Multiple transcription factors in 5'-flanking region of human polymeric Ig receptor control its basal expression

R. Sergio Solorzano-Vargas1, Jiafang Wang2, Lingling Jiang2, Hugh V. Tsai2, Luis O. Ontiveros2, Mukta A. Vazir1, Renato J. Aguilera3, and Martín G. Martín2

2 Department of Pediatrics, Division of Gastroenterology and Nutrition, and 3 Department of Molecular, Cell, and Developmental Biology, School of Medicine, University of California, Los Angeles 90095; and 1 Department of Biology, California State University, Northridge, California 91330


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The polymeric Ig receptor (pIgR) is a critical component of the mucosal immune system and is expressed in largest amounts in the small intestine. In this study, we describe the initial characterization of the core promoter region of this gene. Expression of chimeric promoter-reporter constructs was supported in Caco-2 and HT-29 cells, and DNase I footprint analysis revealed a large protein complex within the core promoter region. Site-directed mutagenesis experiments determined that elements within this region serve to either augment or repress basal activity of the human pIgR promoter. Band shift assays of overlapping oligonucleotides within the core promoter identified eight distinct complexes; the abundance of most complexes was enhanced in post-confluent cells. In summary, we report the characterization of the human pIgR promoter and the essential role that eight different nuclear complexes have in controlling basal expression of this gene in Caco-2 cells.

upstream stimulatory factor; interferon response element; interferon response factor; adaptive immunity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE HUMAN POLYMERIC IG RECEPTOR (pIgR) is a critical component of the mucosal humoral immune system as it transports Igs across the epithelial barrier (1). After synthesis, pIgR is targeted to the basolateral membrane where it binds noncovalently with high affinity to both the IgA dimer and pentameric IgM (19, 28). The Ig-receptor complex then translocates to the apical surface where it is cleaved by an unidentified protease. A portion of pIgR called secretory component remains attached to the Ig and has been shown to protect IgA and IgM from degradation by proteolytic enzymes (1).

The expression of pIgR mRNA is tissue and age specific and is also regulated by several cytokines, hormones, and growth factors (20). During the suckling phase of rodent development, intestinal pIgR expression is nearly undetectable; however, after weaning pIgR levels increase >20-fold (16). pIgR protein and mRNA are highly expressed in the epithelial cells of the small and large intestine and to a lesser extent in the liver, kidney, lung, salivary gland, mammary glands, and endometrium (20). In human and rodent intestine, pIgR expression is most abundant in the less well-differentiated epithelial cells that reside in the crypt compared with the more mature cells of the villus (2, 3, 31).

Nuclear run-on assays demonstrated that interferon-gamma (IFN-gamma ), tumor necrosis factor-alpha (TNF-alpha ), and interleukin-4 (IL-4) augment pIgR mRNA expression at the level of transcription, and the mechanism by which these cytokines regulate pIgR transcription has recently been elucidated (18, 24). An interferon response element (ISRE) located on exon 1 of the human gene was identified (25) as the site that confers IFN-gamma -induced expression via the intermediate synthesis of interferon response factor-1 (IRF-1). The human promoter also contains a nuclear factor-kappa beta site that directs TNF-alpha -induced expression and a signal transducer and activator of transcription (STAT) site located in intron 1 that controls IL-4-induced transactivation (27, 29). An androgen response unit was also identified in the human promoter (31), and an active glucocorticoid response element was identified in the murine gene (13).

The basal expression of the immediate promoter region of the human pIgR gene was recently examined (11). A putative E-box located between nt -65 to -70 relative to the start of transcription was found (11) to activate basal expression in intestinal HT-29, gastric adenocarcinoma (AGS), and Calu-3 cells; however, the identity of the transcription factor(s) that interacted with the element was not elucidated. An inverted repeat sequence (IRS) located immediately downstream of the E-box (-52 to -65) was determined to bind to an unidentified nuclear factor; however, when the IRS site was mutated, basal promoter activity was unaltered (11). Transcriptional activity of nested deletion clones also suggested that the region between -83 and -372 was transcriptionally active; however, these potential sites were not further evaluated in the study (11).

Using the murine pIgR promoter, we (16) identified both a putative activator (-78 to -70) and a repressor (-62 to -52) element in the immediate promoter region in Caco-2 human intestinal cells. Mobility shift assays indicated the presence of an E-box at positions -75 and -70, and overexpression and supershift assays suggested that the basic helix-loop-helix (bHLH) proteins upstream stimulatory factor-1 and -2 (USF1 and USF2) upregulated basal promoter activity (16). The murine promoter also contains an IRS site, and clones containing mutations in the region that overlapped with the IRS site (-62 to -52) identified evidence of repressor activity. The nuclear protein(s) responsible for the repressor activity was not identified by band shift assays.

