Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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Intestinal trefoil factor (ITF)
is selectively expressed in intestinal goblet cells. Previous studies
identified cis-regulatory elements in the proximal promoter
of ITF, but these were insufficient to recapitulate the exquisite
tissue- and cell-specific expression of native ITF in vivo. Preliminary
studies suggested that goblet cell-specific expression of murine ITF
requires elements far upstream that include a silencer element that
effectively prevents ITF expression in non-goblet cells. Transient
transfection studies using native or mutant ITF 5'-flanking sequences
identified a region that restores expression in goblet cells. This
element, designated goblet cell silencer inhibitor (GCSI) element,
enables human and murine goblet cell-like cell lines to override the
silencing effect of more proximal elements. The GCSI has no intrinsic
enhancer activity and regulates expression only when the silencer
element is present. Ligation of GCSI and silencer elements to
sucrase-isomaltase conferred goblet cell-specific expression. Goblet
cells but not non-goblet cells possess a nuclear protein that binds to
the GCSI regulatory element (GCSI binding protein; GCSI-BP). Both
transient transfection and gel mobility shift assay studies localize
the GCSI and GCSI-BP to 2216 to
2204. We conclude that goblet
cell-specific transcription of ITF in vivo depends on a regulatory
element designated GCSI.
intestinal goblet cells; transcriptional regulation; antisilencing
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INTRODUCTION |
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THE TREFOIL PEPTIDE FAMILY encompasses three small peptides that contain one or two trefoil motifs (also called a P domain) composed of six conserved cysteine residues (see Refs. 9, 27, 28, and 31 for review). These peptides are specifically expressed and synthesized by mucin-secreting epithelial cells lining the gastrointestinal tract. The trefoil peptide family includes three different members designated spasmolytic polypeptide (SP) (16), pS2 (25), and intestinal trefoil factor (ITF) (37). SP has two trefoil motifs and is abundant in the mucous neck cells in the body and in antral glands of the stomach (40, 11), although the porcine homologue (PSP) was originally isolated from the pancreas (16). pS2 peptide contains a single trefoil motif and was initially cloned as the product of an estrogen-responsive gene from the breast cancer cell line MCF-7 (25). It is produced predominantly in the proximal stomach (20). SP and pS2 are both expressed by mucous neck cells in the corpus of the stomach (11).
ITF, the third member of the trefoil peptide family, was first cloned
from rat intestinal epithelial cells (37, 32) and subsequently in humans (12, 28) and mice
(24). ITF is selectively expressed in high concentrations
by mucus-producing goblet cells of the small and large intestine
(28, 37, 24). In vitro studies demonstrated that trefoil
peptides promote epithelial migration through a transforming growth
factor -independent pathway (7) and protect intestinal
cell monolayers from a variety of injurious agents in cooperation with
mucin glycoprotein (18). Functional effects observed in
vitro are paralleled by findings in vivo. Oral administration of ITF
was shown to protect the gastric mucosa from injury (3).
Furthermore, ITF-null mice exhibit impaired mucosal healing. In
contrast to wild-type mice, ITF-null mice succumbed to injury resulting
from oral administration of dextran sulfate sodium (23).
Thus ITF plays an important role in repair and healing of the
gastrointestinal tract.
Understanding of the mechanisms through which ITF achieves these
functional effects remains incomplete. Recent studies have demonstrated
that ITF causes tyrosine phosphorylation of -catenin and the
epidermal growth factor receptor in the HT-29 colonic carcinoma cell
line (21), decreases extracellular signal-related protein
kinase activity in IEC-6 cells (17), and stimulates IEC-18
cells to generate nitric oxide via nitric oxide synthase 2 (39).
