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INTRODUCTION |
NaCl absorption occurs in the small intestine and colon
predominantly by coupled Na+/H+ and
Cl
/HCO
exchange (1). In
the absorptive epithelium of the mammalian intestine,
Na+/H+ exchanger isoform 3 (NHE3)1 is a major component
of electroneutral NaCl absorption and is a frequent inhibitory target
in many diarrheal disorders (2). NHE3 also plays an essential role in
maintenance of systemic pH via Na+ and
HCO
reabsorptive processes in the
renal proximal tubules. The importance of the NHE3 gene has been clearly established by gene targeting experiments (3-5); mice
lacking the NHE3 gene exhibit a disease phenotype of chronic diarrhea and altered salt, water and acid-base homeostasis resulting in
mild acidosis and reduced blood pressure (3).
NHE3 activity is regulated at different levels:
phosphorylation-mediated regulation of the transporter dependent on its
linkage to the cytoskeletal protein ezrin with NHERF and E3KARP (6), regulation by trafficking of NHE3 protein on and off the apical membrane via changes in endocytosis and apical membrane recycling (7),
regulation through association with lipid rafts in the brush-border
membrane (8), and through transcriptionally mediated changes in NHE3
mRNA levels. An array of factors has been implicated in
transcriptional regulation of NHE3 gene expression including glucocorticoid hormones (9, 10), thyroid hormone (11), protein kinase C
(12), and sodium butyrate (13, 14). Previous studies from our
laboratory also suggested that transcriptional regulation is a critical
component of maturational changes in intestinal NHE3 activity and gene
expression during rat postnatal development (15). Although laboratory
rodents have become a common model to study physiology and
pathophysiology of Na+/H+ exchange, and the rat
NHE3 gene promoter has been cloned by two independent groups
of investigators (9, 16), a comprehensive analysis of its function and
basal regulation has been lacking to date.
NHE3 exhibits not only a temporal but also a horizontal,
differentiation-specific pattern of expression along the crypt-villus axis. NHE3 expression is very low in the small intestinal epithelium of
suckling animals and drastically increases after weaning (15). Additionally, NHE3 protein is not expressed in undifferentiated crypt
cells of the small intestine or colon, while it is present in high
abundance in differentiated absorptive enterocytes with a detectable
gradient of expression directed toward the tips of the intestinal villi
(17-19). Due to its physiological importance and complex expression
patterns, NHE3 may be considered a marker gene suitable for studying
the role of specific transcription factors and their interactions
during enterocyte differentiation as well as during postnatal
intestinal development, similar to the already established models of
lactase-phlorizin hydrolase (LPH), sucrase-isomaltase (SI), and
intestinal fatty acid-binding protein (iFABP). The body of knowledge
provided by studying regulation of intestinal gene expression provided
by studies of the three latter genes is already significant, but
despite this fact, a complete understanding of the genetic programming
of mammalian intestinal differentiation and maturation is still far
from being achieved.
In recent years, a concept of combinatorial gene regulation has become
a paradigm in understanding complex regulation of gene transcription in
eukaryotes. The activation of eukaryotic genes in vivo often
requires the coordinated binding of multiple transcription factors to
promoter-enhancer regions of genes. Many of these transacting factors
are not only expressed in tissue- and differentiation-specific manners,
but they are also regulated by distinct signaling pathways. Furthermore, in many cases, cooperative binding of multiple
transcription factors to gene promoter requires a unique composition
and spatial arrangement of transcription factor binding sites. These
facts add to the complexity of transcriptional networks participating in regulation of gene expression.
In this article, we describe the first comprehensive analysis of
NHE3 promoter function. We confirm previously disputed
transcription initiation site, describe a potentially novel initiator
sequence, delineate the role of Sp family transcription factors in
regulation of NHE3 promoter activity, and identify a
functional binding site for GATA transcription factors in exon 1. Most
importantly, we also provide evidence for a novel synergistic
interaction between GATA-5 and Sp1 and Sp3 in regulating
NHE3 promoter function.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Human colonic adenocarcinoma (Caco-2) cells,
normal rat small intestinal epithelial (IEC-6) cells, NIH-3T3 mouse
fibroblasts, quail fibrosarcoma (Qt6) cells, and Drosophila
SL2 cells were obtained from American Type Culture Collection
(Manassas, VA). Caco-2 and IEC-6 cells were maintained in high-glucose
Dulbecco's Modified Essential Medium (DMEM) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 µg/ml streptomycin, and
100 units/ml penicillin G (further referred to as antibiotics), and
10% (v/v) fetal bovine serum. NIH-3T3 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% bovine calf serum and
antibiotics. QT-6 cells were maintained in F12K medium with 10%
tryptose phosphate broth, 5% bovine calf serum, and antibiotics. All
of the aforementioned cells were grown at 37 °C in a 5%
CO2, 95% air incubator. Drosophila SL2 cells were maintained in Schneider's insect medium supplemented with 10%
(v/v) fetal bovine serum and antibiotics, and were grown at 25 °C
without CO2.
