Departments of 2 Physiology and Pediatrics, 1 Division of Gastroenterology and Nutrition, UCLA School of Medicine, Los Angeles 90095-1751; and 3 Department of Biology, California State University Northridge, Northridge, California 91330
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ABSTRACT |
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The Na+-glucose cotransporter
(SGLT1) is expressed primarily by small intestinal epithelial
cells and transports the monosaccharides glucose and galactose across
the apical membrane. Here we describe the isolation and
characterization of 5.3 kb of the 5'-flanking region of the
SGLT1 gene by transiently transfecting reporter constructs into
a variety of epithelial cell lines. A fragment (nt 235 to +22)
of the promoter showed strong activity in the intestinal cell line
Caco-2 but was inactive in a nonintestinal epithelial cell line
(Chinese hamster ovary). Within this region, three
cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC
box sites are critical for maintaining the gene's basal level of
expression. The two GC boxes bind to several members of the Sp1 family
of transcription factors and, in the presence of HNF-1, synergistically
upregulate transactivation of the promoter. A novel 16-bp element just
downstream of one GC box was also shown to influence the interaction of
Sp1 to its binding site. In summary, we report the identification and
characterization of the human SGLT1 minimal promoter and the critical
role that HNF-1 and Sp1-multigene members have in enhancing the basal
level of its transcription in Caco-2 cells.
intestine; transcription; glucose-galactose malabsorption; Sp2; Sp3
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INTRODUCTION |
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THE SODIUM-GLUCOSE TRANSPORTER SGLT1, an integral membrane protein, is located in the apical membrane of the small intestinal enterocyte and transports glucose and galactose against their concentration gradients (46). The human SGLT1 gene is located on the long arm of chromosome 22 (22q13.1), spans 70 kb in length, and consists of 15 exons (41, 42).
SGLT1 is the prototype of the Na+-dependent cotransport family of proteins. Mutations in the coding sequence of its gene are responsible for the autosomal recessive disorder glucose-galactose malabsorption [On line Mendelian Inheritance in Man (OMIM) #182380] (14, 20, 21, 43). Expression of SGLT1 in humans is largely limited to small intestinal enterocytes (12). SGLT1 expression in vivo has been shown to be regulated by various dietary, hormonal, and hard-wired stimuli (7). Intestinal glucose transport capacity in the sheep is particularly dependent on dietary glucose, where SGLT1 protein levels are regulated primarily at a posttranscriptional level (16). In rats, glucose transport was shown to be influenced by a diurnal trigger rather than by changes in dietary glucose (7, 33). In support of this, nuclear run-on assays, performed with nuclei isolated from intestinal epithelial cells, revealed a diurnal pattern of rat SGLT1 transcription that fluctuates by as much as sevenfold (33).
The regulation of the SGLT1 gene provides a model system for
evaluation of the mechanism of intestine-specific expression. Compared
with our understanding of hepatocyte- and lymphocyte-specific regulation of expression, relatively little is known about
trans-acting DNA-binding proteins that affect
enterocyte-specific gene expression (6, 40). We do know that expression
of the sucrase-isomaltase gene is controlled by cdx-2 and
hepatic nuclear factor (HNF)-1/
homo- and heterodimers, and the
lactase gene by the homeodomain protein HOXC11 and the GATA family of
nuclear proteins (26, 40). However, transgenic mice expressing these
critical cis-elements failed to show correct tissue- and
age-specific expression (19). Therefore, despite progress, the
transcription factors that can direct expression in epithelial cells of
the small intestine have not been identified (36).
To gain better insight into the tissue-specific, developmental, and diet-induced regulation of human SGLT1, we have evaluated the constitutive regulation of the gene, determined a minimal promoter, and identified its critical elements using in vitro assays, including reporter analysis of deletion- and substitution-mutant clones, band-shift, DNase I footprint, and overexpression assays.
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EXPERIMENTAL PROCEDURES |
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Cloning of the 5'-flanking region of SGLT1.
