfected with (+) or
without ( ) a GATA-6 expression vector (pRSV/GATA-6).
Measurements of luciferase expression were performed as in Table 1. The
reporter activities (relative light units) are normalized to the
activity of LXP/ 112 (WT) in the absence of pRSV/GATA-6.
A: relative luciferase expression by
Caco-2 cell transfectants. B: relative
luciferase expression by Cos-1 cell transfectants.
|
|
Similarly, cotransfection of the LXP constructs with pRSV/GATA-6 into
Cos-1 cells, which do not express GATA-6 mRNA endogenously, stimulated
luciferase expression (Fig. 9B).
Moreover, transcription of the LXP/MutD promoter, which contains
mutated bases 3' of the GATA consensus sequence, was not
stimulated by GATA-6. In contrast to their low levels of activity in
Caco-2 cells, when the GATA-6 expression plasmid was absent, both the
wild-type (LXP/
112) and LXP/MutA promoters were silent in Cos-1
cells (data not shown).
 |
DISCUSSION |
The coincident expression of GATA-6 and LPH mRNA in Caco-2 cells and
the ability of a GATA-6 expression vector to stimulate transcription
from cotransfected LXP plasmids establish a connection between the GATA
binding protein and LPH promoter activation. Consistent with an
activating role for GATA-6, mutations that altered the GATA binding
site also abrogated promoter activation in Caco-2 cells. The GATA
sequence in the LUE, AGATAA, is identical to a sequence to which GATA-6
is known to bind preferentially, as measured by selected and
PCR-amplified binding (J. B. E. Burch, personal communication). When
linked to a minimal promoter, this sequence has been found to
effectively mediate GATA-6-induced transactivation (9). Further
evidence of the activation of the LPH promoter by GATA-6 was
demonstrated by transiently cotransfecting the GATA-6 expression
plasmid and LXP reporter constructs into Cos-1 cells, which ordinarily
do not express significant amounts of GATA-6. In association with the
absence of GATA-6 expression in these cells, the transfected LPH
promoter was transcriptionally silent. However, when GATA-6 was
produced by a transfected expression plasmid, LPH promoters containing
wild-type sequences or a block of substitution mutations 5' to
the GATA motif (LXP/MutA) in the LUE were strongly activated. In
contrast, the promoters with mutations overlapping the GATA site
(LXP/MutB and LXP/MutC) were not stimulated by GATA-6 to produce
significant levels of luciferase.
In both Caco-2 cells and small intestinal enterocytes, the expression
of GATA-6 alone is not sufficient to stimulate LPH transcription. This
is supported by the observation that the level of GATA-6 mRNA
expression is the same in preconfluent and confluent Caco-2 cells,
whereas LPH mRNA expression is low in preconfluent cells and
dramatically increases in 7- and 14-day postconfluent cells. There are
also important differences in the time of onset of both GATA-6 and
GATA-4 mRNA expression and LPH mRNA expression during development of
the mouse. In the primitive gut, the transcripts that encode these GATA
binding proteins are detectable as early as embryonic
day
9.5
(21). In contrast, LPH mRNA only appears after the cytodifferentiation
of endodermal cells into a monolayer of polarized epithelium, which
occurs several days later (29). Thus, in addition to GATA binding
protein expression, another differentiation-associated event(s) is
required to stimulate transcription from the genomic LPH promoter, in
both preconfluent Caco-2 cells and undifferentiated small intestinal
epithelial cells. This may be an alteration(s) in chromatin structure
or a modulation(s) of another nuclear protein(s) that activates or
suppresses LPH transcription.
The inability of GATA-6 to activate the LPH promoter with mutations
downstream of the GATA binding site (LXP/mutD) contrasts with binding
studies that indicate that the GATA-binding CCP is able to form a
stable complex with the LUE containing these mutations (LUE/mutD).
There are two possible explanations that may account for this
discrepancy. First, the binding affinity of a putative GATA-6/LUE
complex in vivo might be modulated by bases 3' to the GATA motif.
Previously, it has been observed that bases flanking the GATA motif
affect the binding of GATA-1, GATA-2, and GATA-3 (16, 18). These
proteins demonstrate base preferences that affect the binding
affinities at bases +3 and +4 on the 3' end of the GAT core.
Moreover, cooperative GATA protein binding to second contiguous sites
composed of a variety of sequences that deviate from the consensus
sequence has been demonstrated. However, it is unlikely that the base
pair substitutions in LUE/mutD cause a significant change in the
affinity of the GATA binding protein-DNA interaction, since the mutated
bases 3' to the GATA motif, which disrupted LPH promoter
transactivation, did not affect formation of the CCP-LUE complex in
vitro. This was demonstrated by the identical efficiency of competition
by titrated amounts of excess LUE/mutD and LUE/wild-type (LUE/WT)
sequences for binding of the CCP with the radiolabeled
LUE/WT.