Plasmid Constructs--
Rat NHE3 promoter constructs
were prepared in pGL-3 basic luciferase reporter vector (Promega,
Madison, WI) as described earlier (14). Since overexpression of Sp or
GATA transcription factors activated promoter-less pGL3-basic but not
p
Gal-basic vector (Clontech, Palo Alto, CA) in
transfected cells, transactivation experiments with these factors were
performed with NHE3 promoter fragments subcloned into
p
Gal-basic. Site-directed mutations were introduced by standard PCR
with mutant forward primers utilizing a high fidelity DNA polymerase
(Pfx, Invitrogen, Carlsbad, CA) or by two-step overlap PCR as
previously described (20). A construct spanning
20/+8 bp of the
NHE3 gene was created by subcloning a double-stranded
synthetic oligonucleotide into XmaI/XhoI sites of
pGL3-basic. All constructs were confirmed by DNA sequencing.
5'-RLM-RACE--
Since two previous reports (9, 16) described
different transcription initiation sites within the rat NHE3 gene, RNA
ligase-dependent rapid amplification of cDNA ends
(5'-RLM-RACE) was employed as an alternative method to identify the
5'-end of the NHE3 transcript utilizing components of FirstChoice
RLM-RACE kit (Ambion, Austin, TX). High quality total RNA was isolated
with trizol reagent (Invitrogen) followed by extraction with acid
phenol. RNA from the rat small intestine and colon was used to confirm
the transcription initiation site in the endogenous NHE3
gene, whereas DNase I- treated RNA isolated from Caco-2 cells
transfected with NHE3 gene reporter plasmid (
118/+58Luc)
was used to confirm the transcription initiation site utilized in
synthetic reporter gene constructs. Briefly, 10 µg of RNA was
dephosphorylated to remove the 5'-phosphate group from RNA or
contaminating DNA molecules. Tobacco acid pyrophosphatase (TAP) was
then used to specifically remove the cap structure from mRNA
molecules. An RNA oligonucleotide was next ligated to newly de-capped
mRNA using T4 RNA ligase and the resulting RNA was
reverse-transcribed using SuperScript III (Invitrogen) and random
primers. Outer and nested hot-start PCR reactions were performed using
proofreading DNA polymerase (Platinum Taq Hi Fidelity,
Invitrogen) with adapter- and gene-specific primers. 1 µl of the
nested PCR product was subcloned into pCR4Blunt-TOPO vector
(Invitrogen). 25-35 independent clones were sequenced for each
5'-RLM-RACE reaction.
Transfections and Reporter Gene Assays--
Caco-2 cells were
transfected in 24-well plates at ~80% confluency with 200 ng of
NHE3 promoter constructs and 2 ng of promoter-less pRL-null
vector containing the Renilla luciferase reporter gene as an
internal control. IEC-6, NIH-3T3, and Qt6 cells were transfected in
6-well plates with 1 µg of NHE3 promoter constructs and 10 ng of pRL-null. In experiments with forced expression of GATA transcription factors, 0.2 or 1 µg of pCDNA3 (Invitrogen) or
pCDNA3-based expression vectors with mouse GATA-4, GATA-5, or
GATA-6 cDNAs (generously provided by Dr. E. Morrissey) were
co-transfected along with NHE3 promoter constructs into
respective cell lines. All mammalian cell lines were transfected with
LipofectAMINE (Invitrogen) and Qt6 cells were transfected with FuGENE 6 (Roche; Indianapolis, IN) according to manufacturer's instructions.
24 h post-transfection, cells were harvested and assayed for
reporter gene activity using a dual luciferase assay (Promega; for
constructs made in pGL3-basic) or with Galacto-Light Plus (Applied
Biosystems, Foster City, CA) and Renilla luciferase assay
system (Promega; for constructs in p
Gal-basic). Endogenous
-galactosidase activity in mammalian cell lines was reduced by
incubating cell lysates at 48 °C for 50 min prior to performing the
-galactosidase assay.
SL-2 cells were seeded at 500,000 cells per well in 24-well plates
24 h prior to transfection with 200 ng of p
Gal
118/+58 and
various amounts of Sp1 or Sp3 and/or GATA-5 expression plasmids containing the Drosophila actin 5 promoter (pPacSp1 and
pPacUSp3 were generously provided by Drs. R. Tjian and G. Suske,
respectively; pAc-GATA-5 construct was made by subcloning a
SmaI/EcoRV fragment of mouse GATA-5 into the
EcoRV site of pAc5.1A plasmid (Invitrogen)).
-galactosidase activity was expressed as relative light units (RLU)
per µg of protein. The protein concentration was determined by BCA
protein assay (Pierce, Rockford, IL).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays--
Nuclear extracts were prepared using a modification
of the method by Dignam (21) as described by Dent and Latchman (22). The obtained nuclear protein was dialyzed in 10,000 MWCO mini dialysis
units (Pierce) against buffer containing 20 mM HEPES (pH
7.9), 100 mM KCl, 1 mM MgCl2, 20%
glycerol, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride and stored at
70 °C until use.
For gel shift assays, PAGE-purified double-stranded oligonucleotides
(Integrated DNA Technologies, Coralville, IA) were end-labeled with
[
-32P]ATP by T4 polynucleotide kinase (Promega).
Assays were performed by incubating 5 µg of nuclear protein with
10,000 to 20,000 cpm of labeled probe for 20 min. Competing unlabeled
oligonucleotides were added 20 min prior to addition of the
radiolabeled probe. Sp protein gel shift assays were performed with
commercially optimized reagents (Sp1/Sp3 Gelshift Kit; Geneka;
Montreal, Canada) with binding reactions carried out at 10 °C.
Binding reactions for GATA transcription factors were carried out at
room temperature in binding buffer containing 10 mM
Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 4% glycerol, 1 µg/reaction poly(dI-dC) and 1 µg/reaction polylysine.