To further characterize the mechanism by which the human pIgR gene is regulated, we focused on defining the elements that control its basal level of expression. Deletion and site-directed mutations of various elements within the core promoter were transiently transfected into human intestinal (Caco-2 and HT-29) cell lines to determine the elements that are required for full promoter activity. DNase I footprints and DNA binding assays were used in combination to further define the critical elements that control the basal expression of the gene.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA cloning and substitution mutagenesis. Four sense oligonucleotides [-600/-579 (5'-gcaatctTACGACAGCCACTCCTGCCACC), -234/-212 (5'-gcaatctAAGCGACAGAAGAGCATCTT), -210/-181 (5'-gcaatctCGCTTGACATTCTGTCCCTCATCCTGAAGC), and -119/-108 (5'-gcaatctCCAGCCAGGAAGGCCAAAAT)] and an antisense oligonucleotide [+55/+76 (5'-ataagcttCAGTTGGTAACCCCTGCCTC TC)] were developed to PCR amplify various size products from human genomic DNA (see Fig. 3) (11). The fragments were digested with HindIII/BglII (restriction site indicated in lowercase lettering) and subcloned into the pGL3-enhancer luciferase reporter vector (Promega), as previously described (16). Clones hpIgR -600/+76/E-Luc, hpIgR -234/+76/E-Luc, hpIgR -210/+76/E-Luc, and hpIgR -119/+76/E-Luc were sequenced to define their size and authenticity.

To determine which elements in the minimal promoter are critical for maintaining the gene's basal level of expression, we produced a series of clones containing 10-bp mutations by standard scanning mutagenesis (see Fig. 3) (17). Various mutagenizing oligonucleotides were designed in which the transversion mutations (Aleft-right-arrowC, Gleft-right-arrowT) were introduced in the central 10 bp and used to perform PCR with the antisense (+55/+76) oligonucleotide (Table 1). The PCR products were gel purified and used as megaoligonucleotides (antisense) with the upstream sense -210/-181 oligonucleotide for the second PCR reaction to generate clones with 10-bp mutations. The full-length fragments were cut with BglII/HindIII, gel purified, and subcloned into the pGL3-E vector. All clones were sequenced to verify the presence of only the desired mutation.

                              
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Table 1.   Oligonucleotides used for mutagenesis

Identification of putative DNA cis-elements. The TRANSFAC database (GBF, Braunschweig, Germany) was used to identify the putative DNA cis-elements in the upstream region of the human pIgR gene (10).

Cell culture, transfection, and transfection procedure. The human intestinal (Caco-2) and Chinese hamster ovary (CHO) cells were maintained and transfected by the calcium phosphate method, as previously described (16). HT-29 cells were obtained from American Type Culture Collection and maintained in DMEM with 20% fetal bovine serum, 200 U/ml penicillin, and 200 µl/ml streptomycin at 37°C in 5% CO2 and switched to L-15 media for 48 h before transfection. Transient transfections were performed using 1 pmol/well of all the luciferase-expressing constructs, and 0.5 µg/well of the pCMV-beta -galactosidase vector was transfected as a control for transfection efficiency and cell number. For comparison of clones containing specific mutations, transfection efficiency was controlled for with the use of Renilla luciferase, a component of the Promega dual luciferase system. Cotransfection of various concentrations of several USF expression vectors was performed as previously reported (16). All samples were rinsed twice and processed 48 h after transfection. Both luciferase and beta -galactosidase were measured as previously described (12). Transfection of HT-29 cells was performed using electroporation with the RF module (Bio-Rad). Each pulse had 10 µg of luciferase construct and 1 µg of either pCMV-beta -galactosidase or Renilla vector for 4 × 106 cells. This mixture was then pulsed at 0% modulation, 0.2 kV, and 50 Hz for 2 ms, with 1-s intervals for 5 bursts. The volume of the DNA-cell mixture was 100 µl. This mixture was immediately transferred into L-15 media with serum using a 1-ml syringe and disbursed over six-well plates. The plated cells were allowed to incubate in the humidifier at 37°C and 5% CO2 for 24 h.

Electrophoretic mobility shift assays and preparation of nuclear extract. Nuclear extracts were obtained from Caco-2 cells either 2 or 10 days after seeding by standard methods (7). Cells seeded for 2 days were subconfluent, whereas those seeded for 10 days were confluent for ~7 days. Electrophoretic mobility shift assays (EMSA) were performed as outlined previously (16), using probes A-G (see Fig. 3). Briefly, various double-stranded oligonucleotides (see Fig. 3) containing a GATC 5'-overhang were labeled with the use of Klenow fragment and [alpha -32P]dCTP (3,000 Ci/mol). Approximately 2-5 × 104 cpm of probe and 2.5-5 µg nuclear extracts were used for each reaction. Standard competition studies were run using excess unlabeled oligonucleotide duplexes. To identify the precise outer limits of the cis-element, competition studies were performed using a series of oligonucleotides that were the same length as the wild-type oligonucleotides and included 5- to 3-bp transversion mutations as specified within each figure (precise oligonucleotides not shown). Antiserum against USF1, USF2, CCAAT/enhancer-binding protein-alpha (C/EBP-alpha ), C/EBP-beta , C/EBP-gamma , IRF-1, and cAMP-responsive element (CRE) binding protein (CREB) were purchased from Santa Cruz Biotechnology and used to perform supershift studies.

