Characterization of the 5'-flanking region of the murine polymeric IgA receptor gene

Martín G. Martín1, Jiafang Wang1, Tony W. H. Li1, Jason T. Lam2, Edgar M. Gutierrez1, R. Sergio Solorzano-Vargas3, and And Hugh V. Tsai1

1 Division of Gastroenterology and Nutrition, Departments of Pediatrics and 2 Physiology, University of California School of Medicine, Los Angeles 90095-1752; and 3 Department of Biology, California State University, Northridge, California 91330

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The regulatory elements that control basal and activated transcriptional expression of the polymeric IgA receptor gene (pIgR) have not been defined. In this study, we performed functional analysis of the murine pIgR 5'-upstream region. Transient transfection studies identified the gene's minimal promoter to reside within 110 nucleotides upstream from the start of transcription. Substitution mutations of this region identified both a putative activator (-78 to -70) and a repressor (-66 to -52) element. DNase I footprint analysis confirmed an area of protection that spans from nucleotides -85 to -62. Mobility shift assays of the putative region confirmed binding of upstream stimulatory factor 1 (USF1) to an E box element at positions -75 and -70, representing the putative enhancer. Overexpression studies using various forms of USF suggest that both USF1 and USF2 enhance activity of the pIgR minimal promoter. We report the identification and characterization of the murine pIgR minimal promoter, as well as the critical role of USF in enhancing its basal level of transcription in Caco-2 cells.

mucosa; transcription; immunoglobulin; upstream stimulatory factor; secretory component

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ROLE OF THE polymeric IgA receptor (pIgR) is to transport immunoglobulins of the IgA isotype across an epithelial barrier to the luminal side of mucosal surfaces (4). Because secretory IgA is intimately involved in reducing bacterial adherence to the epithelial brush-border membrane, pIgR is an important component of the humoral immune system (2). The process through which the dimeric IgA binds to the extracellular domain of pIgR and is subsequently transported across the epithelial layer to be cleaved and to form secretory IgA has been well described (29).

Polymeric receptors have been shown to have an extensive pattern of tissue expression. Secretory epithelial cells of the intestine, liver, lung, biliary tract, mammary glands, endometrium, and salivary glands express varying levels of pIgR (5, 6, 9, 33). The intestine, by virtue of its large surface area, synthesizes the greatest quantity of pIgR (19). Production of pIgR in the intestine has been localized to the villous crypt cells that overlie IgA-producing plasma cells in the lamina propria (8). Polymeric receptor expression can be influenced by many biological effector molecules, such as hormones (6, 38), cytokines (10, 21, 28, 41), and metabolic products (20). Additionally, changes in the physiological status of an organism, including bacterial overgrowth (22), malnutrition (13), and inflammatory conditions (3), can also alter pIgR expression.

An important feature of pIgR is that it is expressed in a development-specific pattern. It has been observed that, in suckling rodents, the synthesis of pIgR protein in hepatocytes and intestinal epithelial cells is undetectable, but at the time of weaning this synthesis increases 10- to 20-fold. The parallel enhancement of pIgR mRNA and protein steady-state concentrations during hepatocyte development suggests that the gene is regulated at least in part at the transcriptional level (7, 16). Interestingly, the increase in expression in postweaned animals corresponds to the time when IgA-secreting cells begin to expand in the lamina propria of the intestine (6, 7, 16).

Several genes that are expressed in the small intestine are either upregulated or downregulated at the time of weaning (15). In rodents, for instance, lactase-phlorizin hydrolase and the Fc receptor of the neonate are both synthesized at high levels in the enterocytes of suckling animals and are downregulated at the time of weaning (15, 27). In contrast, pIgR expression mirrors the upregulation of sucrase-isomaltase at the time of weaning (15). Whether this regulation occurs at the level of transcription has not yet been determined. Moreover, which transcription(s) factor influences the development-specific transactivation of either pIgR or the various other genes expressed in the small intestine is not well understood.

To better understand the molecular mechanism of the complex pattern of regulation of the receptors, we cloned the murine pIgR gene, including nearly 5 kb of its upstream regulatory region (24). Cloning of the human (34, 43) and rat (11) isoforms was recently reported, including identification of the presumed interferon-gamma (IFN-gamma ) response region in the human promoter (34). To study the factors that influence the development- and tissue-specific expression of the murine pIgR gene, we have defined its functional promoter using the Caco-2, Hep G2, and Chinese hamster ovary (CHO) cell lines. In this study, we present our characterization of the gene's minimal promoter identified by deletion and substitution mutations, electrophoretic mobility shift assays (EMSA), DNase I footprinting, and overexpression assays.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RNA isolation, Northern blot, and dot blot analysis. Tissue was resected from proximal intestines of rats at specified ages, and total RNA was isolated according to previous reports (26, 27). Either 5 or 20 µg of RNA were placed on a dot blot or Northern blot, respectively. The blots were hybridized to a previously described murine pIgR cDNA fragment (24). The cDNA fragment corresponds to a portion of exon 3 and was labeled using standard random-primed methods (24). Standardization of RNA loading was performed by hybridizing to a rat ribosomal 18S oligonucleotide according to a previously described method (26). Signals from autoradiograms were measured using a densitometer (Alpha Innotech, San Leandro, CA).

Cloning of the 5'-flanking region of murine pIgR. We have previously described the isolation of several lambda  phage clones from a mouse 129SVJ genomic library (Stratagene, La Jolla, CA) (24). In this study, we used a clone previously identified as PR-lambda -1-1, which includes the most 5'-upstream region of the pIgR gene. A 4.7-kb-Hind III fragment was isolated from the PR-lambda -1-1 clone that contains a portion of exon 1 and extends nearly 5 kb upstream (-4730/+44). This fragment was subcloned into the Hind III site upstream of luciferase in the pGL3-Basic vector (Promega) and is referred to as pIgR-4730/+44/B-Luc.

