1 Canadian Institutes of Health Research Group in Lung Development, Lung Biology Programme, Research Institute, Hospital for Sick Children, and Departments of 2 Paediatrics and 3 Physiology, University of Toronto, Toronto, Ontario M5G 1X8, Canada
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ABSTRACT |
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The
amiloride-sensitive epithelial Na+ channel (ENaC), found in
the apical membrane of Na+-absorptive epithelia, is made up
of three differentially regulated subunits: ,
, and
. We
undertook a study of the 5'-end of the gene encoding the
-ENaC
subunit in the rat. 5'-Rapid amplification of cDNA ends and RNase
protection assays indicated multiple transcription start sites over a
50-bp region. Sequencing 1.3 kb of the 5'-flanking DNA revealed
putative binding sites for PEA3, Sp1, activator protein (AP)-1 and
Oct-1 but neither a TATA box nor consensus sites for steroid hormone
receptor binding. Transient transfections of reporter constructs driven
by
-ENaC 5'-flanking DNA in the representative epithelial cell lines
Madin-Darby canine kidney, MLE-15, and Caco-2 revealed a negative
element present between positions
424 and
311 that affected basal
transcription rates. Gel shift assays showed protein-DNA binding
activity of an AP-1 consensus site in this region; however, mutation of
the AP-1 site did not abrogate the repressive activity of the region in
transient transfections. Deletion of two clusters of Sp1 consensus
binding sites between
1 and
51 bp and between
169 and
211 bp
indicated that the proximal cluster was essential to basal promoter
activity in transfected cell lines. In a comparison of these data with
those in published studies on
- and
-ENaC promoters, the
- and
-subunit promoters appear to be more similar to each other than to
the
-promoter.
ion transport; Sp1; transcription; gene expression
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INTRODUCTION |
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THE EPITHELIAL SODIUM
CHANNEL (ENaC) is an amiloride-sensitive
Na+-permeant ion channel that is found in the apical
membrane of most absorptive epithelia (reviewed in Ref.
13). The ENaC was cloned from the distal colon of
salt-deprived rats and was found to contain three homologous subunits:
,
, and
(6, 7). The channel is expressed in the
distal nephron of the kidney, lung alveoli and airways, distal colon,
skin, salivary and sweat gland ducts, and taste buds (13).
In all of these organs and tissues, the channel has a significant role
in salt and fluid homeostasis. All three ENaC subunits are required for
maximal channel activity in Xenopus oocyte expression
systems (7).
In the kidney, the ENaC contributes to electrolyte balance and is thus
involved in blood pressure control. Loss-of-function mutations cause
the genetic hypotensive syndrome pseudohypoaldosteronism type I
(9, 14), whereas gain-of-function mutations result in an
inherited form of hypertension known as Liddle's syndrome (5,
15, 34). Genetic knockouts of the - or
-subunit of the
mouse ENaC resulted in severe defects in renal Na+ and
K+ transport, leading to death from hyperkalemia within 2 days after birth (4, 24). In the lung, active
Na+ transport across the epithelium helps both in the
reabsorption of fetal lung fluid at birth and in keeping the adult
alveolar spaces free of fluid (23). Pharmacological
inhibition of Na+ channels slowed the clearance of
fetal lung fluid after birth (25), whereas knockout of the
-subunit resulted in mice that were unable to clear their fetal lung
liquid and died from respiratory distress shortly after birth
(16). Pseudohypoaldosteronism type I is not associated
with respiratory distress at birth, but patients have minimal or absent
Na+ absorption from airway surfaces and a volume of airway
surface liquid more than twice the normal value (17). In
the distal colon, the ENaC is expressed in surface epithelial cells
(but not in crypts) and has not been detected in any other part of the
intestine (11, 30). Under basal conditions, only
-subunit mRNA is expressed at a detectable level in the colon,
whereas the
- and
-subunits are induced with exposure to
mineralocorticoid (20, 30).
ENaC mRNA concentrations are subunit and tissue specific during fetal
development and adult life. Promoter studies of the ENaC genes have so
far been reported for the - and
-subunits of both rats and humans
(2, 10, 27, 32, 37, 38). The three
-subunit studies
describing the rat and human genes (10, 27, 32) reported
the absence of a TATA box and the presence of GC boxes near the
transcription start site. Studies of both the human and rat
-subunits (2, 37) also reported the absence of a TATA
box in their promoters and the presence of GC boxes (Sp1 consensus
sites) near their transcription start sites. In one study
(2), deletion of the two GC boxes of the human
-ENaC
severely compromised promoter activity. ENaC subunits display
tissue-specific responses to glucocorticoid stimulation; glucocorticoid
stimulation of adrenalectomized animals produces an increase in mRNA
levels of only the
-subunit in the kidney and lung, whereas in the
colon, only the
- and
-subunit mRNA levels are increased
(1, 8, 12, 22, 30, 35); transient transfections with
-
and
-ENaC promoters have identified an active glucocorticoid
response element (GRE) among the upstream promoter elements in the
-ENaC promoter (10) but not in the
-ENaC promoter
(37).