Southwestern Blotting--
All procedures were carried out at
4 °C. 50 µg of Caco-2 and IEC-6 cell nuclear extract was
fractionated on a 4-20% SDS-polyacrylamide gel and transferred to a
nitrocellulose membrane in a buffer containing 25 mM Tris
and 190 mM glycine for 30 min at 100 V. The membrane was
then incubated with blocking buffer (2% nonfat dry milk, 1% bovine
serum albumin, 50 mM HEPES (pH 7.9), 75 mM
MgCl2, 40 mM KCl, 0.05 mM EDTA, 5%
glycerol, 140 mM
-mercaptoethanol, and 16 µg/ml
sonicated salmon sperm DNA) for 2 h and then incubated with
binding buffer (same as blocking buffer but with 0.2% nonfat dry milk)
containing 32P-labeled double-stranded probe (
20/+8 bp
NHE3 promoter fragment; ~106 cpm/ml) for
16 h, washed, and subjected to autoradiography. An identical
binding reaction was performed with 200-fold excess of unlabeled probe
as a negative control.
Statistical Analysis--
One-way analysis of variance or the
Student's t test were employed for statistical analyses
utilizing StatView software (v. 4.0; SAS Institute, Inc., Cary, NC).
Post-hoc multiple comparisons were carried out with Fisher's PLSD test.
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RESULTS |
Transcription Initiation Site Mapping--
Since previous reports
utilizing primer extension (9) and S1 nuclease protection assay (16)
techniques produced contradictory results, we employed 5'-RLM-RACE as
an alternative method of mapping the 5'-end of the NHE3 transcript.
This technique identified multiple transcription initiation sites
(TIS), overlapping those detected by Kandasamy and Orlowski (16) by S1
nuclease protection assay and localized within the
3ACCTG+2 sequence (Fig.
1). Interestingly, the major
transcription initiation site reported by Kandasamy and Orlowski (16),
which is used for nucleotide numbering throughout this study, was not
seen in the jejunal RNA pool or in the heterologous
NHE3/Luc gene, and only 4% of colonic NHE3
transcripts started at this position. Utilization of TIS in different
tissues varied slightly, as summarized in Fig. 1. None of the sequenced
clones matched the TIS described by Cano (9) by primer extension. Small
proportion of 5'-RLM-RACE products (4% jejunum; 4% colon) started
20-70 bp downstream of the 5'-end of NHE3 transcript identified by
initial cloning experiments with the cDNA library (23). Since
cDNA libraries rarely contain full-length 5'-untranslated regions,
these results were dismissed as likely artifacts.

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Fig. 1.
Verification of transcription initiation
sites in the NHE3 gene by 5'-RLM-RACE. Shown
above is sequence of the proximal rat NHE3 promoter
(plain font) and the 5'-end of cDNA from initial cloning
experiment (Ref. 23; bold letters) with TIS mapped by Cano
(9) and Kandasamy and Orlowski (16). Long vertical arrows
indicate major TIS, short arrows indicate minor TIS from the
two previous studies. Shown in the middle are the results of
5'-RLM-RACE performed with jejunal and colonic RNA and with RNA
isolated from Caco-2 cells transfected with the 118/+58Luc
NHE3 reporter gene construct. The table depicts TIS
utilization expressed as percent of all sequenced clones. Location of
5'-ends of NHE3 transcripts unaccounted for in the table is described
in more detail under "Results." Shown below is gel analysis of the
nested PCR reactions from 5'-RLM-RACE with jejunal and colonic
RNA. TAP+ and TAP lanes represent RACE reactions with RNA
processed with or without tobacco acid pyrophosphatase
(TAP), respectively.
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The same cluster of TIS was utilized to drive expression of the
luciferase reporter gene from the
118/+58Luc NHE3 promoter construct (Fig. 1). While 91% of detected transcripts originated within the same cluster of nucleotides
(
3ACCTG+2), 4% of transcripts of the
reporter gene started at nt
9 and 5% at nt +21. Relevance of these
sites is however, unclear.
Functional Mapping of the Rat NHE3 Promoter--
A series of
reporter plasmids containing various lengths of the NHE3 5'-flanking
regions (from nt
1360 to nt
20) upstream of the firefly luciferase
(Luc) gene was transfected into Caco-2 cells. The levels of
Luc activity were normalized to Renilla luciferase activity.
A short promoter fragment spanning nt
118 to +58 bp conferred 82% of
the activity of the longest cloned promoter fragment (p = 0.015 for
1360/+58 versus
118/+58) (Fig.
2). Minor fluctuations in activity were
observed among constructs including more than 118 bp of the promoter
sequence, suggesting the presence of enhancing or repressive elements,
which conceivably play a role in fine-tuning transcriptional activity.
Also, further deletion to
81 bp only slightly altered the promoter
activity (18% decrease; p = 0.03 for
118/+58
versus
81/+58). Surprisingly, a short construct spanning
20/+58 bp exhibited significant activity (4.9-fold above background;
p = 0.008 for
20/+58 versus pGL3-basic),
whereas extending the sequence further upstream (
35/+58 bp) to
include the reported atypical TATA box (ATTAAA; Ref. 16) resulted in a
decrease to levels not statistically different from the promoter-less vector (Fig. 2). Since extension of the promoter past the TATA-like box
had a negative rather than a positive effect on NHE3
promoter activity, we set out to address its relevance by mutating it
(ATTAAA
ATGCAA; mutated bases in bold) in the
118/+58-bp construct and testing its activity in transiently
transfected Caco-2 and IEC-6 cells. Mutation of this atypical TATA box
did not result in significant changes in promoter activity in either
cell line (Fig. 3A). Similar
results were obtained with an internal deletion construct (not shown).