Development of pIgR nested deletion clones and sequencing. Standard nested deletion methods were employed to develop clones with inserts of sequentially shorter lengths (42). Nested deletions were performed using the murine (m) mpIgR-4730/+44/B-Luc clone linearized with Mlu I (5' overhang) and Kpn I (3' overhang) and subsequently digested with exonuclease III. After mung bean nuclease digestion and DNA ligation, several clones were isolated, and their sizes were determined by restriction digestion and sequencing. Several constructs, including mpIgR-4730/+44/B-Luc, mpIgR-4171/+44/B-Luc, mpIgR-3261/+44/B-Luc, mpIgR-1993/+44/ B-Luc, mpIgR-1023/+44/B-Luc, and mpIgR-574/+44/B-Luc, were used for transient transfection studies. All samples were sequenced with the dideoxy chain-termination method with [alpha -35S]dATP and Sequenase 2 (U. S. Biochemical, Cleveland, OH).

Identification of putative DNA cis-elements. We searched two transcription factor databases for putative DNA cis-elements in the 5'-upstream region of the gene. The databases included MacVector 5.0 subsequence (TFDSITES.SUBSEQ.7.0.aa) and MatInspector Release 2.1 (http://www.gsf.de/cgi-bin/matsearch.pl) (36).

Development of pIgR 5'-deletion fragments subcloned upstream of a heterologous enhancer. To assess the size of the minimal promoter for pIgR, chimeric pIgR promoter-luciferase constructs were developed in the pGL3-Enhancer vector that contains the heterologous SV40 enhancer (Promega). Clones containing different 5'-flanking regions (mpIgR-472/+44/E-Luc, mpIgR-413/+44/E-Luc, mpIgR-293/+44/ E-Luc, mpIgR-246/+44/E-Luc, mpIgR-163/+44/E-Luc, mpIgR-110/+44/E-Luc, and mpIgR-53/+44/E-Luc) were produced using sense oligonucleotides in a standard PCR. Each sense primer contained an upstream Bgl II site that defined the 5'-most regions of each clone (Fig. 1). The antisense oligonucleotide used for PCR, pGL3-3' (5'-CTTTATGTTTTTGGCGTCTTCC-3'), recognizes a region between the multiple cloning site and the luciferase cassette in the pGL3 vector. Amplified fragments were digested with Bgl II and Hind III, purified on an agarose gel, and subcloned into a similarly digested pGL3-enhancer vector. All clones were initially screened for inserts by restriction digestion and subsequently sequenced to determine authenticity and fidelity.


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Fig. 1.   Oligonucleotides used for cloning and gel shift assays. All oligonucleotides are displayed in the 5' to 3' orientation, with antisense (alpha ) primers indicated as such. Oligonucleotides used for site-directed mutagenesis are included, with the mutated nucleotides underlined. Oligonucleotides used for developing the promoter clones of different lengths are listed, with italicized nucleotides representing a Bgl II site.

Substitution mutations of the pIgR minimal promoter. To determine the essential elements within the defined minimal promoter of the murine pIgR gene, standard substitution mutagenesis was performed (18). We designed various mutant primers that contain as their central core a stretch of either ten or three mutated nucleotides (A left-right-arrow  C, G left-right-arrow  T), flanked on either side by a span of about 15-20 wild-type nucleotides (GIBCO BRL, Gaithersburg, MD) (Figs. 1 and 2) (25). PCR was performed using these mutating sense primers and the antisense pGL3-3' primer. The samples were gel purified and used as a "megaprimer" (antisense) with the sense PR-110 primer that recognizes the upstream limits of the minimal promoter (Fig. 1) (25). The amplified full-length fragments were cut with Bgl II/Hind III and subcloned into a similarly cut pGL3-Enhancer vector and sequenced to confirm the presence of the desired mutation.


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Fig. 2.   5'-Upstream sequence of murine pIgR gene. Sequence of nearly 600 nucleotides of the 5'-flanking region is represented (GenBank accession no. pending). Locations of substitution mutations are also displayed for comparison. Large arrow shows location of initiation site (+1), and putative TATA box is depicted as a shaded box. Putative transcription factors and regulatory elements are identified with a box. NF-kappa B, nuclear factor-kappa B. IL-6, interleukin-6. ISRE, interferon-stimulated response elements.

Cotransfection studies using various mammalian expression vectors. The DNA construct containing the minimal promoter (mpIgR-110/+44/E-Luc) or the M4 mutant construct (mpIgRM4-110/+44/E-Luc) was cotransfected with various forms of an upstream stimulatory factor (USF) mammalian expression vector. The USF1 (pN3) and USF2 (pN4) expression vectors were kindly provided by Michèle Sawadogo at the University of Texas. The dominant negative forms of USF1 (bAP4/USF1) and USF2 (USF2db) were kindly provided by Howard C. Towle at the University of Minnesota. Cotransfection experiments were performed with 0.1 µg/well of USF1 and 1 µg/well of USF2, USF1 dominant negative (USF1DN), and USF2 dominant negative (USF2DN) clone.

Cell cultures and transfection procedure. The human intestinal Caco-2 cell line was obtained from American Type Culture Collection (ATCC) and passages 40 to 55 were used for all transient transfections. Cells were maintained in DMEM with bovine serum, 200 U/ml of penicillin, and 200 µg/ml streptomycin at 37°C in 5% CO2. Fetal bovine serum (20%) was used for the maintenance of Caco-2 cells. The rat intestinal epithelial IEC-6 cells (ATCC), human hepatocyte Hep G2 cells (ATCC), and hamster CHO cells (kindly provided by R. Ross, University of California, Los Angeles) were all maintained in DMEM with 10% fetal bovine serum.