Cosmid clone H33 contains 25 kb of the 5'-untranslated region
(UTR) of SGLT1 (42). H33 was digested with restriction
endonucleases, and a Southern blot was probed with an oligonucleotide
corresponding to the region immediately upstream of the promoter
[(10/+10) 5'-CTGGCGAGAGGGAAGGACGC-3']. A
5.3-kb Nco I fragment (
5370/+22) corresponding to the
SGLT1 5'-UTR contiguous to the initiator methionine codon
was subcloned into the luciferase reporter vector pGL3 Basic (Promega)
and is referred to as
hSGLT1
5370/+22/B-Luc.
SGLT1 nested deletion clones.
Nested deletions of the SGLT1 5'-UTR were made by
exonuclease and mung bean nuclease digestion (22). Clones were
isolated, and their sizes were determined by restriction
digestion and sequencing. Constructs
hSGLT15295/+22/B-Luc,
hSGLT1
3852/+22/B-Luc,
hSGLT1
3085/+22/B-Luc,
hSGLT1
1438/+22/B-Luc,
hSGLT1
1041/+22/B-Luc,
hSGLT1
641/+22/B-Luc, and
hSGLT1
243/+22/B-Luc were chosen
for transient transfection analysis. The transcription factor databases
of MacVector 5.0 (TFDSITES.SUBSEQ.7.0.aa), and MatInspector 2.1 (http://www.gsf.de/cgi-bin/matsearch.pl) were searched for
putative DNA cis-elements (32).
Scanning mutagenesis.
Scanning mutagenesis was done to define the essential constitutive
regions of the minimal promoter of SGLT1. Mutagenizing primers
(50-mers), containing a central core of 10 mutated
nucleotides (AC, C
A, G
T, T
G) and flanked on
either side by a span of 20 correct nucleotides, were designed (Fig.
1A). These mutant
oligonucleotides were used with an antisense oligonucleotide from exon
1 (nucleotides +56/+76) to perform the initial PCR amplification. The
purified fragments M3 through M23 served as a "megaprimer," which
together with the
235/
212-Hind III primer were
used to amplify the full-length product. These products were then
digested with Hind III and Nco I, gel purified,
subcloned into the pGL3-Enhancer vector, and sequenced to confirm the
mutations.
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Cell cultures, transfection and nuclear protein isolation.
The cell lines Caco-2 (human intestine), LLC-PK1 (porcine
kidney) and CHO (Chinese hamster ovary) were obtained from ATCC, and
passages 20 to 50 were used for all transfection
experiments (17). Transient transfection experiments and luciferase and -galactosidase assays were performed (17). The HNF-1
and HNF-1
expression vectors (kindly provided by G. Crabtree, Stanford
University), were cotransfected with the specified hSGLT1-Luc
and
-galactosidase vectors. Nuclear extracts were prepared from
Caco-2 cells ~5 days after seeding (17).
DNase I footprinting.
In vitro DNase I footprint analysis was performed with the
hSGLT1330/+22/E-Luc and
hSGLT1
235/+22/E-Luc clones
digested with Hind III, radiolabeled with
[
-32P]dATP (6,000 Ci/mmol), and then cut
with Nco I (5' to 3') (17).
Band-shift assays.
Band-shift analysis was done as previously described, with the
exception of the GC boxes that were analyzed in the presence of 1 mM
ZnCl2 (17). Standard competition studies were run using excess cold oligonucleotides. The primers used included wild-type (WT)
19-21 primer, spanning from 51 to
30,
sense (5'-GATCTGCTGATCATTAACCAGGAGGC) and
-sense
(5'-CTAGGCCTCCTGGTTAATGATCAGCA). The mutant (Mut) 19-21
primers used were sense (5'GATCTGCTGATCATgccaCAGGAGGC) and
-sense (5'-CTAGGCCTCCTgtggcATGATCAGCA), which contained a
mutation in the HNF-1 site (shown in lowercase). Double-stranded
oligonucleotides of the consensus Sp1 site
(5'-ATTCGATCGGGGCGGGGCGAG) and the CTC site
(5'-TTCCCCTCCCCCGGATACTTCACTAGA-3') identified in the
trefoil gene were also used in competition experiments. Supershift
analysis was performed with antisera provided by G. Crabtree (HNF-1
and HNF-1
, 1 µl each) and Santa Cruz Biotechnology (2 µl of
either Sp1, Sp2, or Sp3). Recombinant Sp1 (Promega) was used at a
concentration of 0.5 footprint units (FPU) per gel-shift reaction.