DNase I footprinting. To define the regions of DNA-protein interactions, we performed DNase I footprint analysis according to methods established in our laboratory (16). The hpIgR -600/+22/E-Luc vector was cut with HindIII, kinased with [gamma -32P]dATP (6,000 Ci/mol), and then cut with BglII. Samples were electrophoresed on a 5% polyacrylamide gel, purified, and diluted to 6,000 cpm/µl. All footprints were performed with 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 either nuclear extracts or BSA. Serial dilution of DNase I was added to DNase reaction buffer (40 mM CaCl2, 10 mM HEPES, pH 7.9, 10% glycerol, 0.1 mM EDTA, 0.05 mM KCl, 28 mM 2-beta -mercaptoethanol, and 0.1 mM BSA). Digestion with DNase I occurred for 1 min at 25°C and was terminated with stop solution (10 mM EDTA, 0.2% SDS, 40 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 for 100 min at 50 W. The locations of the protein-DNA binding sites were determined by running a DNA ladder sequenced by the Maxam-Gilbert method.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transient transfection of human pIgR clones into HT-29, Caco-2, and CHO cells identified gene's core promoter. Four clones containing different length fragments of the 5' region of the human pIgR gene (hpIgR -600/+76/E-Luc, hpIgR -234/+76/E-Luc, hpIgR -210/+76/ E-Luc, and hpIgR -119/+76/E-Luc) were transfected into various cell lines (Fig. 1). Transfection of Caco-2 and HT-29 cells with either hpIgR -600/+76/E-Luc or hpIgR -234/+76/E-Luc resulted in promoter activity 10- to 20-fold higher than the promoterless pGL3-E vector. In contrast, in CHO cells, promoter activity was only slightly higher than that observed with the promoter-less vector.


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Fig. 1.   Transient transfection of various polymeric Ig receptor (pIgR) 5'-upstream promoter deletion clones. As described in EXPERIMENTAL PROCEDURES, Caco-2 and Chinese hamster ovary (CHO) cells were transfected by the standard calcium phosphate method, whereas HT-29 cells were transfected by electroporation. Chimeric promoter-reporter constructs were precipitated with 10-fold less beta -galactosidase vector driven by a CMV promoter. After processing 48 h later, relative light units (RLU) and beta -galactosidase activity were measured. Data is shown as fold elevation of the empty pGL3-enhancer vector (pGL3-En). Values are means ± SD of sextuplet data from 2 experiments.

DNA footprint analysis of human pIgR's upstream core promoter identified large footprint with multiple hypersensitivity sites. DNase I footprint experiments of the immediate upstream region of the promoter were performed using only nuclear extracts from Caco-2 cells. The region was analyzed in the sense (5'right-arrow3', Fig. 2A) and antisense directions (3'right-arrow5', Fig. 2B). Footprint analysis in the sense direction identified a rather large footprint containing three hypersensitivity sites, suggesting the presence of DNA-protein interactions located between -135 and -46 (Fig. 2A). When this region was analyzed in the antisense direction, a slightly larger footprint was identified that extends up to 190 nt upstream of the start of transcription (Fig. 2B). Overall, these data suggest that the immediate 5'-upstream region of the human pIgR core promoter contains a large region that is capable of binding to Caco-2 nuclear extracts (Fig. 3).


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Fig. 2.   Bidirectional DNase I footprint analysis of the human pIgR gene. Footprint analysis was performed with nuclear extracts isolated from Caco-2 cells. DNA was labeled in either the sense (A, 5' to 3') or antisense orientation (B, 3' to 5'), in the presence of BSA (-; lanes 1-2 and 5-6) or Caco-2 nuclear extracts (+; lanes 3-4 and 7-8). DNase I was added at 2 different dilutions (indicated by shaded triangles). The filled bar at the right of each image depicts the location of the footprint, and * shows the location of the hypersensitivity sites. The Maxam-Gilbert sequencing ladder is shown adjacent to each panel for comparison.



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Fig. 3.   The 5'-flanking sequences of human, rat, and murine pIgR promoters are shown for comparison. Nucleotides conserved between the human (h) (Ref. 11), rat (r) (Ref. 8), and murine (m) genes (Ref. 15) (indicated at left) are highlighted by a shaded rectangle. Nucleotides are numbered relative to the start of transcription (indicated at right), and the location of the mutation in each clone is labeled M1 to M20, with the boundaries of each mutation identified by short vertical lines. The location of each double-stranded oligonucleotide used for band-shift assays is shown as a horizontal line below its corresponding sequence and labeled probes A to G. A clear rectangular box located on the human stretch of DNA shows the location of each DNA-protein complex identified in this study. The relative locations of the footprints identified in Fig. 2 are displayed as a filled bar, and the hypersensitivity sites are indicated by *. The TATA box, E-box, and inverted repeat sequence (IRS) elements are also highlighted.