Transient transfection experiments were performed using freshly confluent cells treated with trypsin and seeded at 3 × 105 cells/35-mm well. After an overnight incubation, the cells were usually 40-60% confluent before transfection. A standard DNA calcium phosphate method using a commercially available (5 Prime right-arrow 3 Prime, Boulder, CO) protocol was used for all experiments. Transient experiments were done with 1 pmol of the various luciferase-expressing vectors. In all samples, 0.1 pmol of the pCMV-beta -galactosidase vector was cotransfected and served as a control for transfection efficiency and cell number. After a 48- to 72-h incubation, cells were rinsed, lysed, collected, and centrifuged, and extracts were stored at -70°C until analyzed. All experiments were repeated in three- to six-well plates at least twice.

Both luciferase and beta -galactosidase activities were assayed using a Monolight 1500A photon-counting luminometer (Analytical Luminescence, Ann Arbor, MI). Luciferase activity was measured according to the manufacturer's instructions (Analytical Luminescence). The Galacto-Light method and reagents developed by Tropix (Bedford, MA) were used to measure beta -galactosidase activity in cell extracts.

Preparation of nuclear extracts. Nuclear extracts were prepared from newly confluent Caco-2 and IEC-6 cells 3 days after seeding. Cells were washed with phosphate buffer, scraped, and centrifuged, and the supernatant was decanted. Cells were resuspended in 5 vol of hypotonic buffer consisting of 10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM dithiothreitol (DTT) and centrifuged, and the supernatant was discarded.

The pellet was suspended in 3 vol of hypotonic buffer and incubated on ice for 10 min before homogenizing with a Dounce homogenizer and a type B pestle. After centrifugation, the nuclei were suspended in 0.5 vol of low-salt buffer consisting of 20 mM HEPES, pH 7.9, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.2 mM PMSF, and 0.5 mM DTT. The nuclei were mixed at 4°C and centrifuged, and the supernatant was dialyzed for 1 h in 50 vol of dialysis buffer consisting of 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, and 0.5 mM DTT. After centrifugation of the extract, the pellet was discarded and the protein concentration of the supernatant was determined by the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) and stored at -80°C.

EMSA. The putative pIgR enhancer and silencer regions were analyzed by gel mobility shift analysis using complementary sense and antisense PR-M4/5/6 primers (Figs. 1 and 2). The primers were annealed by boiling, labeled with the use of Klenow fragment (Pharmacia, Piscataway, NJ), with [alpha -32P]dCTP (3,000 Ci/mmol) from New England Nuclear Research Products, and purified over a G25 Sephadex column. In competition experiments, various combinations of unlabeled wild-type or mutant oligonucleotides were used at 1,000-fold molar excess (Fig. 1). To determine the sensitivity of DNA-protein complex formation, various amounts (10, 50, and 100-fold excess) of cold wild-type or mutant (FAROUT) oligonucleotides were used in competition experiments.

Approximately 20,000-50,000 cpm of probe were mixed with 4-2 µg of either Caco-2 or IEC-6 nuclear protein and binding buffer for 30 min at 30°C. The master mix consisted of 5.7 mM MgCl2, 53.8 µM 2-beta -mercaptoethanol, 0.38 mM PMSF, 10 mM HEPES, pH 7.9, 10% glycerol, 0.1 mM EDTA, 0.05 M KCl, 2.5 µg BSA, and 0.2 µg of poly(dI:dC) (Pharmacia) in a 13-µl total mixture. USF1 and E47 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used for supershift analysis. After incubation of the master mix, probe, and nuclear protein at 30°C, 1 µl of antisera was added and incubated for an additional 30 min. Samples were loaded on a 4.5% acrylamide gel (30:1) containing 0.5× Tris, borate, and EDTA and 1% glycerol and run for 1 h at room temperature. The gel was fixed in acetic acid, dried, and exposed overnight at -70°C to Kodak XAR film.

DNase I footprinting. To further define the location of the protein-DNA interaction suggested by the substitution mutation studies, DNase I footprint analysis was performed according to established methods (35). Briefly, the pIgR-246 oligonucleotide (Fig. 1) was kinased with [gamma -32P]dATP (6,000 Ci/mmol) and then used with the pGL3-3' primer to PCR amplify a fragment, using the mpIgR-246/+44/E-Luc vector as a template. Samples were electrophoresed on a 5% polyacrylamide gel, purified, and diluted to 6,000 cpm/ml.

All footprints were performed using nuclear extracts isolated from Caco-2 cells and dialyzed at a final concentration of 0.1 M KCl. Approximately 6,000 cpm of probe were mixed with 6 µg of nuclear protein and the master mix specified in EMSA. Serial dilutions of DNase I were added to a buffer consisting of 40 mM CaCl2, 10 mM HEPES, pH 7.9, 10% glycerol, 0.1 mM EDTA, 0.05 M KCl, 28 mM 2-beta -mercaptoethanol, and 0.1 µg/µl of BSA. DNase I digestion occurred over 1 min at room temperature, and the reaction was terminated with stop solution consisting of 10 mM EDTA, 0.2% SDS, 400 mM sodium acetate, and 50 µg/ml yeast tRNA. Samples were purified with phenol and chloroform, precipitated with ethanol, and run on a 6% polyacrylamide gel at 50 W for 100 min. Location of the protected area was determined by Maxam-Gilbert sequencing of labeled probes.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nucleotide sequence of the 5'-upstream region of the murine pIgR gene. The nucleotide sequence of the 5'-upstream region of the pIgR gene is shown in Fig. 2. An additional 3.5-kb section of the 5' region has been sequenced but is not included in Fig. 2 (GenBank no. AF069760). Two transcription factor databases were used to search for the presence of putative cis-elements within this defined upstream region. The 1,300 nucleotides immediately upstream of the start site contain several interesting cis-elements, including several cytokine targets such as five STAT sites (-1294/-1286, -1027/-1019, -677/-669, -247/-239, and -224/-214), one nuclear factor-kappa B (NF-kappa B) site (-237/-227), one IFN-gamma site (-357/-345), and two NF-interleukin-6 sites (-331/-318 and -179/-166). Putative cis-elements for glucocorticoid (-632/-617, -117/-110) and estrogen (-1081/-1076, 419/-401) receptors were also identified in this region. Several other potentially important sites were identified and include four HNF3beta sites (-794/-783, -705/-694, -508/-497, and -208/-197) and four E box sites (-1076/-1069, -749/-744, -297/-292, and -75/-70) (36). These E box sites may bind to an assortment of basic helix-loop-helix/leucine zipper (bHLH/LZ) transcriptional factors (23).