The role of ITF in protecting the epithelium from injury and promoting repair is facilitated by its selective expression in goblet cells that vectorally secrete ITF together with mucin glycoprotein onto the apical surface. As a result, trefoil peptides are present in a continuous layer at the interface between the lumen and mucosal surface. However, the molecular basis of this highly tissue- and cell-specific expression remains unclear. Although promoter sequences of several genes exclusively expressed in the intestinal columnar epithelium have been examined [e.g., fatty acid binding protein (FABP) (38), sucrase-isomaltase (5), and lactase (4)], relatively little is known about the regulatory elements that may drive expression of key goblet cell products. The latter have included preliminary characterization of the MUC2 promoter and initial studies of ITF genes (9). Because goblet cells are major mucus-producing cells of the intestine and are thought to play an important role in mucosal protection (1), detailed exploration of the ITF gene promoter may provide insight into the regulatory mechanism of goblet cell-specific gene expression.
Previous studies (26, 32) of the rat ITF (rITF) gene
promoter identified one cis-regulatory element, designated
the goblet cell response element (GCRE), present in the proximal region
of the promoter, which supported goblet cell-associated expression. The
study of the mouse ITF (mITF) gene identified additional
cis-regulatory elements, including enhancers (181 to
170,
1590 to
1370) and a silencer (
208 to
200)
(14). Although these elements were sufficient to drive
goblet cell-associated gene expression in vitro, collectively these
were found to be insufficient to drive cell-specific expression in
vivo. Thus transgenic mice incorporating constructs with these
relatively short sequences of the ITF promoter showed no evidence of
goblet cell expression of the reporter gene. However, recent studies
(13) have demonstrated the ability of transgenic 6.4 kb of
mITF 5'-flanking sequence to drive highly goblet cell-specific
expression of a reporter transcript (
-galactosidase) in a manner
paralleling native ITF.
These findings indicate that specific gene expression requires additional elements further upstream from the transcriptional start site than those identified during initial studies characterizing ITF promoter sequences in vitro. Indeed, preliminary studies (14) provided initial evidence of both a potent silencer and a nearby element, which in goblet cells overrides the effects of the silencer to result in cell-specific expression in vitro and, more importantly, in vivo. Given the apparent central role of those elements in achieving cell-specific expression in vivo, efforts were directed to identify and characterize these elements. As described in this report, these efforts have resulted in identification of a novel regulatory element called "goblet cell silencer inhibitor" (GCSI), responsible for goblet cell-specific transcription of the mITF gene. These studies demonstrate that goblet cells uniquely express a nuclear protein that binds to this element, counteracting the otherwise universally active silencer and resulting in goblet cell-specific ITF gene transcription.
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MATERIALS AND METHODS |
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Cell culture. All cell lines were obtained from the American Type Culture Collection. The human colon cancer cell line LS 174T exhibits a goblet cell-like phenotype producing significant amounts of secretory mucin (19, 40). CMT-93 is a murine cancer cell line with a rectal epithelial cell-like phenotype. Both HT-29 and Caco-2 cells are human colon cancer cell lines. The human hepatocellular carcinoma cell line Hep G2, human cervix epithelioid cancer cell line HeLa, human fibrosarcoma cell line HT-1080, and mouse immortalized fibroblast cell line NIH/3T3 were used as nonintestinal cell lines. LS 174T cells were grown in MEM and the other cells in DMEM supplemented with 10% or 20% (Caco-2) heat-inactivated FCS, 4 mM L-glutamine, 50 U/ml penicillin, and 50 mg/ml streptomycin in 5% CO2 at 37°C.
RNA preparation and Northern blot analysis.
Total cellular RNA was prepared using TRIzol reagent (GIBCO BRL Life
Technologies), according to the manufacturer's instructions. The
following human cDNA probes were generated by RT-PCR and cloned into
the vector PcDNA3.1 (Invitrogen, San Diego, CA) for use as hybridization probes for Northern blot analysis: human ITF
(28) and mITF (13) and human
(30) and mouse glyceraldehyde-3-phosphate dehydrogenase (13). All probes were prepared using the
random-primed [-32P]dCTP radiolabeling method followed
by removal of unincorporated nucleotides by spin column. Fifteen
micrograms of total RNA were hybridized with probes for 2.5 h at
65°C using rapid hybridization buffer (Amersham Pharmacia Biotech).