EMSA and transfection of reporter clones containing substitution mutations in immediate promoter region defines several active elements. To further define the critical elements in the core promoter region of the hpIgR -210/+76/E-Luc vector, a series of 20 scanning mutagenesis clones was generated (some data not shown). Each clone contained a different stretch of 10 mutated contiguous nucleotides within the 286-bp minimal promoter (from -210 to +76) (Fig. 3). Transfection of the majority of mutant clones in Caco-2 and HT-29 cells had a level of activity that was similar to the nonmutated wild-type clone (data not shown). The M2 region corresponded to the TATA box (-26 to -30), and the promoter activity of the M2 clone was significantly lower than the wild-type vector, indicating the important role of general transcription factors in controlling promoter activity (data not shown).

The clone contained a mutation in the M4 region, which increased promoter activity fivefold in Caco-2 cells (Fig. 4B). Probe A, which spans -22 to -65, was used to assess this region for DNA-protein complexes in the M4 region. For the sake of clarity, the EMSA performed in Fig. 4C was with a form of probe A (M4) that contains a mutation in the M4 region (-40 to -49) (Fig. 4A). EMSA with the mutant oligonucleotide resulted in three specific complexes (Fig. 4C, A1-A3, lanes 1 and 2). However, when competed with the wild-type oligonucleotide (Fig. 4C, lane 3), only A2 and A3 competed for binding, suggesting that A1 is unique to the M4 probe. The remaining competition studies (Fig. 4C, lanes 4-8) were consistent with the hypothesis that the mutation in the M4 region creates an actual E-box. Supershift studies using USF1 and USF2 antiserum confirmed that A1 consists of USF1 and USF2 (Fig. 4C, lanes 10-15). Overall, these data are consistent with the hypothesis that the M4 mutation creates an E-box (CACGTG; Fig. 4A) and therefore the induction of promoter activity with the M4 clone (Fig. 4B) was not secondary to a loss of a repressor element, but instead the introduction of an activator. A2 and A3 are specific to the wild-type oligonucleotide; however, their identity and the location at which they bind probe A have not been elucidated.


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Fig. 4.   Transient transfection and electrophoretic mobility shift assay (EMSA) of the probe A region. A: the probe used for EMSA was M4, which contains the M4 mutation and spans -22 to -65. The rectangle highlights the location of the E-box (CACGTG) introduced by the M4 mutation (inner rectangle). The locations of the nucleotides altered by the M4 mutation are indicated above the large rectangle. B: Caco-2 cells were transiently transfected with either the wild-type (WT; hPR-210/+76) or M4 clone, and luciferase activity was measured 48 h later. Data are displayed as fold elevation over hPR -210/+76 wild type. C: competition was performed with several duplex oligonucleotides that were either wild type or mutants (lanes 2-8). Specifically, the Ba oligonucleotide corresponds to probe B (-74 to -35) and contains a mutation in the E-box (-71 to -66). In contrast, the Bb oligonucleotide contains a mutation in the putative IRS element (-61 to -53). The A' oligonucleotide scans -79 to -60, while the A" oligonucleotide includes bases -59 to -29. Antiserum to upstream stimulatory factor-1 (USF1) and -2 (USF2), E47, or nonreactive serum (NRS) was used to perform supershift experiments (lanes 11-15).

Clones containing mutations in the M6 and M7 region have promoter activities that are <10% of the wild type (Fig. 5B). Probe B (-74 to -35) is located in the region of M6 and M7 and in the presence of nuclear extracts forms two large prominent and specific complexes (B1 and B2) and a faster migrating complex of much lower intensity (B3; Fig. 5C, lanes 1-4). To determine the precise location of the DNA-protein complex, we performed EMSA competition experiments. The sequence of the most critical region of probe B is displayed in Fig. 5A (the location of the nucleotides mutated in the competition experiment is given by the lowercase letter bracketed by two parallel lines). Competition experiments with oligonucleotides (Ba) containing mutations in the putative E-box (-71 to -66) failed to bind to the proteins that form the three complexes (Fig. 5C, lanes 5-7), and supershift studies (Fig. 5C, lanes 16-21) confirmed that the complexes represent USF1 and USF2 homo- and heterodimers. Competition with the oligonucleotide (Bb) that contains a mutation within the previously described IRS region demonstrates that this region fails to form complexes (Fig. 5C, lanes 8-10). Both complexes were equally abundant in nuclear extracts isolated from pre- and postconfluent Caco-2 cells (Fig. 5C, lanes 12-15). These data suggest that the bHLH proteins USF1 and USF2 are equally abundant in pre- and postconfluent cells and that they form the B1 and B2 complexes that activate basal expression of the immediate pIgR promoter region.