Polymeric mRNA expression in the intestine of rats at various ages is increased at weaning. Quantitative dot blot and Northern blot analyses of RNA isolated from the proximal intestine of rats of various ages are shown in Fig. 3, A and B, respectively. When steady-state pIgR mRNA levels were standardized to ribosomal 18S, receptor expression was nondetectable in the intestine before 14 days of age but increased more than 30-fold in postweaned animals. The Northern blot demonstrates a single predominant transcript that is ~4 kb. These data are consistent with earlier reports that pIgR protein content increases in the intestine at the time of weaning and suggest that the induction is controlled at least in part at the level of transcription.


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Fig. 3.   Polymeric receptor mRNA expression in small intestine is upregulated at time of weaning. A: quantitative dot blot analysis of total RNA isolated from small intestine of rats at various ages was performed. Each dot represents RNA isolated from the proximal intestine of a single animal, with 4-9 animals per group. Five micrograms of RNA were hybridized with both pIgR and 18S probes. Densitometry data are expressed as arbitrary units (AU) ± SE. B: Northern blot analysis of RNA isolated from the proximal intestine of rats at various ages, hybridized with pIgR probe (top). The relative abundance of RNA can be seen (bottom), demonstrating 28S and 18S ribosomal RNA on the gel.

Transiently transfected Caco-2 and Hep G2 cells support the promoter activity of pIgR 5'-deletion clones. To evaluate the role of the various upstream elements in controlling the basal expression of the pIgR gene, deletion clones were developed. These clones contain sequentially shorter pIgR promoter regions located upstream of the luciferase reporter in the pGL3-Basic vector. Transient transfection of the mpIgR-4730/+44/ B-Luc clone in Caco-2 cells resulted in a 17-fold higher level of luciferase activity compared with the promoterless basic luciferase vector (Fig. 4A). Moreover, transfection of the clone of shortest length (mpIgR-574/+44/ B-Luc) resulted in promoter activity that was nearly sixfold greater than empty basic luciferase vector. Transient transfection of these clones into Hep G2 cells resulted in lower promoter activity that mirrored the trend seen with Caco-2 cells. In contrast, whereas CHO cells were transfectable, there was only a marginal level of promoter activity over the promoterless basic luciferase vector. In addition, the data suggest a tissue-specific pattern of pIgR expression when tested in Caco-2 and Hep G2 cells. These data suggest that the promoter activity as assessed by in vitro transfection assays in Caco-2 and Hep G2 cells is controlled primarily by cis-regulatory elements located within a short distance of the gene's transcriptional start site.


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Fig. 4.   Transiently transfected Caco-2 and Hep G2 cells support expression of several pIgR 5'-upstream promoter deletion clones. A: deletion clones were developed by nested deletion methods, and relative size of 5'-upstream region is displayed on left. Arrow, location of transcriptional start site. Transfection efficiency and basal luciferase expression were controlled for by transfection of pGL3-Control (containing SV40 enhancer and promoter) and pGL3-Basic. Using a calcium phosphate method, we cotransfected 1 pmol of vectors of various sizes with 0.1 pmol of CMV-beta -galactosidase vector. Transiently transfected Caco-2, Hep G2, and Chinese hamster ovary (CHO) cells are shown. Samples were processed and standardized for efficiency of transfection 2 days after incubation. B: deletion fragments subcloned into pGL3-Enhancer vector were transiently transfected with beta -galactosidase into either Caco-2 or Hep G2 cells. Two days after transfection, cells were processed, and luciferase and beta -galactosidase activity were measured. Results are given as degree of increase over either pGL3-Basic (A) or pGL3-Enhancer (B); means ± SE of data from 2-4 experiments are shown.

Identification of the minimal promoter of pIgR in Caco-2 and Hep G2 cell lines. To further assess the size limits of the gene's minimal promoter, seven clones (mpIgR-472/+44/E-Luc, mpIgR-413/+44/E-Luc, mpIgR-293/+44/E-Luc, mpIgR-246/+44/E-Luc, mpIgR-163/+44/E-Luc, mpIgR-110/+44/E-Luc, and mpIgR-53/+44/E-Luc) were developed that contain different lengths of the immediate 5' region of the gene subcloned upstream of the heterologous enhancer SV40 in the vector pGL3-Enhancer (Fig. 4B). Transient transfection of the longest fragment (mpIgR-472/+44/E-Luc) resulted in nearly sevenfold higher reporter activity compared with the promoterless pGL3-Enhancer vector in Caco-2 cells. In the clones with the fragment of the longest length (mpIgR-472/+44/E-Luc, mpIgR-413/+44/E-Luc, mpIgR-293/+44/E-Luc, mpIgR-246/+44/E-Luc, mpIgR-163/+44/E-Luc, or mpIgR-110/+44/E-Luc), promoter activity differed only slightly between clones when transfected into Caco-2 cells. However, promoter activity declined fourfold with the mpIgR-53/+44/E-Luc clone, which contained only -53 nucleotides upstream of the transcriptional start site. The 57 nucleotides deleted from the longer -110 clone contained two NF-1 sites (-110 to -99 and -66 to -51) and an E box (-75 to -70). In contrast, when these same clones were transfected into Hep G2 cells, only a minimal level of expression was identified. Overall, these data were interpreted to suggest that in Caco-2 cells, the minimal promoter of pIgR is limited to the initial -110 bp upstream of the gene's transcriptional start site. Moreover, because transiently transfected Caco-2 cells supported stronger pIgR promoter activity, all subsequent studies were limited to this cell line.