The hybridized membranes were washed under high-stringency conditions
and exposed to X-ray film.
Reporter plasmid constructs.
The promoterless pGL3-basic (Promega, Madison, WI), which contains a
luciferase structural gene immediately downstream of a polylinker, was
used for reporter constructs. All reporter plasmid constructs were
generated from a 6353WT construct, which contains the 6353/+24 mITF
gene, linked to a luciferase gene as previously described
(14). A
201/+24 mITF-luciferase construct (201WT) was
prepared by digestion of 6353WT with Mlu I and Bgl
II and blunt-end formation by Klenow enzyme (Promega), taking
advantage of a convenient Bgl II site at position
201,
because a Bgl II site in the polylinker was disrupted by
ligation with a BamH I site when subcloning. The deletion
constructs, 6353M1, were made by ligating the Mlu I- and
BamH I-digested PCR products corresponding to the position
of
6353/
1848 into the Mlu I- and Bgl
II-digested 6353WT plasmid, respectively, using the Bgl
II restriction site at position
201. Additional internal
deletion constructs were made using the 6353M1 construct by the
exonuclease III (Promega) deletion method or by subcloning of Mlu
I- and BamH I-digested PCR products into a Mlu
I- and Bgl II-digested 6353WT construct. Human
sucrase-isomaltase (hSi) promoter gene construct was the kind gift of Dr. Peter Traber (43). A Sma I-
and Hind III-digested hSi gene, corresponding to
a position of
183/+54, was subcloned into a PGL3-basic vector named
PGL3/hSi. Mlu I- and Sma I-digested PCR products from the mITF gene were ligated into Mlu I and
Sma I sites of PGL3/hSi. Mutant constructs were
made by the Quikchange site-directed mutagenesis kit (Stratagene, La
Jolla, CA).
Transient transfections and promoter analysis.
Transient transfection was performed using Lipofectamine Plus reagent
(GIBCO BRL, Life Technologies), according to the manufacturer's protocol. Sixteen to thirty-six hours before transfection, cells were
plated out in triplicate in 35-mm wells of a six-well cell culture
plate so that they were 50-80% confluent on the day of transfection. DNA-lipofectin complexes were added to each well containing fresh serum-free medium and incubated in 5% CO2
at 37°C for 3 h. After incubation, medium containing the
complexes was replaced with fresh, complete medium. Cells were
harvested after an additional 48-h incubation. To correct for
variations in transfection efficiency, pSV -galactosidase control
vector (Promega) was cotransfected as an internal control. For
determination of luciferase and
-galactosidase activities, cells
were lysed and assayed immediately using a commercial luciferase assay
system (Promega) and luminescent
-galactosidase genetic reporter
system II (Clontech, Palo Alto, CA), respectively, measured in a
Monolite 2010 luminometer (Analytical Luminescence Laboratory).
Luciferase activity was adjusted for transfection and harvesting
efficiencies by dividing the value of luciferase activity by that of
-galactosidase activity. From the normalized
luciferase/
-galactosidase activity for each plasmid, the activity of
promoterless PGL-basic vector was subtracted for an enzyme blank and
then expressed as a percentage of the expression of the maximal
promoter construct, PGL3-control vector (Promega), consisting of the
SV40 promoter and enhancer connected to the luciferase gene.
Reproducibility of results was confirmed by at least three independent
transfections, and each transfection was done in triplicate. Values are
expressed as means ± SE.
Nuclear protein preparation and gel mobility shift assay.
Nuclear extracts were prepared by Nonidet P-40 detergent lysis and 0.5 M NaCl extraction performed as described previously by Schreiber et al.