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Fig. 5.   Transient transfection and EMSA of probe B region. A: nucleotides essential for binding nuclear protein are depicted within the rectangular box with the outer nucleotides numbered. The locations of the M6 and M7 mutations are shown above the rectangle, while those in the oligonucleotides used in the EMSA are displayed below the rectangle. All oligonucleotides were annealed to an antisense counterpart (sequence not shown). B: Caco-2 cells were transiently transfected with M6, M7, or wild-type clones, and luciferase activity was measured 48 h later. C: crude Caco-2 nuclear extracts and probe A were used for lanes 1-11. Competition experiments were performed with 50-, 100-, and 250-fold excess (indicated by triangle) of either cold wild-type probe B (lanes 2-4) or mutant clones Ba (lanes 5-7), Bb (lanes 8-10) or unrelated U (lane 11). The arrows indicate the location of the 3 specific complexes (B1 to B3). In lanes 12-15, nuclear extracts were isolated from Caco-2 cells either 2 (2d) or 10 days (10d) after seeding. Supershift studies were performed with either or both anti-USF1 or USF2 antiserum (lanes 17-20) or nonreactive serum (lane 21). D: either the wild-type clone (hPR-210/+76) or mutant M6 clones were cotransfected with various concentrations of the USF1, USF2, USF1 dominant negative (USF1 DN), and USF2 DN expression vectors. Luciferase activity was measured 48 h later with data shown relative to the promoter activity of the wild-type vector transfected in the absence of expression vectors.

Transactivation with expression vectors containing USF1 and USF2 augmented promoter activity of the wild-type hpIgR -210/+76 vector in a dose-dependent manner, while the dominant negative (DN) constructs significantly diminished activity (Fig. 5D). Transfection of the USF1 and USF2 expression vectors failed to alter promoter activity of the M6 clone, indicating that the sole E-box in the promoter resides between nt -71 and -66.

Probe C spans nt -122 to -82 and forms three relatively small complexes (C1-C3) with Caco-2 nuclear extracts (Fig. 6C). Competition with duplex oligonucleotides that contained specific mutations in the Cf to Ci regions (nt -104 and -93) was capable of only partially binding to the three complexes (Fig. 6C, lanes 9-12; Fig. 6A). Mutagenesis of the region that disrupts the upstream portion of this complex (M10) resulted in a threefold increase in promoter activity, whereas mutation of the M9 region failed to influence reporter activity (Fig. 6B). Although the element resembles an interferon-stimulated response element, it contains several sequence polymorphisms that would interfere with its ability to interact with the IRF family of proteins (14). In fact, anti-IRF-1 antibodies failed to supershift the complexes (data not shown). To determine whether the abundance of the C1 -C3 complexes varied by the stage of Caco-2 cell confluency, we isolated nuclear extracts 2 (subconfluent) and 10 (postconfluent) days after the cells were seeded on plastic plates. The complexes were significantly more abundant in nuclear extracts isolated from postconfluent vs. subconfluent cells (Fig. 6C, lanes 17-20). Because transient transfection analysis is limited to subconfluent dividing cells, the role of a transcription factor that is primarily expressed in the postconfluent state may be indiscernible by this analysis. These data were interpreted to suggest that the proteins forming C1 -C3 have a distinct repressor role and are more abundant in quiescent compared with proliferating Caco-2 cells.


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Fig. 6.   Transient transfection and EMSA of probe C region. A: the locations of the nucleotides mutated in each oligonucleotide are shown below as lowercase letters, and those of the M10 and M9 clone are shown above the rectangle. B: promoter activity was assessed in Caco-2 cells by transiently transfecting M9, M10, or wild-type clones and measuring luciferase activity. C: competition with 100-fold excess of duplex oligonucleotides of probes C (lane 2) or D (lane 3) or containing mutations in various regions (Ca, Cl) (lanes 4 and 15). Competition with unrelated duplex oligonucleotides is shown in lane 16. In lanes 17 to 20, nuclear extracts were isolated from Caco-2 cells either 2 or 10 days after seeding. The arrows indicate the location of the 3 complexes.