Substitution mutation analysis of the minimal promoter of pIgR identifies an enhancer and repressor element. Deletion analysis revealed that the critical upstream region of pIgR is within the first -110 bp of the promoter when tested in Caco-2 cells. To dissect the critical elements within the minimal promoter, the 80 nucleotides from -110 to the TATA box at -32 were mutated using a standard substitution mutation method. Initial mutagenesis resulted in the development of eight clones (M1-M8), each containing a consecutive span of 10 nucleotide mutations (Figs. 1 and 2). Transient transfection experiments in Caco-2 cells are displayed in Fig. 5A, demonstrating that with the exception of clones M4, M5, and M6 (nucleotides -81 to -52), the relative promoter activities of the other mutant clones were similar to the wild-type clone. More specifically, mutations within the M4 and M5 regions (nucleotides -81 to -62) result in more than a 50% decline of promoter activity relative to the wild-type mpIgR-110/+44/E-Luc clone. These data suggest the presence of a putative enhancer element located within nucleotides -81 to -62 of the promoter. The sequence disrupted by substitution mutations contains several potential cis-elements, including a classic E box (consensus: CANNTG) (23) and a degenerative NF-1 site [consensus: TGG(N5)GCCA] (17).


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Fig. 5.   Substitution mutagenesis of the minimal promoter of pIgR transiently transfected into Caco-2 cells. A: promoter activity of transiently transfected clones containing 10-bp substitution mutations is shown. Actual location of each mutation within the minimal promoter of the gene is shown in Fig. 2. Cells were transfected with 1 pmol of the specific mutant clone and 0.1 pmol of CMV-beta -galactosidase vector. B: within the context of the gene's minimal promoter (-110 bp), 3-bp substitution mutant clones were transiently transfected into Caco-2 cells with 0.1 pmol of CMV-beta -galactosidase vector. Location of each mutation is shown in Fig. 2. Cells were processed 2 days later, and beta -galactosidase and luciferase activity was measured as previously described. Data are given as degree of increase over wild-type mpIgR-110/+44/E-Luc clone.

In contrast, a mutation within the M6 region (nucleotides -61 to -52) results in more than a sixfold elevation of promoter activity compared with the native mpIgR-110/+44/E-Luc clone. These data are consistent with the identification of a putative silencer located between positions -61 and -52 within the promoter region of the gene. The only previously identified cis-element within this 10-nucleotide fragment disrupted by substitution mutagenesis is the same NF-1 site that slightly overlaps with the M5 region of the promoter.

To further dissect this region of the promoter (nucleotides -81 to -52), a set of 10 clones (A-J) were produced that contain mutations within the M4 to M6 region in the context of the minimal -110-bp promoter. Each clone contains a three-nucleotide mutation located within a 30-nucleotide stretch of DNA encompassing the pIgR putative enhancer (-81 to -62) and silencer (-61 to -52). The data shown in Fig. 5B demonstrate that only clones B, C, and D (nucleotides -78 to -70) result in more than a 50% decline in promoter activity relative to the wild-type mpIgR-110/+44/E-Luc clone. Moreover, clones (F, G, H, I, and J) with mutations of nucleotides -66 to -52 in the putative silencer regions resulted in a significant elevation of promoter activity compared with the wild-type mpIgR-110/+44/E-Luc minimal promoter. Unfortunately, experiments were not performed to define the downstream limits of the repressor element. In summary, these data were interpreted to suggest that the critical element for the pIgR promoter is located between positions -78 and -52 and consists of at least two elements that bind to a putative transcriptional factor(s) that activates and represses pIgR promoter activity.

DNase I footprinting of the upstream minimal promoter of pIgR identifies a single site. To confirm our mutation analysis of the promoter, DNase I footprint experiments were performed on the immediate upstream region of the promoter. The entire minimal promoter was examined bidirectionally for protein-induced footprints, using nuclear protein isolated from Caco-2 cells. Footprint analysis using both 5'- and 3'-end-labeled probe identified a single site of DNA-protein interaction that corresponds to the region defined by M3, M4, and M5 (nucleotides -85 to -62) (Fig. 6). No other footprints were identified within the gene's immediate promoter region using unstimulated Caco-2 cell extracts (-226 to -32). Interestingly, the footprint (-85 to -62) overlaps only slightly with the putative repressor region (-66 to -52) identified by transfection of mutagenesis clones. In summary, the DNase I footprint experiment confirms our mutation analysis that identified nucleotides in the region between positions -78 and -70 as the location of the protein-DNA interactions.


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Fig. 6.   DNase I footprinting of the immediate 5' promoter region of pIgR. DNase I footprinting analysis of murine pIgR gene region from -226 to -32 is shown. DNA fragment labeled in the sense orientation was incubated with (+) or without (-) nuclear extracts from Caco-2 cells and subjected to DNase I digestion. Varying concentrations of DNase I were used, with the left lane (lanes 1 and 4) of each group containing the most enzyme as depicted by the triangle. Samples were electrophoresed with a Maxam and Gilbert reaction as described in MATERIALS AND METHODS. Shaded box labeled -85 to -62 indicates sequences protected between the reported limits.

EMSA of activator and repressor regions of pIgR. EMSA were used to define the binding characteristics of the putative transcription factor(s) that interacts with the enhancer and silencer region of the murine promoter. Double-stranded (PR-M456) oligonucleotides (nucleotides -81 to -52) representing the presumed enhancer and repressor region were end labeled and used as the probe for all gel shift assays (Fig. 7A). Either crude human Caco-2 (Fig. 7, B and D) or rodent IEC-6 (Fig. 7C) nuclear extracts were used for all EMSA. Figure 7, B and C, is a representative EMSA and shows the presence of a single protein-DNA complex (Fig. 7, B and C, lane 2) competed with a 1,000-fold molar excess of unlabeled (PR-M456) DNA (Fig. 7B, lane 6, and Fig. 7C, lane 3). Moreover, the complex could not be competed with a 1,000-fold molar excess of either an unrelated double-stranded primer (Fig. 7B, lane 12) or DNA representing wild-type nucleotides from regions M6 and M7 (Fig. 7B, lane 8) (nucleotides -61 to -42).