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RESULTS |
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The promoter region of human SGLT1 gene.
The 5'-untranslated sequence of the human SGLT1 isoform
is shown in Fig. 1B and is aligned for direct comparison with
the rat (GenBank accession no. 9AF007832) and sheep (GenBank accession no. AJ223077) isoforms (33, 42, 45). Analysis of the 1.5-kb region
immediately upstream of the initiator codon for putative
cis-acting elements revealed putative binding sites for a
single HNF (HNF-1) (51/
37), three HNF-3
sites
(
187/
175,
707/
696,
1253/
1265), five CCAAT/enhancer binding protein
(C/EBP)
sites (
325/
312,
684/
670,
1035/
1022,
1300/
1287,
1464/
1448), and a single signal transducer and activator
of transcription (
346/
338) and cAMP-responsive element
binding protein site (
132/
121) (32).
Transiently transfected Caco-2, LLC-PK1, and CHO cells.
Reporter constructs containing shortened lengths of the promoter region
were cloned into the luciferase reporter construct pGL3 Basic and were
transfected into Caco-2, LLC-PK1, and CHO cells. In Caco-2
cells, the longer clones had promoter activities ~10-fold higher than
the promoterless vector, pGL3 Basic (Fig. 2A). Transfection of the shortest
clone (hSGLT1243/+22/B-Luc) into
Caco-2 cells resulted in a 16-fold higher level of promoter activity
compared with pGL3 Basic. In contrast, promoter activity was generally
lower (eightfold above the empty vector) when tested in
LLC-PK1 cells (Fig. 2A). Finally, CHO cells could not support the activity of any size promoter construct. We interpreted these results to suggest that Caco-2 and LLC-PK1 cells are
capable of supporting the basal promoter activity of a limited region of the human SGLT1 promoter. These data are consistent with
reports that SGLT1 is expressed in Caco-2 and
LLC-PK1 cells (18, 29).
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DNase I footprint analysis identifies two DNA-protein complexes.
Using 5' to 3'-labeled
hSGLT1330/+22 as a template
revealed a single DNA-protein complex in the region corresponding to
229 through
206 (named FP-I; Fig.
3). Similarly, a DNase I digest of the hSGLT1
235/+22 vector labeled in
the same orientation revealed a second complex located at
49 to
31 in the upstream region of the gene (named FP-II; Fig. 3). No
other footprints were identified within bases
270 to +30.
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Scanning mutagenesis indicates several binding sites for
transcriptional activators.
Figure 1 shows the location of the mutations in 23 clones named M1 to
M23. Each contained a different stretch of 10 mutated contiguous
nucleotides within the 260-bp minimal promoter. Transient transfection
in Caco-2 cells revealed that the promoter activity of all but two
clones (M22 and M17) was significantly lower than the WT
hSGLT1235/+22/E-Luc clone (Fig.
4), suggesting either the existence of
numerous cis-acting elements or a pronounced interdependence of
regional secondary structure for optimal promoter function. Clones M2, M18, and M20 displayed the most pronounced declines in promoter activity (<20% of WT), suggesting that three exceptionally active cis-elements are located in the gene's minimal promoter
region. Indeed, footprints I and II, identified in Fig. 3, correspond to the regions mutated in the M2 (
225/
216) and M20
(
45/
36) clones, respectively.
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HNF-1 is a potent activator of the SGLT1 minimal promoter.
The nucleotide sequence from 51 to
30 accords with a
transcription factor HNF-1 consensus
(5'-GGTTAATnATTAACCa/c-3'). This domain, corresponding to
the M20 clone and footprint II, was further evaluated by band-shift
analysis. The WT 19-21 primer (nucleotides
51 to
30)
was radiolabeled and found to compete with the Mut 19-21 primer,
which contains a critical 4-bp mutation in the HNF-1 site. Figure
5 is a representative band-shift study
using the HNF-1 probe that reveals a DNA/protein complex that runs as a smear. This complex was specific because it was competed with a 10-fold
excess of the unlabeled HNF-1 oligonucleotide (Fig. 5) but not with
either the Mut 19-21 or an unrelated oligonucleotide. Supershift
experiments showed a shift of a portion of the specific complex with
antiserum to the HNF-1
isoform (Fig. 5) and its complete removal by
antiserum to the HNF-1
isoform (Fig. 5).