Probe D is composed of nt -137 to -107 and by EMSA forms two vastly different size complexes (Fig. 7C, lanes 1-5). In competition experiments, the upper complex (D1) failed to be competed by duplex oligonucleotides containing mutations at positions Dc, Dd, Df, Dg, and Dh (Fig. 7C, lanes 8, 9, and 11-13). These data suggest that the protein complex forming D1 specifically interacts with DNA located between nt -130 and -114. Probes D, G, and G' specifically competed for formation of the nuclear extract forming D2 in these assays (Fig. 7C, lanes 17-20). These data indicate that D2 is identical to the complex formed with probe G that is located between nt -174 and -169 (Fig. 3, data not shown). A mutation in the M12 region disrupts both complexes (D1 and D2) and leads to a threefold increase in promoter activity (Fig. 7B). However, mutations M11 and M13 selectively prevent the formation of D1 and fail to influence basal activity (Fig. 7B). The D1 and D2 complexes were most abundant in postconfluent Caco-2 nuclear extracts and were barely detectible in rapidly dividing subconfluent cells (Fig. 7C, lanes 25-28). The cis-acting element (ATGGAG) that binds to the D2 complex appears to be unique, since it does not resemble any previously identified transcription factor binding site (10). In contrast, the upper complex (D1) is formed by a transcription factor that binds an element resembling a putative binding site (nt from -122 to -133) for C/EBP transcription factors (21). However, the protein responsible for forming the slower migrating D1 complex clearly interacts with nucleotides as far downstream as -114, making it unlikely that a member of the bZIP family of proteins forms the complex. Moreover, supershift studies with anti-C/EBP-alpha , anti-C/EBP-beta , and anti-C/EBP-gamma antisera failed to shift D1 (data not shown). Together, these data suggest that the novel D1 and D2 complexes are formed by composite factors that are present in highest abundance in quiescent cells and repress basal promoter activity of the human pIgR promoter.


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Fig. 7.   Transient transfection and EMSA of probe D region. A: the region of DNA responsible for forming the 2 complexes is contained within the 2 rectangles and the outer limits of the complexes are numbered, whereas those of the M11 to M13 clones are shown above the rectangle. B: Caco-2 cells were transiently transfected with clones M11-13 or wild-type clones, and luciferase activity was measured 2 days later. C: Caco-2 extracts identified 2 specific complexes indicated by the arrows (D1 and D2, lanes 1-5). Competition with unlabeled oligonucleotides containing mutations (Da--Dj) is specified below the sequence surrounded by a rectangle. In lanes 25-28, nuclear extracts were isolated from Caco-2 cells either 2 or 10 days after seeding.

Probe E was located just upstream of probe D between nt -150 to -132, and EMSA analysis with this probe results in two intermediate size complexes (E1 and E2; Fig. 8C). Competition with oligonucleotides containing specific mutations revealed that only the Eb-Ed oligonucleotides failed to compete for the two specific complexes, suggesting that the nuclear extract interacts with nucleotides located between -144 and -136 (Fig. 8C, lanes 13-15). Whereas the abundance of E1 and E2 was barely detectable in nuclear extracts isolated from the dividing, subconfluent cells, levels were significantly higher in postconfluent cells (Fig. 8C, lanes 18-21). The relative absence of these complexes in nuclear extracts isolated from dividing Caco-2 cells may explain why site-directed mutagenesis of this region (M13 and M14) failed to alter basal promoter activity (Fig. 8B). The protein(s) that forms these complexes appears to weakly resemble a CRE (10); however, anti-CREB antibodies fail to supershift the complex, indicating that a transcription factor other then CREB forms the complex (data not shown).


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Fig. 8.   Transient transfection and EMSA of probe E region. A: the region of DNA responsible for forming the 2 complexes is contained within the rectangle and the outer limits of the complexes are numbered, whereas those of the M13 and M14 clones are shown above the rectangle. B: promoter activity was assessed in Caco-2 cells by transiently transfecting M14, M13, or wild-type clones. C: two specific complexes (E1 and E2) are indicated by arrows, and the location of the nucleotides responsible for a rectangular box surrounds protein interactions. Competition experiments were performed with 100-fold excess of either wild-type (lanes 2-8, 10, 11) or mutant (lanes 12-16) duplex oligonucleotides. In lanes 18-21, nuclear extracts were isolated from Caco-2 cells either 2 or 10 days after seeding.

Probe F is a duplex probe composed of nt -171 to -142, and EMSA with Caco-2 nuclear extracts formed both large and small complexes (F1 and F2) that were competed by an excess of unlabeled probe F (Fig. 9C, lanes 2 and 3) but not by an unrelated double-stranded oligonucleotide (Fig. 9C, lane 9). Competition with unlabeled oligonucleotides that contained mutations in the Fa-Fd region (Fig. 9C, lanes 4-7) failed to compete for F1, indicating the location of the nucleotides (-165 to -154) that interact with the nuclear extracts. Both complexes were slightly more abundant in nuclear extracts isolated from postconfluent Caco-2 cells (Fig. 9C, lanes 14-17). Site-directed mutagenesis of this element results in a barely detectable 40% increase in promoter activity (Fig. 9B). Although this element appears to be unique, it weakly resembles cis-elements recognized by two homeobox proteins (Cdx-2 and Nkx-2.5); however, the identities of the proteins that bind to these sites have not yet been determined.