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Fig. 7.   Binding of upstream stimulatory factor (USF) to the E box element in the enhancer region in the minimal promoter of pIgR. A: oligonucleotides used for electrophoretic gel mobility shift assay (EMSA) are shown. Top: sequence from M4 to M7; the E box of interest is boxed. Annealed antisense and sense primers were used for all experiments. Oligonucleotide PR-M456, which spans from M4 to M6, was labeled with 32P and used for all experiments. Competition experiments were performed with either PR-M456, PR-M45, PR-M67, IN, OUT, or FAROUT. Wider bar represents nucleotides mutated in each circumstance. B: EMSA were performed with nuclear extracts (NP) isolated from Caco-2 cells (lanes 2-12). DNA-protein complex identified by arrow labeled b is specific, whereas that labeled c is nonspecific. Lane 1, labeled probe without nuclear extract or antisera. Antisera for USF1 (lane 3) and E47 (lane 4) and nonimmune antisera (lane 5) were added for the supershift reactions. Supershift complex is labeled a. One thousand-fold excess of unlabeled PR-M456 (lane 6), PR-M45 (lane 7), PR-M67 (lane 8), FAROUT (lane 9), IN (lane 10), OUT (lane 11), or nonrelated (lane 12) oligonucleotides are shown. EMSA were performed as described in MATERIALS AND METHODS. Comp, competed. C: crude nuclear extracts isolated from rodent IEC-6 cell line were used to perform gel shift assays. Lanes 1 and 2 correspond to those described in B, lanes 3 and 4 correspond to lanes 6 and 7 from B. D: EMSA using either M456 or FAROUT oligonucleotides was competed either 10- (lanes 7 and 11), 50- (lanes 8 and 12), 100- (lanes 9 and 13), or 1,000-fold (lanes 10 and 14) excess (from left to right) of the unlabeled M456 (lanes 7-10) or FAROUT (lanes 11-14) DNA. Lane 5, without nuclear extract. Lane 6, Caco-2 extract. Specific DNA-protein complex is shown by arrowhead on left.

To determine the sensitivity of this DNA-protein interaction, competition experiments were performed with various molar excesses of unlabeled PR-M456 and FAROUT oligonucleotides. The data displayed in Fig. 7D demonstrate the ability to compete DNA-protein interactions with as little as 10-fold excess of cold probe. We interpret these data to suggest that the complex is specific to this stretch of DNA, and more importantly, that the wild-type M6 (nucleotides -61 to -52) portion of the DNA does not contribute to the observed protein-DNA complex. Moreover, the complex could be competed with an excess of DNA representing the M4 and M5 regions (nucleotides -81 to -62) (Fig. 7B, lane 7, and Fig. 7C, lane 4). From these data, we infer that the identified protein-DNA complex results from the relatively sensitive binding of a protein(s) to nucleotides located between -81 and -62 of the promoter.

To further define the precise location of the protein-DNA complex within nucleotides -81 to -62, unlabeled double-stranded oligonucleotides from this region that contained transversion mutations were used in 1,000-fold molar excess. More specifically, three sets of mutant oligonucleotides, named IN, OUT, and FAROUT, were used that contain mutations in different regions within this 20-bp fragment (Figs. 1 and 7A). The M456 protein-DNA complex was competed by the addition of the FAROUT primer (Fig. 7B, lane 9), which contains mutations in the 10 outermost nucleotides of the M4 and M5 complex (Fig. 7A). This suggests that the central core, from nucleotides -76 to -68, contains the putative cis-element that accounts for the protein-DNA complex. A classic E box (CACGAT) that binds to a group of bHLH transcriptional factors is located at position -75 to -70 within this stretch of DNA. The OUT (Fig. 7B, lane 11) (mutation of nucleotides -76 to -74 and -69 to -67) and IN (Fig. 7B, lane 10) (mutation of nucleotides -73 to -70) oligonucleotides, which contain mutations within the E box, failed to compete for the protein-DNA complex. Identical gel shifts were obtained with nuclear extracts from IEC-6 cells (Fig. 7C; other data not shown). In summary, these data indicate that the basal expression of the pIgR promoter in Caco-2 and IEC-6 cells is due at least in part to binding of a bHLH/LZ protein to the E box located at position -75 to -70 within the gene's minimal promoter.

Supershift experiments were done to further define which bHLH/LZ transcriptional factor binds to the upstream region of the pIgR gene. Two candidate bHLH/LZ proteins that may bind to this region of the promoter are USF and E47 (23). To distinguish between these and other members of the large bHLH/LZ family of genes, supershift assays were performed using antisera that specifically recognize either E47 or USF1. Figure 7B demonstrates that only the USF antiserum supershifts the protein-DNA complex (Fig. 7B, lane 3), whereas neither the E47 antiserum (Fig. 7B, lane 4) nor preimmune serum (Fig. 7B, lane 5) alters the mobility of the complex. Similar supershift complexes were obtained using nuclear extracts from rat IEC-6 cells (data not shown). These data strongly suggest that either a homo- or hetero-oligomerized member(s) of the USF family of proteins binds to the E box located within nucleotides -75 to -70 of the minimal promoter and is responsible for basal transcriptional transctivation of the pIgR gene.