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Two regions form specific DNA-protein complexes.
A double-stranded oligonucleotide named WT 1-3 was made that
encoded domains M1, M2, and M3 (see Fig. 1B; bases 235
to
206). A double-stranded oligonucleotide named WT 17-18
was developed that encodes for domains M17 and M18 (see Fig.
1B; bases
75 to
56). The WT 1-3 probe
revealed two prominent DNA-protein complexes (arrowheads) that could be
competed entirely with the addition of 90-fold excess of cold WT
1-3 oligo (Fig. 7). Two DNA-protein complexes were also seen with the WT 17-18 probe, but this complex could not be competed with as much as 250-fold excess of cold WT
17-18 duplex (Fig. 7).
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GC boxes 1 and 2 bind recombinant Sp1.
Because the WT 1-3 and WT 17-18 duplexes share DNA-protein
complexes of similar size and sequence (g/tCCCCTCCCC), we hypothesized that the complexes may be attributed to binding of the transcription factor Sp1 to the GC box. To compare the ability of the WT 1-3 (GC
box 1) and WT 17-18 (GC box 2) duplexes to bind recombinant Sp1,
the WT 1-3 primer was labeled and competition studies were performed with various multiples of excesses of either itself (Fig.
8) or the WT 17-18 duplexes (Fig. 8).
Recombinant Sp1 and the WT 1-3 primer form a prominent complex
that corresponds in size to that of the slower migrating complex seen
with crude nuclear extracts (Fig. 8). Overexposed autoradiograms
revealed an additional complex whose molecular weight was approximately
twice that of the main complex and may represent Sp1 dimers (Fig. 8).
Competition with a 90-fold excess of either the consensus Sp1 or WT
1-3 duplexes was more effective than with WT 17-18 (Fig. 8).
In addition, the CTC duplexes failed to compete (Fig. 8). Recombinant
Sp1 formed a complex with the labeled WT 17-18 fragment, and this
complex could be competed with a 240-fold excess of cold probe (Fig.
8). The slower migrating complex seen with Sp1 and WT 1-3
was not visualized in even the overexposed autoradiogram of the WT
17-18 probe (Fig. 8). Overall, these data were interpreted
to suggest that the WT 1-3 (GC box 1) duplexes were capable
of binding to Sp1 as efficiently as a consensus Sp1 duplexes and
significantly better than the WT 17-18 (GC box 2)
duplex.
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Sp1, Sp2, and Sp3 bind to GC boxes 1 and 2.
To determine whether other members of the Sp1 could account for the
DNA-protein complex, supershift assays were performed. A representative
gel of GC box 1 demonstrates that the lower portion of the slower
migrating complex is shifted by the Sp1 antiserum (Fig.
9). Similarly, Sp2 antiserum shifted a
complex that originated in a location that was similar to Sp1 (Fig. 9).
In contrast, Sp3 antiserum shifted the entire faster migrating complex
and a portion of the slower complex when added with the Sp1 antiserum
(Fig. 9). Finally, the addition of all three antisera resulted in
residual complex formation that is very similar to what was seen with
the addition of both the Sp1 and Sp3 antisera (Fig. 9). These
supershifted complexes were specific since the same quantity of
preimmune rabbit serum failed to produce a similar supershifted complex
(Fig. 9).
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GC box 1 is flanked downstream by nucleotides critical for binding
recombinant Sp1.
To determine which nucleotides of the WT 1-3 oligonucleotide are
necessary for binding Sp1, band-shift assays were performed with a
500-fold excess of a series of cold oligonucleotide duplexes containing
specific mutations (Fig. 10A).
Each competing oligonucleotide contained a three contiguous base pair
mutation, the exact location of which is displayed in Fig. 10A.
Figure 10B shows a band-shift study with labeled WT 1-3
oligonucleotide and 7 µg of Caco-2 nuclear extracts. Two complexes
were identified that could be competed with 500-fold excess of the WT
1-3 primer (Fig. 10B). Primers that contained mutations in
regions a, b, l, and m were
capable of binding the protein(s) that form both complexes (Fig.