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Fig. 2.
Deletion analysis of the rat NHE3
promoter. Firefly luciferase reporter constructs containing
various lengths of the NHE3 promoter were generated as
described under "Experimental Procedures" and were cotransfected
into Caco-2 cells along with a promoter-less pRL-null plasmid.
Twenty-four hours later, the cells were harvested and firefly and
Renilla luciferase activities were measured. The results are
the means (±S.D.) of at least three independent experiments. The
sequence of the NHE3 gene spanning 118/+58 bp with
annotated putative cis-elements is depicted below. Predicted
binding sites shown to be important for basal promoter activity are
depicted in bold letters. The arrow at
T+1 represents the major TIS identified previously (16),
whereas other arrows mark the 5'-ends of promoter fragments
studied in reporter gene assays.
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Fig. 3.
The role of the TATA-like sequence and
initiator in NHE3 promoter activity. A,
caco-2 (open bars) or IEC-6 cells (black bars)
were transfected with wild-type NHE3 promoter construct
( 118/+58) or with analogous construct containing mutated TATA-like
box ( 118/+58mTA), or with promoter-less pGL3-basic vector
(basic). No statistical differences were detected between
activities of wild-type and mutated promoter constructs within
respective cell line. Data are means ± S.D. (n = 4). Different letters indicate statistical differences within
respective cell lines. B, the 20/+8 bp sequence
surrounding the major transcription start site promoted low level
expression of the reporter gene when transfected into Caco-2 cells. *,
p < 0.05; n = 6. C, the
20/+8 bp sequence used as a probe in gel mobility shift assay formed
a specific complex with Caco-2 nuclear protein. This complex could be
competed in a dose-dependent manner with unlabeled probe
containing a canonical TATA-box (fold excess indicated) but not with a
nonspecific (ns) double-stranded oligonucleotide. No
NE indicates reaction with no nuclear extract (probe only).
D, in Southwestern blotting experiments, 20/+8 bp
radiolabeled probe interacted weakly (2-week exposure) with a Caco-2
and IEC-6 nuclear protein of ~40 kDa (upper panel);
200-fold excess of unlabeled probe in analogous experiment effectively
competed formation of labeled protein/DNA complex (lower
panel).
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Additional studies showed that 28 bp immediately surrounding the
transcription start site (
20/+8 bp), were able to promote low level
transcription (Fig. 3B). In gel mobility shift assays with
Caco-2 cell nuclear extract and a labeled
20/+8 bp oligonucleotide as
a probe, we were able to demonstrate a specific protein-DNA complex,
which could be competed in a dose-dependent fashion with a
consensus TATA box containing oligonucleotide, which was previously demonstrated to bind TATA-box binding protein (TBP; Ref. 24) (Fig.
3C). Additionally, in Southwestern blotting experiments with
nuclear protein from Caco-2 and IEC-6 cells, we demonstrated weak
binding of labeled
20/+8 bp probe to a protein of ~40 kDa, which is
consistent with the molecular weight of the p36 TFIID protein (Fig.
3D).
Downstream Elements--
Deletion of 50 nt of exon 1 in construct
118/+8 resulted in a significant reduction in NHE3
promoter activity (Fig. 4B). Computer prediction analysis (25) of putative cis elements
located in this exonic region suggested binding sites for AP4 and GATA transcription factors (Fig. 2) and an overlapping binding site for AP1
and CREB. Mutation of the AP1/CREB site resulted in no significant
change in promoter activity (data not shown). We therefore focused on
the sequence spanning nt +8 to +30 and performed gel mobility shift
assays with this region as labeled probe and with competing
double-stranded oligonucleotides with scanning mutations in blocks of
four base pairs (Fig. 4A). Only mutant 4 (M4), which included a mutated GATA box was not able to compete for nuclear protein
binding, suggesting that GATA transcription factors bind to this
element. Furthermore, this shifted complex could also be competed with
a commercially obtained consensus GATA box oligonucleotide (Geneka)
from the tal-1 gene (26). The importance of this GATA-box in
the NHE3 gene was further demonstrated by introducing an
internal deletion (
GATA) into the
118/+58 construct. In
transfected Caco-2 cells, the activity of this construct was
significantly lower than that of the wild-type
118/+58-bp plasmid,
and was not significantly different from the construct with 3' deletion
of 50 nt of exon 1 (Fig. 4B).

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Fig. 4.
The role of GATA-box (nt +20/+23) in
NHE3 gene promoter activity. A, gel
mobility shift assays utilizing scanning mutations in competing
oligonucleotides demonstrated specific binding of Caco-2 nuclear
protein to a GATA box at nt +20/+23. In the top panel,
WT is the sequence derived from NHE3 gene exon 1 and was used as a labeled probe and as a cold competitor. M1 through M6
are sequences representing progressive mutations introduced into the WT
sequence in blocks of four base pairs. M4 represents a competitor with
a mutated GATA box. GATA is an oligonucleotide containing a consensus
GATA box derived from the tal-1 gene (see "Experimental
Procedures"). All competitors were used in 50-fold excess. Below is a
typical gel shift experiment. Sc and ns indicate
specific and nonspecific protein/DNA complexes, respectively.