USF overexpression studies. To further analyze the role of USF in activating the transcriptional activity of the pIgR minimal promoter, cotransfection experiments were performed. Cotransfecting a USF1-containing expression vector with the mpIgR-110/+44/E-Luc construct resulted in nearly a modest twofold enhancement of luciferase activity over the minimal promoter alone (Fig. 8). On the other hand, cotransfection of a USF2-expressing vector had no discernible effect on promoter activity of the mpIgR-110/+44/E-Luc vector. Neither USF1 nor USF2 expression vectors were capable of activating transcriptional expression of the mpIgRM4-110/+44/E-Luc construct, which contained a mutation in the putative E box (M4). When the dominant negative forms of either USF1 or USF2 were cotransfected with the -110 promoter construct, luciferase activity was significantly lower compared with mpIgR-110/+44/E-Luc and was indistinguishable from the promoterless luciferase vector. Overall, the data obtained from overexpressing the dominant negative forms of USF1 and USF2 were interpreted to suggest that both transcriptional factors play a critical role in activating the minimal promoter of pIgR. In contrast, the inability of the ectopically expressed wild-type form of USF2 to enhance promoter activity presumably may have occurred if endogenous levels USF2 were saturated in Caco-2 cells. Alternatively, since both USF1 and USF2 have a relatively weak transactivation domain, we may not have been able to measure a modest elevation in USF2-induced promoter activity with the particular conditions used in the experiment (23).


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Fig. 8.   Effect of overexpressing various forms of USF1 and USF2 on promoter activity of the pIgR gene. Transfection experiment was performed using 1 pmol of either the mpIgR -110/+44/E-Luc vector or a construct of the minimal promoter containing a mutation (mpIgRM4-110/+44/E-Luc) in the putative E box. Either one of the vectors was cotransfected with an expression vector containing either USF1, USF2, or USF1 (USF1DN) or USF2 dominant negatives (USF2DN). Cells were also transfected with CMV beta -galactosidase vector and processed 48 h later. Results are given as degree of changes from the promoterless pGL3-basic vector.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have performed a thorough analysis of the immediate promoter region of the murine polymeric receptor in a human intestinal cell line. Data obtained from deletion analysis reveals that a large portion of the gene's basal promoter activity is controlled by DNA cis-regulatory elements located within a region defined as the minimal promoter, located immediately upstream of the gene's transcriptional start site. The promoter activity was supported best in transiently transfected Caco-2 cells and to a smaller extent in Hep G2 cells (Fig. 4). Although several putative cis-elements reside within the minimal promoter (Fig. 2), transiently transfected clones containing substitution mutations provide convincing evidence that nucleotides -81 to -62 (M4/M5) are solely responsible for the enhancer activity of the minimal promoter (Fig. 5A). Similarly, clones with 3-bp substitution mutations decrease promoter activity by more than 50% compared with wild-type, confirming that the putative enhancer element in this region is represented by an E box at nucleotides -75 to -70 (Fig. 5B). Overexpression studies using wild-type and dominant negative USF1 and USF2 suggest that both transcriptional factors activate promoter activity in this model system (Fig. 8).

In contrast, substitution mutation of nucleotides -61 to -52 (M6) results in a sixfold elevation in promoter activity, suggesting that a transcription factor binding domain in this region silences wild-type promoter activity (Fig. 5A). Clones containing 3-bp mutations also detected evidence of silencer activity from nucleotides -66 to -52, containing a novel symmetric CCTGGCTGGCC element (Fig. 5B). DNase I footprint analysis using Caco-2 nuclear extracts detected a single area of protection from nucleotides -85 to -62, and gel shift analysis of this region demonstrated that only a single protein complex interacts with this stretch of DNA (Figs. 6 and 7). Several wild-type and mutation oligonucleotides were used to show that the complex was limited to the binding of a transcriptional factor(s) to the E box region that comprises the enhancer element. Moreover, the wild-type oligonucleotides represented by the M6 region failed to compete with this complex, providing further evidence that under the conditions used in this in vitro analysis, nuclear proteins failed to complex with the silencing M6 region of DNA. One possible explanation for this discrepancy is that we failed to identify the optimal conditions for binding of the putative silencing transcription factor(s). Another plausible explanation is that instead of the substitution mutation removing a repressor wild-type element, the mutation actually creates an artificial enhancer element. In fact, although the M6 mutation abolishes the wild-type NF-1-like site (-66 to -55), it also creates a CEBPbeta site (TTAGCAAGGT) (36). However, the ability of the series of 3-bp mutations to augment promoter activity (Fig. 5B) would most likely occur if the mutation disabled a wild-type repressor. Moreover, the conservation of nucleotides within this region of the rat and in human isoforms of the pIgR gene suggests that this element (CTGGCTGGC) may play an important physiological role in receptor expression. Further analysis of this putative repressor is currently underway.

The upstream region of the murine polymeric receptor contains several E box sites (-1076/-1069, -749/-744, and -297/-292) that correspond to the classical consensus core motif (CANNTG). The E box element is recognized by a diverse group of well-described bHLH transcriptional factors, including a distinct group with leucine zipper proteins (bHLH/LZ) (23). The tissue distribution of the bHLH/LZ factors may be either ubiquitous (class A) or restricted (class B) to a specific cell type. Moreover, class A proteins usually form homodimers, whereas the restricted class B proteins generally form heterodimers with other class A proteins. The transcription factors E12/E47, ITF-1/2, and HTF4/HEB are the main ubiquitous bHLH family members, but a growing group of class B proteins that include MyoD, Mash-1/2, and Lyl-1/2 have been isolated from a wide range of tissues. The ubiquitous group of family members with a leucine zipper includes USF-1/2, Max, Mad, Mxi1, and TEF3, which generally form homodimers.

The specific E box element at position -75 to -70 in the murine pIgR promoter could potentially be recognized by either USF, Max homodimers, or Max heterodimerizing with either c-Myc, Mad, or Mxi1 (23). However, Max heterodimers with c-Myc or Max/Max homodimers were less likely candidates to bind because the surrounding 5' and 3' nucleotides (5'-<UNL>GT</UNL>CACGTG<UNL>A</UNL>-3') would disrupt heterodimer binding (1). The ubiquitous E box bHLH proteins E47/E12 were also unlikely candidates because of their low affinity for CACGTG binding sites (1). This assumption is supported by a supershift assay that failed to shift the DNA-protein complex with E47/E12 antisera (Fig. 7). In contrast, antisera specific to USF1 entirely supershifted the DNA-protein complex. Although no other USF binding sites (CACGTG) were identified in the upstream 4.7-kb region, substitution mutations and footprint analyses have not been performed on the other E box elements further upstream to assess their role in pIgR promoter activity. Interestingly, the immediate upstream region of the human pIgR promoter contains two noncanonical E boxes (-71 to -66 and -53 to -48), and although these have not yet been analyzed, it is possible that USF can bind to the -71 to -66 element and enhance promoter activity (1) (Fig. 9). The rat pIgR gene has an identical E box (-73 to -68) just upstream of the TATA box, suggesting that the reported role of USF in controlling pIgR basal expression may be limited to rodents (40).


