1 Division of Gastroenterology
and Nutrition, 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 (
mucosa; transcription; immunoglobulin; upstream stimulatory factor; secretory component
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- 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 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 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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(IFN-
) 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
phage clones
from a mouse 129SVJ genomic library (Stratagene, La Jolla, CA) (24). In
this study, we used a clone previously identified as PR-
-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-
-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.
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 [
-35S]dATP and
Sequenase 2 (U. S. Biochemical, Cleveland, OH).
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.
View larger version (72K):
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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 ( ) 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 C, G
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.
|
Cotransfection studies using various mammalian expression vectors.
The DNA construct containing the minimal promoter
(mpIgR110/+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 PrimePreparation 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 atEMSA.
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
[-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.
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 [-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.
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RESULTS |
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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-
B (NF-
B) site
(
237/
227), one IFN-
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 HNF3
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.
|
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
mpIgR4730/+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.
|
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
(mpIgR472/+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).
|
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.
|
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).
|
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
mpIgR110/+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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DISCUSSION |
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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 CEBP
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'-
CACG
-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).
|
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- 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--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-
. However, no substitution mutation analysis was
performed to further characterize the relative contribution of each of
these sites to IFN-
-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.
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ACKNOWLEDGEMENTS |
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We thank S. Smale for helpful discussions.
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FOOTNOTES |
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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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