B, in transiently transfected Caco-2 cells, deletion of 50 bp of exon 1 from NHE3 promoter construct ( 118/+58 118/+8) resulted in a similar decrease in reporter gene expression as
compared with a construct with internal deletion of the GATA box in
position +20/+23 ( 118/+58 GATA). Different letters next to
bars indicate statistical differences (p < 0.05; data are means ± S.D.; n = 4-6).
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Proteins Binding to a +20/+23 NT GATA-Box--
GATA-4,
-5, and -6 subfamily proteins are expressed in an overlapping pattern
in the developing heart and endoderm-derived organs of the
gastrointestinal tract including the stomach, intestine, liver,
and pancreas (27). To address binding specificity of the reported
GATA box, a 20-bp GATA box-containing probe (nt +10/+30) was used
in gel mobility shift assays. Nuclear protein from Caco-2 (Fig.
5A) and IEC-6 cells (not
shown) formed a specific complex which could be competed with excess
unlabeled probe (Comp. A) and a tal-1 consensus GATA box (Comp. B), but
not with a probe containing a mutation in the GATA box from the
NHE3 gene (Comp. C). Also, the complex could be supershifted
with an antibody specific for GATA-6 (Geneka), the GATA isoform
predominantly expressed in Caco-2 cells. Gel shift experiments with
IEC-6 cell nuclear protein showed identical results (data not shown).
Although IEC-6 cells were found by RT-PCR to express GATA-4, -5, and -6 (data not shown), we were not able to demonstrate their binding in
supershift experiments due to lack of reliable antibodies for GATA-4
and GATA-5. Instead, we used nuclear protein from Qt6 cells transfected with empty expression vector or with GATA expression plasmids. Gel
mobility shift assays with Qt6 cell nuclear extract confirmed that
these cells lack the endogenous nuclear proteins, which bind to
mammalian GATA sequences. Moreover, nuclear protein from
GATA-transfected Qt6 cells formed specific complexes with the NHE3 GATA
probe (+10/+30 nt), which upon longer separation corresponded with
differences in molecular weight of respective GATA proteins (Fig.
5B). This experiment indicated that all three intestinally
expressed GATA transcription factors were capable of binding to the
NHE3 GATA probe.

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Fig. 5.
Proteins binding to the GATA-box.
A, in-gel mobility shift assays with Caco-2 nuclear
protein, specific protein/DNA complexes (lane 2) could be
supershifted with anti-GATA-6 antibody (Ab; lane
3) and could be competed with excess of unlabeled probe
(Comp. A; lane 4), or a consensus GATA probe from
an unrelated gene (Comp. B; lane 5), but not by a
cold probe with a mutated GATA box (Comp. C, mutation
underlined; lane 6). B, nuclear
proteins from Qt6 cells transfected with GATA-4, -5, and -6 expression
vectors form specific complexes with the NHE3 gene GATA box
(radiolabeled probe as in panel A), which upon longer
separation correspond to differences in molecular weight of respective
GATA transcription factors. C, forced expression of
GATA transcription factors in Qt6 cells resulted in induction of
co-transfected 118/+58- Gal reporter gene construct activity.
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Transactivation of NHE3 Promoter by Overexpressed GATA
Transcription Factors--
Forced expression of GATA-4, -5, or -6 in
transiently transfected Caco-2 and IEC-6 cells resulted in increased
NHE3 promoter activity (Fig.
6A), with GATA-5 being the
strongest stimulator. Similar results were obtained in the
non-intestinal cell lines, NIH-3T3 (data not shown) and Qt6 fibroblasts
(Fig. 5C). Surprisingly, overexpression of GATA-5 did not
affect the activity of a short promoter fragment (nt
20/+58),
despite the fact that it contained an intact GATA box (nt +20/+23)
(Fig. 6B), suggesting that elements upstream of nt
20 are
necessary for GATA-5 stimulation of the NHE3 promoter.
Extending the 5'-flanking region to nt
81 restored the
transactivation of the promoter to levels observed with the
118/+58
construct. Furthermore, mutation of three putative Sp family
transcription factor binding sites, with core cis elements located at nt
71/
68 (SpA),
58/
55 (SpB; complementary strand), and
46/
43 (SpC), again eliminated the stimulatory effects of GATA-5
on NHE3 promoter activity (Fig. 6B). This
suggested cooperative or synergistic effects of GATA-5 and Sp proteins
interacting at one or more of the three putative Sp binding sites.

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Fig. 6.
Transactivation of NHE3
promoter by GATA transcription factors. A,
activity of an NHE3 promoter construct ( 118/+58) in Caco-2
(black bars) or IEC-6 (open bars) cells
contransfected with empty vector (pCDNA3) or with respective GATA
cDNA cloned into the same vector. B, in transfected
Caco-2 cells, the shortest active promoter construct ( 20/+58)
containing an intact GATA box in exon 1 did not respond to
overexpression of GATA-5 (black bars) when compared with
cells contransfected with empty expression vector (gray
bars). However, extension of the 5'-flanking region to include
putative Sp transcription factor binding sites in construct 81/+58
restored the response, which was again abolished by mutating all three
Sp binding sites (81/+58 SpMutABC). Depicted below is a scheme
representation of NHE3 promoter constructs used in this
experiment. +1 indicates TIS (16). Data presented in panels
A and B are means ± S.D. (n = 4-6).