(34). Protein concentration was determined by the Bradford
assay (Bio-Rad, Hercules, CA). For gel mobility shift assay (GMSA),
complementary oligonucleotides with overlapping ends were synthesized.
After annealing, they were labeled by Klenow fill-in reaction in a
buffer consisting of 10 mM Tris · HCl, 5 mM MgCl2,
7.5 mM dithiothreitol (DTT), 33 mM of dATP, dGTP, and dTTP, 0.33 mM [-32P]dCTP, and 1 U Klenow enzyme, as described
previously (14). GMSAs were carried out by incubating 10 µg of nuclear extract with 5 fmol of probe (20,000 cpm) in 20 µl of
binding reaction containing 10 mM Tris · HCl (pH 7.5), 5 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 5% glycerol, and
1 mg poly(dA/dT). After incubation at room temperature for 20 min,
samples were loaded onto 6% polyacrylamide-0.25× TBE
(Tris-borate-EDTA) gels and electrophoresed at 10 V/cm for 2 h.
Competition experiments were carried out by preincubating the nuclear
extracts with a 25- to 200-fold excess of unlabeled competitor
oligonucleotides before addition of the probe. The gels were dried for
30 min and exposed to Kodak X-AR film for 6-24 h at
80°C.
Reproducibility of GMSAs was confirmed by at least three independent assays.
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RESULTS |
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ITF expression in various cell lines.
We characterized the specificity of ITF expression in various cell
lines. Northern blot analysis used total RNAs derived from human and
murine intestinal epithelial cell lines and nonintestinal cell lines.
As shown in Fig.1, ITF mRNA is abundantly
expressed in LS 174T cells. CMT-93 cells also express lesser amounts of endogenous ITF compared with LS 174T. However, no ITF expression was
observed in other intestinal (HT-29 and Caco-2) and nonintestinal (Hep
G2 and HeLa) cell lines.
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Transient transfection gene analysis identifies silencer and GCSI.
Transient transfection of constructs containing 5'-flanking sequences
of the mITF gene were used to localize promoter regions required for
cell-specific transcriptional activity. Because preliminary studies
(14) suggested that a silencer element and GCSI are present in the 6353 to
1848 region of the mITF gene (Fig.
2), a series of 5' unidirectional
deletion mutants of mITF-luciferase PGL reporter plasmid constructs was
generated from a 6353WT construct. All constructs also
contained the
201 to +24 region (demonstrated previously to encompass
elements contributing to ITF transcription, including a required
positive regulating element designated GCRE) and an internal deletion
between
1847 and
202, which includes an additional enhancer and
silencer element as previously reported (Ref. 14; Fig. 2). These
constructs were transiently transfected into representative intestinal
goblet cell lines (LS 174T and CMT-93), which express ITF mRNA, and
nonintestinal cell lines (Hep G2, HeLa, HT-1080, and NIH/3T3). These
nonintestinal cell lines do not express ITF mRNA (Fig. 1; HT-1080 and
NIH/3T3 data not shown). As shown in Fig.
3A, extension of the promoter
up to
2136 from
1848 resulted in no significant increase in
transcriptional activity when transfected into cell lines (HeLa,
HT-1080, and NIH/3T3 data not shown). In contrast, the 2161
1848/201
construct containing the region
2161 to
1848 ligated to
201 to
+24 resulted in a marked reduction of transcriptional activity compared
with constructs encompassing
2136 or smaller amounts of the
5'-flanking sequence. This result suggested that a potent negative
regulatory element (silencer element) is present in the
2161 to
2136 region. A slightly larger construct, designated 2193
1848/201,
exhibited transcriptional activity that was essentially comparable to
the 2136 construct.
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Goblet cells contain nuclear protein, which specifically binds to
GCSI.