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Fig. 9.   Transient transfection and EMSA of probe F region. A: the region of DNA responsible for forming the 2 complexes is contained within the rectangle and the outer limits of the complexes are numbered, whereas those of the M15 and M16 clones are shown above the rectangle. B: Caco-2 cells were transiently transfected with the M15, M16, and wild-type clones, and luciferase activity was measured 48 h later. C: competition mobility shift assays (100-fold excess) were performed with either wild-type probes F, G', G, E (lanes 2-3, 10-13, and 15) or mutant oligonucleotides Fa-Fe (lanes 4-8) and unrelated oligonucleotide (lane 9). In lanes 14-15, nuclear extracts were isolated from Caco-2 cells either 2 or 10 days after seeding. The arrow indicates the location of the complex.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we analyzed the 5'-flanking region of the human pIgR gene using transfection studies, DNase I footprint analysis, and DNA binding assays. Transfection into both intestinal (Caco-2/HT-29) and the nonexpressing CHO cell lines with the human pIgR-210/+76 promoter regions reveals the specificity of the activity of the promoter (Fig. 1). DNase I footprint analysis confirmed the presence of multiple footprints and hypersensitivity sites located between nt -46 and -190 relative to the start of transcription and within the minimal promoter of the gene (Fig. 2). The pattern of footprints identified in the human promoter is distinct from what we (16) have previously reported with the murine gene. Regulation of pIgR expression does differ between humans and rodents, because rodent and rabbit pIgR is expressed in hepatocytes, whereas human pIgR is expressed in the biliary epithelium (6). Moreover, expression in the intestinal epithelium of humans occurs as early as 20 wk of gestation and at 3 wk of postnatal life in rodents (20).

We employed an unbiased approach (scanning mutagenesis) to determine the regions of the core promoter that influence its activity. One drawback to such an approach is that the mutations can create an active region by introducing a cis-element. Such was the case with the M4 mutant, which increased promoter activity not by the removal of a repressor element, but instead by the introduction of an active E-box, which binds USF1 and USF2 and augments promoter activity (Fig. 4).

Probe B overlaps with both an E-box (-71/-66) and the IRS element (-65/-50), and competition studies shown in Fig. 5 demonstrate that the three complexes (B1 to B3) interact with the putative E-box. In an analysis of the human pIgR promoter, Johansen et al. (11) demonstrated the importance of the E-box in conferring basal expression in AGS epithelial cells. Similarly, we (16) demonstrated that the E-box located in a similar region of the murine gene was also essential for driving basal promoter activity in Caco-2 cells. The role of the leucine zipper proteins (bHLH/LZ) USF1 and USF2 in augmenting basal expression of the murine pIgR gene was confirmed by supershift and overexpression assays using both wild-type and their corresponding DN vectors. Similar studies were performed with the human core promoter and confirm the role of USF1 and USF2 in transactivating the basal expression of the pIgR gene (Fig. 5D).

Transfection of a clone (M5) that disrupts the IRS site (-65/-50) failed to significantly alter transcriptional activity (Fig. 3 and data not shown). A similar mutation in the murine promoter results in a sixfold elevation of promoter activity compared with that of the wild-type vector (16). Three base pair mutations covering the entire IRS element (including an additional 7 nt downstream) augmented basal expression in Caco-2 cells (Ref. 16 and unpublished data). However, in the human promoter transfection of the M5 clone only increased activity by 50% (data not shown). Despite developing several overlapping oligonucleotides, we have not been able to consistently detect a specific complex that binds to the IRS region (data not shown). In contrast, Johansen et al. (11) identified a complex that they attributed to binding at the IRS; however, mutation of the site failed to alter transcriptional activity in AGS cells and did not disrupt complex formation. Johansen et al. (11) speculated that their failure to alter promoter activity might have occurred because they introduced transition mutations, which may have been insufficient to completely disrupt binding or alter transcriptional activity. In the current study, we altered the IRS by substituting with transversion mutations, yet these mutations still failed to change transcriptional activity, raising the possibility that this site is only transcriptionally active in the murine promoter (16).

The C complexes bind to a DNA element that is an insertion in the human promoter and not present in the murine gene (Fig. 3). Mutagenesis of this region suggests that the proteins that form the C1 -C3 complexes have a role in repressing basal promoter activity (Fig. 6). The EMSA competition studies indicate that mutations of nt -107 to -99 (region e-g) most substantially altered the formation of the C3 complex, and these data are most consistent with the transfection data demonstrating that the promoter activity of the M10 clone was more significantly altered compared with M9. Because mutations located in M9 should disrupt binding of the C1 and C2 complexes, the transfection data indicate that these two complexes may not alter promoter activity. Although the transfection data clearly suggest that the alterations in the upstream portion (M10) of the element most dramatically influence promoter activity, the three C complexes are not as abundant in nuclear extracts of subconfluent Caco-2 cells. Transient transfection studies performed in this study occurred in subconfluent, dividing cells. Therefore, it is possible that the magnitude of the C complexes' influence on pIgR promoter activity is more significant in differentiated, nondividing cells than can be measured by standard transfection assays.

Piskurich et al. (25) previously studied this region of the human pIgR promoter and determined that the complexes interacting in this area failed to be supershifted by antiserum to IRF-1, IRF-2, interferon-stimulated gene factor-3, and STAT1. However, the influence of this element on basal promoter expression was not evaluated, and the single complex detected previously does not resemble the complex detected in our analysis.