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The Role of the Putative Sp Binding Sites in NHE3 Promoter
Activity--
Gel mobility shift assays were performed with a 42-bp
DNA probe spanning all three putative Sp consensus elements and nuclear protein purified from Caco-2 and IEC-6 cells. We demonstrated that this
sequence forms a specific shifted complex, which could be competed with
excess unlabeled probe (Fig.
7A). We also tested the
ability of individual and composite mutants of the three Sp sites to
compete for binding as an indirect measure of their affinity for Sp
transcription factors. Single site A and C mutants effectively competed
for binding with labeled wild type probe, although not as efficiently
as the unlabeled wild type probe, suggesting that these sites have very
low affinity for Sp proteins. Site B mutant oligonucleotide was much
less effective as a competitor, which signifies the importance of site
B in interacting with Sp transcription factors. Double mutation of
sites A and C did not result in decreased competition; however, double
mutations of sites A and B as well as B and C reduced their ability to
compete for binding. Furthermore, mutation of all three Sp binding
sites resulted in complete loss of competition. Identical results were
obtained with Caco-2 and IEC-6 nuclear protein (Fig. 7A).
These data suggest that while site B is the predominant site of Sp/DNA
interaction, sites A and C may also play a supportive role, with Sp
binding to site B being a prerequisite and possibly preceding
event.

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Fig. 7.
The role of putative Sp binding sites in
NHE3 gene promoter activity. A, gel
mobility shift assay with Caco-2 and IEC-6 cell nuclear protein and a
wild-type oligonucleotide containing a cluster of putative Sp sites
(A, B, and C; nt 77/ 36) in the
proximal promoter of the NHE3 gene. Sequences depicted below
represent the labeled probe (WT) and competing
oligonucleotides used in the assay. Core Sp cis-elements are
emphasized by rectangles and bold letters
indicate mutations introduced to one or more of the Sp sites of
competing oligonucleotides (all competitors were used in 50× molar
excess). B, similar gel mobility shift analysis with
oligonucleotides containing individual NHE3 gene promoter Sp
sites used as competitors and Caco-2 nuclear protein. SpA, B, and C
sequences were slightly modified on the 5'- and 3'-ends to prevent
overlap and to provide sufficient length for binding. Sp and SpM are
consensus Sp1 elements from an unrelated gene and its mutated form,
respectively. ns indicates a consensus TATA-box-containing
oligonucleotide, which was used as a nonspecific competitor.
C, functional analysis of individual or composite mutants of
Sp sites in the NHE3 promoter in transiently transfected
Caco-2 cells. The 81/+58 construct was used with mutations introduced
as depicted in panel A. Data are means ± S.D.
(n = 4).
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The relevance of site B in nuclear protein binding is further
demonstrated in Fig. 7B, where individual sites A, B, and C from the NHE3 promoter were used as competitors along with a
commercial Sp consensus probe and a nonspecific double-stranded
oligonucleotide (TATA-box). Competitor probes with individual Sp sites
were slightly modified at their 5'- and 3'-ends to avoid overlap and to
provide sufficient probe length. In this series of experiments, site B was able to compete for binding along with excess wild type unlabeled probe and Sp consensus probe. Furthermore, functional data from Caco-2
cells transfected with the
81/+58 bp constructs with individual or
composite mutations of the putative Sp sites presented in Fig. 7C, correlated well with the results of gel mobility shift assays.
To identify proteins binding to the cluster of Sp sites in the
NHE3 promoter, supershift assays were performed with the
same labeled probe (see Fig. 7) and Caco-2 or IEC-6 nuclear protein. As
shown in Fig. 8A, Sp1 appeared
to be the predominant protein binding to this sequence in Caco-2 cells,
since the majority of the specific complex was supershifted by
Sp1-specific antibody. A blocking antibody specific for Sp3 only weakly
reduced the intensity of the complex. In IEC-6 cells, however, Sp3 was
the predominant isoform found in the protein-DNA complex, as the band
intensity was significantly reduced by anti-Sp3 antibody, while Sp1
antibody only very weakly supershifted the formed complex. Binding of
Sp1 and Sp3 to this region of the NHE3 promoter was further
demonstrated by gel mobility shift assay with nuclear protein from
Drosophila SL2 cells transfected with empty expression
vector or Sp1 or Sp3 expression constructs (Fig. 8B).

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Fig. 8.
Proteins binding to the cluster of Sp sites
in the NHE3 promoter. Gel mobility shift assays
were performed with Caco-2 and IEC-6 cell nuclear protein and WT
sequence (see the legend to Fig. 7) as a labeled probe. Supershift
analysis (sc, specific complex; ss, supershifted
complex) indicated that Sp1 is the predominant Sp transcription factor
binding this promoter region in Caco-2 cells, while Sp3 is the
predominant protein in IEC-6 cells. B, similar analysis with
nuclear protein from SL2 cells transfected with empty vector or Sp1 or
Sp3 expression plasmid further confirmed that both Sp1 and Sp3 were
capable of binding the probe.
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Synergistic Interaction between GATA-5 and Sp1 and
Sp3--
Results reported in Fig. 6 suggested that GATA-5 functionally
interacts with Sp transcription factors to regulate NHE3
promoter activity. To determine if these transcription factors indeed
act synergistically, we utilized Sp-deficient SL2 cells. Cotransfection of an NHE3 promoter construct (nt
118/+58 in
p
Gal-basic) with increasing amounts (5-200 ng) of GATA-5 expression
plasmid under control of the Drosophila actin 5 promoter,
did not result in a significant increase in reporter gene activity in
the absence of Sp proteins (data not shown). In order to determine the
amount of transfected Sp1 or Sp3 expression plasmids that would result in submaximal stimulation of NHE3 promoter activity, we
cotransfected SL2 cells with the
118/+58
Gal promoter construct
along with increasing amounts of pPacSp1 or pPacUSp3 vectors. The
stimulatory effect of overexpressed Sp1 saturated with a lower amount
of cotransfected plasmid (Fig.