Transient transfection studies utilizing a series of internal deletion
constructs were used to localize the GCSI more precisely. GMSAs were
performed on the basis of these findings to determine whether any
nuclear extracts from intestinal epithelial cells contain proteins that
bind this element. An oligonucleotide probe designated probe
S1 was prepared containing the nucleotide sequence present
between 2230 and
2194 (Fig.
4A). After radiolabeling, this
oligonucleotide probe was used in binding assays with nuclear extracts
from the gobletlike LS 174T cells. These nuclear extracts contained a
protein that strongly bound the probe. Binding of GCSI to LS 174T
nuclear protein was efficiently competed by an excess of unlabeled
oligonucleotides but not by unrelated oligonucleotides (Fig.
4A). Another DNA-protein complex appeared to reflect
nonspecific binding with an entirely unrelated probe yielding the same
complex (data not shown). Subsequently, GMSAs were carried out using
nuclear proteins from CMT-93, Hep G2, and HeLa cells. In contrast to LS 174T cells, Hep G2 and HeLa extracts lacked any nuclear protein that
specifically bound the GCSI (Fig. 4B). However,
CMT-93 cells (cells from another intestinal goblet cell-like line)
contained a nuclear protein that formed a specific complex that could
be efficiently competed by cold wild-type probes (Fig. 4B).
Of note, the strong DNA-protein complex formed by extracts from the
mouse-derived CMT-93 line was slightly smaller than that found in the
human LS 174T cell line. Despite use of 5-20 µg of nuclear
extracts derived from LS 174T and CMT-93 cells, no other complexes were identified in either cell line (data not shown).
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Binding of GCSI binding protein requires region 2216 to
2204.
To define the binding requirements in greater detail, additional
oligonucleotide probes, designated probes S2 to
S5, were prepared. As depicted in Fig.
5A, probe S2 covers
the entire GCSI and probe S3 is partly overlapping with
S2, whereas probes S4 and S5 cover the
sequence in GCSI that is outside of probe S3 from
2230 to
2194. These probes were used as cold competitors in binding assay
with nuclear extract from LS 174T and cells. As demonstrated in Fig.
5B, specific protein-DNA interaction with radiolabeled
probe S1 was competed by cold probes
S2 and S3 but not probes S4
and S5. This result suggested that the
2216 to
2204
region is required for the specific protein-DNA interaction at the
GCSI.
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Region 2216 to
2204 contains responsive element for interaction
with GCSI protein.
To further define the sequence necessary for protein-DNA interaction, a
series of mutated probe S1 oligonucleotides, represented in
Fig. 6A, was constructed and
used as unradiolabeled competitor to the radiolabeled probe
S1. GMSA with nuclear extract from LS 174T revealed that formation
of the complex could be competed by an excess of mutant
oligonucleotides mut-1, mut-2, and mut-5 to mut-7, but not by the mut-3
or mut-4 oligonucleotide (Fig. 6B). These results indicate
that the cis-responsive element for specific interaction
with the GCSI binding protein (GCSI-BP) is present between
2216
and
2204.
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Confirmation of sequence requirement for functional regulation of
ITF transcription between 2216 and
2204.
To verify whether the presence of nuclear proteins binding the GCSI in
GMSAs actually correlates with promoter activity of these elements, the
effects of various mutations of the mITF promoter on gene expression
were assessed using transient transfection assays. The 2216
1848/201
construct, which contains the silencer and silencer inhibitor element,
was used as a template for production of various additional mutant
constructs by site-directed mutagenesis. The various mutations
introduced into the WT construct are depicted in Fig.
7A. As shown in Fig.
7B, Mut 1, Mut 2, and Mut 6 had no effect on the
transcriptional activity of the WT construct in LS 174T cells. However,
Mut 3, Mut 4, and Mut 5 resulted in >60% reduction in expression.