10B). Oligonucleotides that contained mutations in regions
c-k failed to compete for binding (Fig. 10B),
suggesting that the nucleotides mutated in the c-k
primers are essential for binding to the protein(s) that account for
the two complexes (Fig. 10A). Moreover, it appears that mutant
primers g-k were able to slightly compete for
binding since the intensity of both complexes was less compared with
complexes competed with primers c-f (Fig.
10B).
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Binding of Sp1 to the WT 17-18 primer is limited to GC box 2.
Similar studies were performed with GC box 2 (the WT 17-18
oligonucleotide) and crude Caco-2 extracts. Figure
11B demonstrates that primers
containing mutations in regions a-d and h
could compete for binding of the nuclear proteins that are responsible
for complex formation. Furthermore, primers with mutations in the
e-g region (Fig. 11B) failed to compete,
suggesting that nucleotides 51 to
59 (boxed nucleotides
in Fig. 11A) are critical for the formation of the DNA-protein
complex. The WT 1-3 primer could compete for binding of the
complex formed with the WT 17-18 primer (Fig. 11B). Additional evidence suggesting that the complex is related to binding
of Sp1 is provided by the ability of consensus Sp1 primer to compete
for the two complexes (Fig. 11B). Similarly, the CTC oligonucleotide was able to partially compete for binding (Fig. 11B).
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Proteins of the Sp1 multigene family and HNF-1 synergistically
activate transcriptional activity of SGLT1.
To determine whether the identified HNF-1 and GC boxes influence the
activity of a heterologous promoter, we subcloned these elements just
upstream of the SV40 promoter in the reporter vector pGL3 Promoter. All
constructs contain a single copy of the identified element oriented in
the 5'-to-3' direction. Because of the close apposition of
the downstream GC box 2 (63 to
55) to the HNF-1 element
(
51 to
37), we also tested whether or not the two
elements altered promoter activity in a synergistic manner. Compared
with the enhancerless SV40 promoter construct, the addition of the GC
box 1 (WT 1-3) failed to alter promoter activity (Fig.
12). Similarly, neither the HNF-1 site
nor GC box 2 (WT 17-18) influenced luciferase production above
what was seen with the enhancerless SV40 promoter vector. However, the
addition of both the GC box 2 and the HNF-1 element enhanced promoter
activity by 60% compared with control, suggesting that proteins
binding to both of these sites synergistically influenced
transcriptional activity in the context of the heterologous SV40
promoter.
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DISCUSSION |
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In this study we analyzed the basal pattern of expression of the
promoter region of the human SGLT1 gene in the human intestinal cell line Caco-2. Deletion analysis of various lengths (5295/+22 to
27/+22) of the promoter inserted upstream of the luciferase reporter indicated that nucleotides
235/+22 represent the
gene's minimal promoter (Fig. 2). Within this region of the promoter, several DNA-protein complexes were identified by in vitro footprinting (Fig. 3), and mutagenesis identified three distinct sites that were
responsible for enhancing the promoter activity of SGLT1 (Fig.
4).
We determined that an HNF-1 element and two GC boxes control SGLT1 basal expression in the Caco-2 cell. Two active cis-elements were found that bind members of the Sp1 family of proteins within the minimal promoter of SGLT1, and scanning mutagenesis revealed that both elements function to enhance the gene's basal expression (Fig. 4). The components of the complexes were defined by supershift analysis and revealed that portions of the two complexes are formed by binding of Sp1, Sp2, and Sp3 or a related protein(s) (Fig. 9). More specifically, the fastest-migrating DNA-protein complex was completely shifted by Sp3 antisera, whereas the slower-migrating complex appears to be at a minimum a closely migrating triplet, composed of Sp1, Sp2, Sp3, and another undefined complex.