A second explanation for the discrepancy between preservation of CCP
binding to LUE/mutD and the loss of transactivation by LXP/MutD is that
GATA-6 may require the presence of a downstream accessory factor to
activate the LPH promoter. Analogously, GATA-1 has previously been
shown to functionally interact with SP1 and erythroid Kruppel-like
factor (12). It has recently been demonstrated that GATA-6 functionally
cooperates with other transcription factors (9). In the vitellogenin II
promoter, estrogen-induced transcription depends on the contribution of
this GATA binding protein, which binds a site flanking the estrogen
receptor element. In the case of the LUE, accessory factor binding may
be a necessary step for LPH promoter transactivation. By EMSA analysis,
we have found that a second Caco-2 cell protein binds a separate site
that is immediately 3' of the GATA binding site (data not shown).
This site (GTTAAATATTAAG) includes 11 of 13 bases that are identical with the reported consensus motif for HNF-1 (GTTAATNATTAAC) (28, 31).
In vitro binding of the second Caco-2 cell nuclear protein at the
HNF-1-like site in the LPH promoter occurs independently of binding at
the GATA element by CCP and is selectively disrupted by the base
substitutions identical with those in LUE/mutD. Both the related
homeoproteins HNF-1
and HNF-1
have been identified in primary
enterocytes and Caco-2 cells (31). These transcription factors, along
with CDX homeodomain proteins, were shown to regulate the small
intestine- specific product of the sucrase-isomaltase gene (SI) (31).
Consistent with a role in the activation of LPH expression in the small
intestine during murine development, HNF-1
mRNA is expressed by
embryonic day
12.5
(19), which precedes LPH mRNA expression by a few days. The possibility
that a GATA binding protein(s) cooperates with a member(s) of the HNF-1
family to stimulate the LPH promoter awaits further studies.
The region between the GATA motif in the LUE and the TATA box of LPH
contains other elements known to bind nuclear transcription factors. A
second GATA binding site located 77 to 72 bp upstream of the LPH
translation start site, which has previously been designated as +1 and
is ~62 to 57 bp upstream of the LPH transcription start site (4),
does not stimulate transcription from the LPH promoter in Caco-2 cells,
as measured in the transfection analysis. This element, whose
orientation is opposite of the GATA element in the LUE, may not be
positioned to effectively interact with other critical accessory
protein binding sites and/or the RNA polymerase complex. The
CE-LPH1 element is located 63 to 51 bp upstream of the LPH translation
start site (27). This element has been shown to bind an intestinal
nuclear protein (NF-LPH1), whose expression in pig enterocytes changes
in parallel to lactase expression. Although it may also play a role in
LPH activation, the range of NF-LPH1 expression does not parallel the
cell type-restricted pattern of LPH mRNA synthesis (6).
Whether GATA-4, which similar to GATA-6 is expressed in the small
intestine, stimulates or regulates LPH transcription has yet to be
studied. Overlapping expression of GATA proteins in cells may lead to
additive, synergistic, or inhibitory effects on LPH transactivation.
There is precedent for such regulatory interactions between GATA
factors. GATA-1 and GATA-2 show signs of mutual cooperativity. In
addition, GATA-1 has been shown to repress GATA-2 expression (30).
Studies of the ranges of expression and functional interactions of
intestinal GATA binding proteins will lead to a clearer understanding
of how they modulate one another in the gut. Not surprisingly, there
are GATA motifs upstream of other genes whose expression is restricted
to enterocytes, including those which encode the intestinal fatty acid
binding protein (25) and SI (32). It will be of interest to determine whether an intestinal GATA binding protein(s) plays a functional role
in regulating the transcription of these and/or other promoters that are active in intestinal cells.
 |
ACKNOWLEDGEMENTS |
We thank Dr. N. Mantei for providing the LPH promoter-containing
phage
LPH 7, Dr. J. B. E. Burch for providing a GATA-6 expression vector, and Dr. T. Evans for providing a GATA-6 cDNA probe. In addition, we acknowledge the excellent technical assistance of M. Potts, D. Meighen, the help of P. D. Avigan, and the generous sharing
of reagents by Dr. I. Sunitha. Finally, we also thank Drs. S. Irving,
A. Riegle, B. Jenson, and R. Glazer for helpful discussions and their
critical reviews of this manuscript.
 |
FOOTNOTES |
M. I. Avigan was supported by National Cancer Institute Grant CA-54818.
Address for reprint requests: M. I. Avigan, Depts. of Pathology and
Medicine, Georgetown Univ. School of Medicine, 3900 Reservoir Rd. NW,
Washington, DC 20007.
Received 30 January 1997; accepted in final form 6 November 1997.
 |
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