This reduction in transcriptional activity almost correlated with the
observed effects on binding of nuclear proteins. Of note, the reduction
in the Mut 5 construct was also observed and is not correlated with the
results of GMSA. We assume that the sequence mutated in Mut 5 construct
is necessary for transcriptional activation; however, the weak binding
affinity resulted in competition observed in GMSA. These
results demonstrate the correspondence between sequences
attributable to GCSI in their ability to specifically bind nuclear
proteins and their ability to regulate promoter activity in transient
transfection assays. Subsequently, the same mutations as those found in
the Mut-4 construct were introduced into 6353M1 and 6353WT, which
contain the full length (
6353 to +24) of the mITF gene (Fig.
7C). Both constructs showed the same activity as Mut-4 (Fig.
7C), further confirming the significance of the GCSI sequence.
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GCSI confers goblet cell expression when ligated to a
non-goblet cell gene promoter.
The data above suggest GCSI-BP binding to GCSI permits goblet
cell gene expression. To confirm this conclusion, we assessed the
ability of GCSI and the adjacent silencer to confer comparable goblet
cell expression to a gene not normally expressed in this cell
population. For this purpose, we utilized a construct containing promoter sequences of the hSi gene, a product normally
expressed by columnar small intestinal epithelial cells but not goblet
cells (42). The hSi-PGL3 reporter construct
(PGL3/hSi) contains the hSi gene (183 to +54),
sufficient specific regulatory elements to yield columnar epithelial
transcription as previously described (43). Two constructs
were generated containing the regulatory sequence of hSi
ligated to luciferase reporter gene construct. The
PGL3/hSi-S construct contains the silencer element of the mITF gene, whereas the PGL3/hSi-GCSI construct contains the
silencer element and GCSI. These three constructs were transiently
transfected into LS 174T, non-goblet intestinal cell line HT-29,
Caco-2, and nonintestinal cell line Hep G2. As shown in Fig.
8, significant reduction in luciferase
activity was observed in the cells transfected with
PGL3/hSi-S compared with those transfected with
PGL3/hSi, demonstrating the silencer effect. However,
addition of GCSI in PGL3/hSi-GCSI restored the same activity
as the mITF 2216
1848/201 construct in LS 174T cells but not in
non-goblet cells. These results demonstrate that the GCSI can regulate
expression of hSi gene activity in a goblet cell-specific
manner.
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DISCUSSION |
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Among the growing list of cloned genes with intestine-specific products (4, 5, 38), ITF represents the first exclusively expressed by goblet cells. Tissue- or cell-specific regulation of transcription, especially of genes with highly restricted expression, often involves cooperative interactions between several regulatory elements. Previously, we (26) reported the identification of a goblet cell-specific enhancer element in the rITF gene promoter, designated GCRE, bound by a goblet cell nuclear protein. In addition to GCRE, previous study of the mITF gene identified enhancer and silencer elements, which are not goblet cell-specific regulatory elements in the ITF (14). However, constructs limited to these proximate 5'-flanking sequences were insufficient to recapitulate tissue- and cell-specific expression of a reporter transgene in mice. These observations suggested that additional upstream regulatory elements are required for goblet cell-specific transcription of the mITF gene. In this report, we demonstrate the presence of a potent silencer and goblet cell-specific silencer inhibitor (GCSI) element that contributes to goblet cell-specific transcription of mITF, correlating with the ability of the larger construct to drive goblet cell transgene expression in vivo (14). The present findings suggest that a nuclear protein specifically expressed by goblet cells binds a regulatory element upstream of silencers functionally inactivating them and allowing increased transcriptional activity driven by specific and nonspecific enhancers in an intestinal cell-specific manner, as represented in Fig. 2.
This analysis revealed that a region is present in 2216 to
2193
that binds a protein that functionally blockades adjacent silencer activity (and designated GCSI). Goblet cells express a nuclear
protein that binds to the GCSI (GCSI-BP), restoring gene
activity. In contrast, nonintestinal cells lack GCSI-BP, resulting in
sustained silencing of ITF transcription. Furthermore, non-goblet
intestinal cells also lack the protein.