Sp1, Sp3, and Sp4 have highly conserved zinc finger DNA binding domains and recognize the consensus GC box (5'-KRGGMGKRRY) with similar specificity and affinities, whereas Sp2 binds with much lower affinity (9). Moreover, whereas Sp1 and Sp4 have only been implicated as transcriptional activators, Sp3 usually functions as a transcriptional repressor and on occasion as an activator (1, 8, 10, 11, 13). Although Sp1 is ubiquitously expressed in all cell types examined, its level of expression varies by as much as 100-fold, and this variability has been implicated in specifying tissue and developmental-specific regulation of several genes (15, 37). Similarly, other members of the zinc finger Sp1 multigene family, Sp2 and Sp3, are ubiquitously expressed, whereas Sp4 expression is limited to the brain (9). However, an exhaustive analysis of the tissue distribution, particularly in the intestine, has not been performed for any member of the growing family of Sp1-like proteins (37). Because of the disparate levels of expression, affinity, and function of the Sp1 multigene family members, the overall impact that they may have on SGLT1 expression may be dramatic and is currently under investigation.
Gel-shift assays of GC box 1 revealed that nucleotides immediately
downstream of the box (5'-ATTCGCAGGACAGCTC) were critical for
complex formation with both crude and recombinant Sp1 protein (Figs.
10 and 13). Interestingly, although GC
box 1 is conserved in both rat and sheep promoters, this adjacent
sequence is not conserved and does not resemble a GC box (Fig. 13).
Most good Sp1 binding sites are 10 nucleotides in length and do not
differ from the consensus sequence at more than one position.
Furthermore, Sp1 is only able to bind simultaneously to adjacent Sp1
sites if the central portions of the elements are more than 10 nucleotides apart (2). Since Sp1 is clearly capable of binding to the
GC box 1, we would expect that if two Sp1 sites are occupied
simultaneously, only the furthest downstream portion of the sequence
would be able to bind Sp1. In fact, we failed to identify evidence that two Sp1 monomers could bind simultaneously to the GC box 1 and the
adjacent downstream sequence. The faint and slowly migrating complex
seen with recombinant Sp1 (Figs. 8 and 10) probably represents the
multimerized form of Sp1, a process that can occur at high protein
concentrations (2, 27). Therefore, it would be rather surprising if
this downstream sequence could bind Sp1 independently of GC box 1. What
remains unclear is how Sp1 and related proteins are capable of
interacting with the adjacent nucleotides, since they do not resemble a
GC box (Fig. 13). It is conceivable that the specificity of binding,
including the process of multimerization, may be governed by the
nucleotides that flank GC box 1.
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Can the Sp1 family of proteins that bind to the two GC boxes interact with one another to synergistically activate expression of the SGLT1 gene? In many genes, GC boxes frequently occur as multiple repeat sequences (2). The repeat Sp1 sites may be either adjacent to one another or widely separated and at either distance are capable of inducing synergistic transactivation (2). Sites that are at close proximity may undergo protein-protein interactions that influence cooperative binding, whereas distal GC boxes may form multimeric complexes that enhance DNA binding and bring elements closer to the core transcriptional machinery (2). This form of synergistic activation between distal and proximal GC boxes may result from self-association of Sp1 and enhanced activity of the transcriptional complex.
Scanning mutagenesis identified that the putative HNF-1 element was
also active in inducing SGLT1 basal expression. HNF-1 induces
the expression of several intestinal genes, including sucrase-isomaltase, aminopeptidase, apo B, lactase, -fetoprotein,
1-antitrypsin, and aminopeptidase N (26, 40). HNF-1
is expressed in the small intestine, kidney, stomach, and liver,
whereas HNF-1
is produced in the ovary, lung, and small intestine;
expression of both proteins is limited to villus epithelial cells (23, 38). The relative concentrations of HNF-1
and -
differ markedly from tissue to tissue and may be developmentally regulated (44).
Rhoads et al. (33) have implicated HNF-1 in altered SGLT1
expression during the normal day and night cycles of rodents. Nuclear
run-on experiments in rat intestinal epithelial cells showed that this
regulation occurs at the level of transcription. Moreover, band-shift
assay with nuclear extracts isolated from rat intestine identified a
HNF-1 protein-DNA complex that migrated as a smear in the evening and
as a faster complex in the morning. Antiserum to the HNF-1 isoform
supershifted all complexes, implicating HNF-1
at all time intervals.