9A) as compared with Sp3,
which increasingly activated reporter gene expression throughout the
range of transfected plasmid amount (Fig. 9B). In subsequent
experiments, the
118/+58 bp NHE3 promoter construct was
contransfected into SL2 cells with 20 ng of GATA-5 expression vector, 5 ng of Sp1 or 10 ng of Sp3 vector, or with a combination of GATA-5 and
Sp1 or Sp3 plasmids (Fig. 9C). In agreement with previously
obtained data, overexpression of GATA-5 alone did not stimulate
NHE3 promoter activity. However, the same amount of GATA-5
expression plasmid cotransfected with Sp1 or Sp3 resulted in
synergistic activation of NHE3 promoter activity. Additive,
synergistic, or antagonistic interactions among transcription factors
were identified by calculating the interaction response (IR), which is
a measure of comparing the effect of two expression vectors
cotransfected together to the additive effect of each of the expression
vectors cotransfected separately (28) as shown in Equation 1.

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Fig. 9.
Transactivation of the NHE3 promoter by
overexpressed Sp1, Sp3, and GATA-5 transcription factors in SL2
cells. The 118/+58 Gal NHE3 promoter construct was
cotransfected with increasing amounts of Sp1 (panel A) or
Sp3 expression plasmid (panel B) into SL2 cells and reporter
gene activity was analyzed 24 h later. Based on the obtained data,
amounts of Sp expression plasmids resulting in submaximal activation of
NHE3 promoter were selected (5 ng of pPacSp1, 10 ng of pPacUSp3) and
cotransfected with NHE3 promoter vector with or without a
GATA-5 expression construct (panel C). The dashed
lines indicate the sum of the transcriptional activities of
NHE3 promoter in cells transfected individually with GATA-5
and Sp1 or GATA-5 and Sp3. Values extending upward of the dashed
lines indicate synergistic activation of the NHE3
promoter. Data in all panels are means ± S.D. (n = 6).
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(Eq. 1)
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Values of
0.1 to 0.1 are defined as additive effects, greater
than 0.1 represent synergistic effects, and less than
0.1 represent
antagonistic effects. According to these criteria, both Sp1 and Sp3
activated the NHE3 promoter synergistically with GATA-5 (IR = 0.44 and 0.19, respectively).
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DISCUSSION |
The rat NHE3 promoter was cloned in 1996 by two
independent laboratories (9, 16). Both reports provided genomic
sequence of approximately the same region and discussed several
putative, prediction analysis based, transcription factor binding
sites. However, no detailed characterization of the promoter or
functional mapping of cis-acting elements was presented.
Moreover, different transcription initiation sites were demonstrated by
both groups. Kandasamy and Orlowski (16) used S1 nuclease protection
analysis and mapped it to a cluster of five nucleotides with a major
site at
97 (T), and two minor sites at
100 (A) and
96 (G) nt
relative to the translation start codon. Cano (29) mapped the
transcription initiation site to the atypical TATA box
(GGATTAAA; +1 nt in bold) located at
128 nt 5' of the AUG
initiation codon. To address this discrepancy, we utilized an
alternative method to map the NHE3 gene transcriptional
start site, 5'-RLM-RACE. Sequencing data obtained from RACE products
obtained with RNA isolated from the small intestine, colon, as well as
from Caco-2 cells transiently transfected with the
118/+58 Luc
construct, mapped the transcription initiation site to a cluster of
nucleotides reported by Kandasamy and Orlowski (16). Also, functional
data from transiently transfected Caco-2 and IEC-6 cells described in
this paper, argue against the transcription initiation site reported by
Cano (29). A reporter construct containing
20 to +58 bp of the
NHE3 gene lacking the TATA-like sequence (positioned at nt
30/
27) was transcriptionally active (5-fold higher then promoter-less vector). Extending the 5'-flanking region of the gene to
include the only start site reported by Cano (in construct
35/+58
Luc) in fact decreased the activity of the promoter to background
levels. Many A/T-rich sequences can convey TATA box activity, which is
partly because TATA-box-binding protein (TBP) recognizes the minor DNA
groove, where protein-DNA interactions are more often influenced by T/A
content than by a specific sequence (30). Therefore, we also mutated or
deleted this atypical TATA box in constructs
118/+58mTA (Fig.
3A) and
118/+58
TA (not shown). Both promoter constructs
exhibited the same activity as the wild-type sequence in transfected
Caco-2 and IEC-6 cells. Taken together, these data suggest that
transcription intiation occurs independently of the presence of this
TATA-like sequence, and that sequence surrounding the transcription
start site as mapped by S1 nuclease protection assays and 5'-RLM-RACE
may form the actual NHE3 gene core promoter. In support of
this hypothesis, we demonstrated that the sequence spanning
20/+8 bp
of the NHE3 gene was able to promote low level
transcription, consistent with typical low activity of isolated
initiator (Inr) elements.