Regulatory elements that similarly activate transcription overriding negative regulatory elements have been described recently. Thus the GAGA factor that binds a GA-rich sequence of Drosophila Ultrabithorax and Kruppel genes activates transcription only when the general repressor H1 protein is present, leading to its designation as an antirepressor (6). Similarly, "anti-silencer" elements have been identified in human and chicken vimentin genes. Similar to the present findings, these other factors serve to override silencer elements and regulate transcription (13, 36). An anti-repressor element is also present in the carbamyl phosphate synthetase I gene (8). Finally, a similar type of regulation has been suggested in plasminogen activator inhibitor type-2 enhancers (2) or platelet-derived growth factor (PDGF)-induced MCP-1 expression (35).
GMSAs demonstrated that the GCSI is strongly and specifically bound by a protein present in nuclear extracts from intestinal goblet cells but not nonintestinal cells. Of note, a GCSI-BP was present both in the human-derived LS 174T and murine CMT-93 goblet lines. The protein in the two lines differed in apparent molecular mass, presumably reflecting species differences.
The putative regulatory effects of the GCSI and adjacent silencer were confirmed by the ability to confer comparable goblet cell-specific transcriptional effects on the non-goblet cell intestinal specific gene, human sucrase-isomaltase. Normally, it is restricted to small intestinal absorptive enterocytes and absent in the goblet (22), enteroendocrine (42), and Paneth cell lineages. Transient transfection assays using the human sucrase-isomaltase gene constructs fused with the silencer or the silencer together with GCSI revealed that GCSI can restore gene activity in goblet cells that is reduced by silencer effect in the same manner observed for the mITF gene.
The GCSI does not have sequence similarity to known promoter elements.
A search for 5'-flanking regions of genes that are expressed in
non-goblet cells of the intestine, including rat and human intestinal
FABP (22), hSi (31), and human
lactase (4), did not reveal elements that are similar to
GCSI. Of note, a sequence (ATTCAGGCTA; 231/
222) resembling
probe S1 is present downstream from a GCRE-like sequence
(CCCCTCCCC;
298/
289), whereas a sequence similar to GCSI
(GGGCAGCTT;
1580/
1572) is present upstream of these sequences in
the human MUC2 gene, another goblet cell product (10). In
this study, a highly homologous sequence with GCSI was observed in the
human ITF gene at the nucleotide position between
2240 and
2227,
which is upstream of the sequences similar to enhancer and silencer
elements (14). Recently, the mechanisms regulating
intestinal cell-specific expression of MUC2, sucrase-isomaltase, and
FABP gene have been partially characterized. It has been suggested that
specific sequences required for suppressing inappropriate expression
are important for each differentiated cell-specific gene expression in
the intestine (9, 22, 10). Thus the antisilencing
mechanism of ITF transcription appeared to be essential for goblet
cell-specific expression. Presumably, expression of GCSI-BP is closely
associated with cellular differentiation.
In conclusion, we have shown that goblet cell-specific transcription of the mITF gene is regulated by GCSI, a "new" regulatory element that collaborates with GCRE and other enhancer and silencer elements. The GCSI-BP that binds to GCSI is an intestinal goblet cell-specific nuclear factor and blocks the silencer effect on transcription. This study shows that intestinal goblet cell-specific transcription is regulated by antisilencing mechanisms. Further characterization, including purification and/or cloning of GCSI-BP, may provide insight into ITF gene transcription as well as regulation of other intestinal genes and goblet cell differentiation.
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ACKNOWLEDGEMENTS |
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We thank K. Lynch-Devaney for expert assistance and Dr. P. G. Traber for the gift of the human sucrase-isomaltase promoter construct.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43351 and DK-46906.
Address for reprint requests and other correspondence: D. K. Podolsky, Gastrointestinal Unit, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114 (E-mail: Podolsky.Daniel{at}mgh.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 July 2000; accepted in final form 5 January 2001.
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