Through sequence homology, Piskurich et al. (25) also identified another element that resembles an ISRE located between nt -128 and -138. This putative element spans the complexes identified with probes D and E. The previous analysis (25) of this region may have not detected D1-D2 or E1-E2 because the studies did not use a probe of sufficient length and the protein responsible for the D1 and D2 complex is found in low concentrations in HT-29 nuclear extracts (data not shown). Nevertheless, this element is clearly not binding members of the C/EBP family of proteins, since antiserum toward C/EBP-alpha , C/EBP-beta , and C/EBP-gamma fails to supershift the complex (21).

The faster migrating complex (D2) formed with probe D interacts with the ATGGAG element located from -125 to -120 and does not resemble any previously described cis-element (Fig. 7A). An identical element located from -173 to -168 was identified with probe G, which itself was capable of competing for binding of the nuclear extract with probe D (Fig. 7C, lane 20). In Fig. 7, competition with an oligonucleotide containing a transversion mutation at location Df could only partially compete for binding to the complex (Fig. 7, lane 11). Similar results were obtained with probe G; however, when transition mutations were substituted (GAG to AGA) the probe failed to compete for the complex (data not shown). Whereas mutations (M11 and M13) that selectively prevent the formation of the upper complex with probe D only marginally increase basal expression, the M12 clone that results in a disruption of both complexes increases promoter activity more than threefold (Fig. 7A). Therefore, the significant upregulation of basal expression that occurs with the clones containing mutations that disrupt both complexes suggests that the protein(s) that binds to these elements functions in a cooperative manner to repress promoter activity. Because the D complexes are most abundant in postconfluent cells, their influence in controlling pIgR expression may be more dramatic in quiescent cells.

The most abundant complex that we identified in this study was the E complex, which also formed a very specific interaction with the identified cis-element (Fig. 8C). However, transfection studies failed to identify a role for this element in controlling pIgR expression (Fig. 8A). Nuclear extracts from subconfluent cells contained only a marginal amount of the complex and may have made it difficult to discern whether this complex has a positive or negative influence on pIgR promoter activity in the nondividing state. Of all the complexes identified in this study, only USF1 and USF2, which bind to the E box and augment promoter activity, are expressed in similar concentrations in pre- and postconfluent cells (Fig. 5C, lanes 12-15). Interestingly, pIgR expression in the intestine is most abundant in the less well-differentiated cells of the crypt and is decreased in the mature cells of the villus (2, 3, 31). Dai et al. (5) and Rottman and Gordon (26) have argued that subconfluent in vitro cells resemble cells in the intestinal crypt, whereas postconfluent cells are a more appropriate model for the differentiated and quiescent cells that populate the villus. Because complexes C and D appear to have an inhibitory effect on pIgR promoter activity, their role in the intestinal villus may be to diminish pIgR expression in vivo.

Site-directed mutagenesis of the element that forms the F complexes (TCCCAAGTAACA) results in a marginal 40% enhancement of basal promoter activity (Fig. 9B). Although this element appears to be unique, it resembles cis-elements recognized by two homeobox-containing proteins, Cdx-2 and Nkx-2.5 (9, 22). Whereas expression of Nkx-2.5 is limited to cardiac mesoderm and is essential for cardiac development, its counterpart, Nkx-2.3, is also expressed in gut mesenchyme during early development (22). Targeted disruption of the Nkx-2.3 gene results in early postnatal death and a marked reduction in the proliferation of epithelial cells of the intestinal villus (23). However, the element identified with probe F does not have a well-preserved Cdx-2 core element (ATTTACA), which is believed to be critical for its function (9). Nevertheless, Cdx-2 plays a critical role in controlling basal expression of several genes that are developmentally regulated in the small intestine, including sucrase-isomaltase, lactase-phlorizin hydrolase, and apolipoprotein B (9). While the murine pIgR gene is also developmentally regulated in a similar pattern to both sucrase-isomaltase and the apolipoprotein B genes, with the exception of the E-box, the murine promoter does not appear to contain any of the active elements identified in the human gene (11). The precise identification of the various protein complexes that bind to these distinct regions in the human promoter and their role in controlling basal and stimulated activity await further analysis.


    ACKNOWLEDGEMENTS

We thank Drs. M. Sawadogo and H. Towle for providing the USF1, USF2, USF1DN, and USF2DN expression clones.


    FOOTNOTES

This study was supported by National Institute of Child Health and Human Development Grant HD-34706 and Crohn's and Colitis Foundation of America Grant 016714.

Address for reprint requests and other correspondence: M. G. Martín, Dept. of Pediatrics, Division of Gastroenterology and Nutrition, School of Medicine, Univ. of California, 10833 Le Conte Ave., 12-383 MDCC, Los Angeles, CA 90095 (E-mail: mmartin{at}mednet.ucla.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.

March 20, 2002;10.1152/ajpgi.00420.2001

Received 28 September 2001; accepted in final form 14 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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