In mammals, the diversity of core promoters is quite significant,
although precise analyses on a genome-wide scale are complicated by the
lack of accurate descriptions of transcription initiation sites for the
majority of genes. Promoters can be classified into those that contain
a functional TATA-box, TATA-box paired with an Inr, Inr element with
downstream promoter elements (DPM), and CpG island-rich promoters which
apparently lack all three core elements (31). These core elements are
believed to serve as recognition sites for the TFIID complex, which
contains TBP and various TBP-associated factors (TAFs) (32). Although
far from being conclusive, our data obtained from gel mobility shift
assays as well as Southwestern blotting (Fig. 3, C and
D) suggests that TBP bind to the NHE3 core
promoter Inr element, despite the fact that it has a weak homology to
the otherwise loose Inr consensus sequence
(Py-Py-A+1-N-(T/A)-Py-Py) (33).
Downstream control elements located 3' of the transcription initiation
site within Inr-containing promoters have been found essential for
promoter activity in vivo, e.g. in the adenoviral major late (AdML) (34) and murine terminal transferase
(TdT) (35) genes. Although some studies indicated that
TFIID, TFII-I, cap-binding protein (CAP), or USF proteins may bind to
these regions, downstream elements remain poorly understood. In the
NHE3 promoter, deletion of nt +9/+58 resulted in a
significant loss of promoter activity. Further studies unequivocally
demonstrated that a GATA-box, located at position +20/+23, was critical
for promoter function, since deletion of it was equivalent to the gross
deletion of 50 bp of exon 1. We further demonstrated that all three
GATA isoforms expressed in the intestinal epithelium, GATA-4, -5, and
-6 are capable of binding to this GATA sequence. Of these three
transcription factors, however, GATA-5 was capable of the strongest
NHE3 promoter transactivation when overexpressed in cell
lines of both intestinal and non-intestinal origin. Strikingly, shorter
constructs or mutated constructs that lacked putative Sp transcription
factor binding sites upstream of TIS, yet had preserved GATA-box in
exon 1, failed to respond to overexpression of GATA-5. This suggested
that the presence of Sp transcription factors binding their upstream
cis-elements is necessary for GATA-5 to exert its
stimulatory effect. We further confirmed this observation by
demonstrating that overexpression of GATA-5 in Sp-deficient SL2 cells
had no effect on NHE3 promoter activity.
To delineate the role of Sp transcription factors in regulating
NHE3 promoter activity, we performed a series of gel
mobility shift and functional analyses of individual or composite
mutations of the three putative Sp binding sites in transiently
transfected Caco-2 cells. From these experiments, we concluded that
site B, with core binding sequence located at nt
58/
55, represents
the highest affinity Sp protein binding site. We also observed that on
a functional level this site acts in concert with the two neighboring Sp elements to control basal activity of the NHE3 promoter.
Furthermore, this cluster of Sp binding sites was shown to bind both
Sp1 and Sp3 proteins, as demonstrated by supershift analysis with
nuclear protein from Caco-2 and IEC-6 cells and specific antibodies, as well as by gel mobility shift assays with transfected SL2 cells. Both
Sp1 and Sp3 stimulated NHE3 promoter activity in cotranfected SL2 cells
in a dose-dependent manner.
Functional interaction between GATA-5 and Sp1 or Sp3 was confirmed in
experiments with SL2 cells, in which GATA-5 considerably potentiated
the stimulatory effects of Sp1 or Sp3. Such synergy has not been
described before and therefore represents a novel mechanism of
transcriptional regulation. Within the intestinal epithelium, GATA-5
has recently been shown to act synergistically with hepatocyte nuclear
factor 1
(HNF1
; Ref. 28), and both transcription factors were
shown to physically interact in regulating the activity of the human
lactase-phlorizin hydrolase gene (36). It remains to be determined
whether GATA-5 physically interacts with Sp1 or Sp3. The proximity of
GATA sites and the HNF1
binding site in the lactase promoter (both
core cis-elements within 23 bp) may facilitate physical
interaction. In the NHE3 promoter, however, the cluster of
Sp binding sites and the GATA box are fairly distant (~100 nt) and
divided by the putative initiator and binding of RNA polymerase II
holoenzyme. If such an interaction occurred, whether direct or
indirect, it would most likely result in looping the initiator sequence
out, and it may facilitate recruitment or assembly of the basal
transcriptional machinery. Interestingly, in cardiac myocytes, GATA-5
has been found to physically interact with p300 (37). p300 proteins are
negative regulators of cell proliferation, which function as
transcriptional coactivators that mediate the interaction between
transcription factors binding on enhancer DNA sequences and the basal
transcriptional complex formed on the core promoter (38). The
acetyltransferase region of p300 can also functionally and physically
interact with Sp1, stimulating its DNA binding properties (39). It is
therefore conceivable that GATA-5 may indirectly interact with Sp1 or
Sp3 via an adaptor protein such as p300, linking them to the basal transcriptional complex to stabilize or enhance its activity.
In conclusion, we present a functional model of basal regulation of the
rat NHE3 gene promoter. In this model GATA-5, through binding to a GATA element in exon 1, enhances transcription through functional interaction with Sp1 or Sp3 binding upstream of an atypical
initiator sequence. Intestinal GATA-5 is believed to function during
differentiation to activate genes in fully differentiated absorptive
cells of the villi (40). Therefore, the cooperative activation of an
intestinal promoter through GATA-5 and Sp family transcriptional
factors likely represents a novel mechanism of regulating expression of
genes along the crypt-villus axis of the intestinal epithelium.