However, antiserum for the
isoform abolished the migration of
primarily the evening complex, indicating that HNF-1
was a component
of the evening complex. The authors suggested that this effect could be
explained if the epitope recognized by the antibody is part of the
HNF-1 DNA binding site. However, previous reports showed that the
identical
-antiserum did not disrupt complex formation but rather
supershifted HNF-1
when tested by band-shift technique (24). Our
data showed that the HNF-1
antiserum also abolished the slower
migrating complex (Fig. 5). However, other data from our laboratory
suggested that this effect was nonspecific, as other unrelated
DNA-protein complexes were also disrupted by this antiserum (data not
shown). Thus these data underscore the difficulty of assessing the
contribution of the
-isoform by supershift assay using currently
available antisera.
The critical role of HNF-1 in controlling the regulation of genes in
vivo was clearly shown in HNF-1 knockout mice. These mice developed
profound multiorgan effects that resulted in a dramatic decline in
survival after weaning (30, 31). They also experience failure to
thrive, dramatic hepatic enlargement, severe phenylketonuria, and
Fanconi syndrome, including severe glucosuria. The HNF-1 knockout mice
had reduced phlorizin-binding to renal brush-border membranes,
suggesting a decline in the yet-to-be-identified renal
high-affinity/low-capacity cotransporter (SGLT2) (46). Interestingly,
the authors did not describe diarrheal symptoms in these mice. If HNF-1
is critical in controlling SGLT1 expression, one would have
expected evidence of glucose/galactose malabsorption (and consequent
diarrhea) on a lactose-based diet (breast milk) (21). Similarly, the
HNF-1 knockout mice have been shown to develop a form of Laron
dwarfism and non-insulin-dependent diabetes (5). In humans,
heterozygous germline mutations (autosomal dominant) of either HNF-1
or -
result in a poorly defined form of diabetes whose onset
begins in late adolescence (MODY3) (47). Diarrhea and glucose
malabsorption have not been reported in patients with MODY3.
Functional analysis suggested that proteins that bind to GC box 2 and
the HNF-1 element synergistically enhanced SGLT1 promoter activity (Fig. 12). Numerous transcription factors have been shown to
interact with Sp1, including GATA1, gut-enriched kruppel-like factor,
AP1, NF-B, GATA, and HNF-4 (35, 48). Although Sp1 enhances
HNF-4-induced transcription of the apoCII promoter, the nature of the
synergy between these factors was not actually defined (39). Analysis
of other transcription factors suggests that the Sp1 COOH-terminal
domain is involved in both synergistic activation and protein-protein
interaction (39). HNF-1, on the other hand, has been shown to interact
only with C/EBP
and to synergistically activate expression of the
human albumin promoter (25). A serine-threonine- and
proline-glutamine-rich region of HNF-1 is critical for its interactions
with C/EBP
and its functional synergy at the albumin promoter.
Although transcriptional synergism between Sp1 and HNF-1 has never been
reported, this type of interaction may explain how a ubiquitous
transcription factor like Sp1 may direct intestinal-specific expression. Interestingly, both HNF-1 and Sp1/Sp3 have been implicated in mediating glucose activation of various promoters (3, 4, 34).
Overall, this study represents the first detailed analysis of the SGLT1 promoter and the critical role that HNF-1 and Sp1 have in controlling basal transcription. In the analysis of the promoter, we have identified several unique features that deserve further attention. Although Sp1, Sp2, and Sp3 influence the basal expression of the gene via two separate GC boxes, the precise role that these and other members of the Sp1 multigene family have in altering expression of the gene has not been determined. Sp1 was shown to interact with GC box 1, but the nature of how the 16-nucleotide element immediately downstream of the box confers specific binding to Sp1 remains unexplored. Finally, the mechanism by which Sp1 and HNF-1 synergistically influence SGLT1 gene expression should be further analyzed since it may be a general model for understanding the mechanism of intestinal-specific gene expression.
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ACKNOWLEDGEMENTS |
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This research was supported by grants from the National Institutes of Health (HD-34706 and DK-44582), the Robert Wood Johnson Foundation Faculty Training program, and the American Gastroenterology Industry Training Award.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. G. Martín, Dept. of Pediatrics, Div. of Gastroenterology and Nutrition, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1751 (E-mail: mmartin{at}mednet.ucla.edu).
Received 2 November 1999; accepted in final form 9 December 1999.
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