A silencer inhibitor confers specific expression of intestinal trefoil factor in gobletlike cell lines

Dai Iwakiri and Daniel K. Podolsky

Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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 (beta -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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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 [alpha -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).

The number used in the name of each internal deletion construct indicates the absolute position of the mITF gene 5'-flanking sequence. The composition of each construct was verified by direct DNA sequencing. Plasmid preparation purity was confirmed by an absorption ratio (260/280 nm) of >1.6, and supercoiling of DNA was confirmed by appearance on agarose gel electrophoresis before use in transfection experiments.

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 beta -galactosidase control vector (Promega) was cotransfected as an internal control. For determination of luciferase and beta -galactosidase activities, cells were lysed and assayed immediately using a commercial luciferase assay system (Promega) and luminescent beta -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 beta -galactosidase activity. From the normalized luciferase/beta -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 [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Northern blot analysis of intestinal trefoil factor (ITF) expression in various cell lines. Total RNAs were extracted from various cell lines and blotted. The membrane was hybridized with both human and murine ITF (mITF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes.

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 2161Delta 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 2193Delta 1848/201, exhibited transcriptional activity that was essentially comparable to the 2136 construct.


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Fig. 2.   Model of mITF gene transcription. The regulatory elements including enhancer and silencer elements identified in previous studies (14) and their locations in mITF 5'-flanking region are indicated within boxes. E, enhancer element; S, silencer element; PIC, preinitiation complex; GCRE, goblet cell response element; GCSI, goblet cell silencer inhibitor; X, transcriptional repression by silencers or silencer inhibition. Transcriptional regulators in goblet cells and nonintestinal cells are compared.



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Fig. 3.   A: transient transfection assays of mITF promoter deletion constructs in intestinal-derived LS 174T and CMT-93 cells and nonintestinal Hep G2 cells. The normalized transcriptional activity (luciferase/beta -galactosidase units) from each plasmid construct is shown as relative activity compared with the activity obtained for the PGL3 control plasmid construct after subtracting activity of promoterless PGL basic vector (described as PGL) used as an enzyme blank (see MATERIALS AND METHODS). Regions of the mITF promoter that were cloned upstream of the luciferase reporter gene are shown at left. Constructs are designated by the nucleotide positions relative to the transcription initiation site. Deletions are marked by Delta  followed by the numbers of the nucleotide position of the deleted segment. B: transient transfection assays in human non-goblet intestinal HT-29 and Caco-2 cells as well as gobletlike LS 174T cells. The normalized transcription activity is as shown in A. Deletion constructs transfected into these cell lines are indicated at left. All transfections were done in triplicate and repeated at least 3 times. A and B: values are means ± SE.

Constructs containing further extensions of the 5'-flanking sequence resulted in a similar lack of transcription when transfected into any of the nonintestinal cell lines used in these studies, e.g., Hep G2 cells. However, when these larger constructs were transfected into the LS 174T goblet cell line, a marked increase (3-fold) in transcription relative to the 2161 or 2193 constructs was observed. Similar increases were found when these larger constructs were transfected into CMT-93 cells, which also express ITF. These results demonstrate that the inclusion of the -2216 to -2193 sequences was sufficient to abrogate the silencing effects of the element present between -2161 and -1848 in goblet cell lines. The addition of further 5'-flanking sequence up to -6353 had no additional modulatory effect on transcriptional activity. As noted, similar constructs containing the additional 5'-flanking sequence did not further modify promoter activity observed after transfection into nonintestinal cell lines. These data suggested that an element that inhibits a silencer in goblet cells (GCSI) is present in the -2193 to -2216 region. Interestingly, the lesser absolute transcription observed in CMT-93 cells compared with LS 174T corresponds to the difference in endogenous ITF expression between these cells (Fig. 1).