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Fig. 9.   Comparison of the immediate 5'-upstream region of murine, rat, and human pIgR gene. Nucleotide sequence of murine, rat, and human pIgR immediate promoter is shown, with the more 3' portion placed on the bottom section. Shaded region corresponds to the location of perfect homology between the three species. The E box, NF-1, and TATA box are delineated by a clear thin box for comparison.

Both substitution mutation analysis and EMSA provide good evidence that either homodimers or heterodimers of the USF family of proteins bind to the enhancer element. USF1 and USF2 are both expressed in liver, kidney, lung, and in most other tissues except for muscle (40). USF1 is a 43-kDa protein; the 44-kDa protein USF2a is encoded by a separate gene that undergoes alternate splicing to form the USF2b isoform (38 kDa) (40). Although recombinant USF1 and USF2 have binding characteristics nearly identical to that of the CACGTG motif (40), USF2b is threefold less active in enhancing promoter activity as USF1 or USF2a homodimers (44). Although the antisera used in the supershift experiments distinguish USF1 from USF2, its potential cross-reactivity with other as yet unidentified USF family members cannot be ruled out at this time. Because of the propensity of USF1 and USF2 to form heterodimers with one another, the supershift data suggest that USF1 binds to the DNA element either as a homodimer or a heterodimer. Moreover, overexpression experiments using both isoforms of the dominant negative forms of USF1 and USF2 suggest that both may play an important role in transcriptional activity of the pIgR promoter.

Although USF is ubiquitously expressed, it has been implicated in the regulation of several tissue-regulated and developmentally regulated genes, including surfactant protein-A (12, 31). Other tissue-specific bHLH proteins have been implicated as central transcriptional control elements in the development of various tissue (30). The data provided in this study cannot definitively rule out a tissue-specific transcription factor that shares DNA binding elements and is recognized by the USF antisera. However, recent attempts to screen mouse intestinal epithelium for a tissue-specific (class B) bHLH transcription factor using degenerative oligonucleotides and PCR have failed (data not shown) (32).

USF is believed to activate promoter activity by interacting with the TFII portion of the initiation complex via a PC5 cofactor (14, 39). Stimulated activation of USF binding has also been described with hormones and cytokines (37). Because of the proximity of the NF-1-like binding site to USF, activation of the NF-1 pathway by transforming growth factor-beta stimulation may influence USF-induced transactivation. In fact, a similar activation of the plasminogen activator inhibitor gene has been described where an NF-1 site just downstream of a USF element alters promoter activity (37). One may speculate that pIgR promoter activity is controlled by the combinatorial action of USF and an as yet unidentified tissue or a ubiquitous transcriptional factor such as NF-1.

The 5'-upstream sequence of the human, rat, and murine isoforms of the pIgR gene have only recently been identified (11, 34, 43). Piskurich et al. (34) analyzed the IFN-gamma -responsive region of the human promoter using HT-29 cells and identified several IFN-stimulated response elements (ISRE) within the immediate 5'-upstream region of the gene. Interestingly, only the ISRE located on exon 1 was found to bind IFN regulatory factor-1 when stimulated by IFN-gamma . However, no substitution mutation analysis was performed to further characterize the relative contribution of each of these sites to IFN-gamma -induced activation of pIgR promoter activity.

Dot blot analysis of the ontogeny of pIgR mRNA expression confirms a pattern of upregulation similar to that previously described in rat hepatocytes (Fig. 3) (6, 7). What specific transcriptional regulatory event accounts for the upregulation of pIgR expression seen in the intestine and liver at the time of weaning? Could USF synthesis be regulated during ontogeny of the intestine and liver? A careful assessment of USF tissue-specific expression and the developmental pattern of expression have not been reported. Furthermore, although the relative abundance of the USF family members differs between cell lines and tissues, its expression has not been tested in the intestine and other tissues that express pIgR (40). Interestingly, the level of the less potent USF1/2b heterodimer is threefold higher in fetal compared with adult liver, suggesting that the upregulation of pIgR may be in part related to a change in the relative abundance of the USF isoforms (44). More specifically, the augmentation of pIgR expression at weaning could be secondary to a reduced concentration of the 1/2b heterodimer relative to the more potent enhancing 2a/2a homodimer. What role, if any, does the identified repressor have in controlling the normal developmental pattern of expression of pIgR? Could this as yet unidentified repressor be downregulated or inactivated at the time of weaning? Although the use of in vitro cell lines helps in the careful dissection of naked DNA, the system has several well-described shortcomings, especially when attempting to address a development-specific process. Both transgenic animal studies or nuclear extracts isolated from these various tissues would be particularly informative in defining the specific in vivo role of USF in pIgR regulation.

    ACKNOWLEDGEMENTS

We thank S. Smale for helpful discussions.

    FOOTNOTES

This study was supported by National Institutes of Health Grants HD-34706, DK-44582, and DK-41301 and by fellowships to M. G. Martín from the Robert Wood Johnson Foundation Minority Faculty Training Grant and the American Gastroenterology Industry Training Award. E. M. Gutierrez was supported by the National Science Foundation-California Alliance for Minority Participation.

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: M. G. Martín, Division of Gastroenterology, Dept. of Pediatrics, 12-383 MDCC, University of California School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1752.

Received 9 February 1998; accepted in final form 5 June 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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