Subsequently, similar transient transfection assays were performed using other intestinal epithelial cell lines. As shown in Fig. 3B, similar marked reduction of transcriptional activity by silencing effect was observed when the 2161Delta 1848/201 construct was transfected into HT-29 or Caco-2 cells and no increase in activity appeared when the 2261 or 6353M1 constructs were transfected. These two cell lines did not show endogenous ITF expression (Fig. 1); therefore, these results demonstrated that the ability of the -2216 to -2193 element to inhibit a silencer is specific to goblet cells. The pattern of results after transfection of the ITF promoter into HT-29 and Caco-2 cells paralleled the results in nonintestinal cells.

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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Fig. 4.   Goblet cells contain a nuclear protein that binds to the GCSI. A: analysis of LS 174T cell nuclear extract with GCSI was carried out using probe S1. Nucleotide sequences of probe S1 are shown at top. Specific protein-DNA complexes are marked by arrow. Competitions were performed with 25- to 150-fold molar excess (×25, ×50, × 100, and ×150) of probe S1. Competition with 150-fold molar excess of unrelated (UR) oligonucleotide was also carried out, and the result is shown at right. B: analysis of various cell lines for GCSI binding protein expression. Nuclear extracts were prepared from the gobletlike colon cancer-derived cell lines LS 174T and CMT-93 and nonintestinal cell lines HeLa and Hep G2. Probe S1 was used. Competitor used was 150-fold molar excess of unlabeled probe S1 double-strand oligonucleotide. The specific complexes are marked by arrows as is the free DNA probe.

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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Fig. 5.   Characterization of protein interactions with GCSI DNA. A: the nucleotide positions of probe S1 and competitors (probes S2-S5) are represented. B: radiolabeled probe S1 was mixed with nuclear extracts prepared from LS 174T cells in the absence or presence of 150-fold excess of unlabeled double-strand oligonucleotides probes S1-S5. Specific protein-DNA complexes and free DNA probes are indicated by arrows.

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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Fig. 6.   Determination of the sequences required for the GCSI protein-DNA interaction. A: probe S1 and block-mutated probe S1 oligonucleotides (m1-m7) used in competition assays are represented. Nucleotide sequences and positions of probe S1 are indicated at top, and the mutated sequences, designated m1 to m7, are indicated below. B: competition assay with nuclear extract from LS 174T cell line. Competitor oligonucleotides are in 100-fold molar excess. Specific protein-DNA complexes and free DNA probes are indicated by arrows.

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 2216Delta 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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Fig. 7.   Delineation of the mITF GCSI by transient transfection. A: site-specific mutations were introduced in the internal deletion construct 2216Delta 1848/201 (named WT) described at top. LUC, luciferase. Mutated constructs (mut1-mut6) are represented below with the mutated sequences; mutations correspond to the sequences shown at top. B: transient transfection assays of mutated constructs were carried out in LS 174T cells. Data are presented as %activity of the WT (2216Delta 1848/201) construct and are means ± SE of 3 independent assays. C: functional analysis of GCSI mutant. The normalized transcriptional activity from each plasmid construct is shown as luciferase activity relative to that obtained for the PGL3 control plasmid construct as given in Fig. 3 legend. The composition of each plasmid is represented at left. Symbols are as described in Fig. 2 legend. Names of the constructs are indicated at right and represent the nucleotide positions described in Fig. 3 legend. 201+S2 is a construct in which probe S2 is fused with 201 WT. All assays were done in triplicate and repeated at least 3 times. Values are means ± SE.

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 2216Delta 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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Fig. 8.   GCSI confers goblet cell expression of the non-goblet gene sucrase-isomaltase (hSi). GCSI and one silencer element were fused with human sucrase-isomaltase gene construct PGL/hSi. Composition of the reporter plasmids is given at left. Symbols and values are as described in legends for Figs. 2 and 3. All constructs were transiently transfected to LS 174T, HT-29, Caco-2, and Hep G2 cells. The normalized transcriptional activity from each plasmid construct is shown as in Fig. 3. Results are means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank K. Lynch-Devaney for expert assistance and Dr. P. G. Traber for the gift of the human sucrase-isomaltase promoter construct.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(6):G1114-G1123
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