The genomic organization of the - and
-ENaC genes from both rats
and humans have been reported along with initial characterizations of
their promoters (2, 10, 21, 27, 32, 37, 38). The
organization of the gene encoding the human
-ENaC has been reported
(31) and is highly homologous to the other two subunits. We present here the first report describing the sequence and
transcriptional activity of the
-ENaC promoter, using a clone
isolated from a Sprague-Dawley rat genomic DNA library.
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MATERIALS AND METHODS |
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Cell culture.
Madin-Darby canine kidney (MDCK) epithelial cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum (FBS). Human colon adenocarcinoma cells (Caco-2) were maintained
in -MEM without ribonucleosides and deoxyribonucleosides and with
20% FBS. Mouse distal lung epithelial cells (MLE-15) were maintained
in HITES medium with 2% FBS. HITES medium consists of RPMI 1640 medium
supplemented with 5 µg/ml of insulin, 10 µg/ml of transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM
-estradiol, 10 mM
HEPES, and 2 mM L-glutamine. MLE-15 cells were a gift from
Dr. J. Whitsett (Children's Hospital Medical Center, Cincinnati,
OH); MDCK and Caco-2 cell lines were obtained from American
Type Culture Collection (Manassas, VA). All lines were maintained in
the presence of 100 U/ml of penicillin G and 100 µg/ml of
streptomycin sulfate.
Rapid amplification of cDNA ends.
Rat total RNA was isolated from whole kidneys with TRIzol reagent (Life
Technologies, Burlington, ON). RNA ligation-mediated (RLM) rapid
amplification of cDNA ends (RACE) was used to amplify cDNAs from the
rat -rENaC (
-rENaC) mRNA (First Choice RLM-RACE kit, Ambion,
Austin, TX). The manufacturer's protocol was followed with the
gene-specific primer
sp4 (5'-GGCTGGACTTCGGAGCCAGAATCT-3') and the
kit's outer adapter primer. The amplified cDNAs were cloned into
pGEM-T easy for sequence analysis of multiple subclones.
Library screening.
A commercial rat genomic DNA library from kidney cells of a 16-mo-old
male Sprague-Dawley rat in a Lambda FIX II vector (Stratagene, La
Jolla, CA) was screened for genomic sequences encoding exon I and the
5'-flanking region of -rENaC. A probe specific to the 5'-end of the
-rENaC gene was generated by PCR with a
-rENaC RACE product as a
template.
-Recombinants (1.0 × 106) were
screened as previously described (27).
RNase protection assay.
Due to the small size of exon I, the DNA template for the RNA probe was
constructed by joining genomic and cDNA sequences (see Fig.
2A). The genomic fragment was amplified with primers LEI
[5'-CTGCTCCAATGTGCAGTGATGGCAGCTAA-3'; nucleotides (nt) +91 to +63],
and
RPAG5' (5'-CCCAAGCTTAGTCCCCTGTCGTTGCTTT-3'; nt
239 to
221), and a
-clone containing exon I of
-rENaC as a
template. The cDNA fragment was amplified with primers
5'EI
(5'-ACCACCTTAGCTGCCATC-3'; nt +57 to +74) and
RPAcDNA3'
(5'-CCCCTCGAGATGCGTTTGGGGCCGTGTGT-3'; nt +233 to
+214), with a 5'-RACE product as a template. (Underlined sequences represent nonhomologous regions, including restriction enzyme
recognition sites for subcloning.) The full-length RNase protection
assay (RPA) template was generated by PCR from the overlapping cDNA and
genomic PCR products. The 490-bp product was digested with
MspI, blunted, and digested with XhoI; the
resulting 279-bp fragment was cloned into pBluescript II KS(
); and
the sequence was verified. This DNA template was transcribed in vitro following standard protocols (3) with 8,000 µCi/mmol (10 µCi/µl) of [
-32P]CTP (ICN) and T3 RNA polymerase
(Ambion). The resulting transcript is 309 bp in length, of which 266 bp
are homologous to
-rENaC. The probe was gel purified before RPA with
the RPA III kit (Ambion) as per the manufacturer's instructions. Rat
total RNA was isolated from primary cultures of fetal distal lung
epithelial (FDLE) cells, whole lungs, and whole kidneys with TRIzol
reagent (Life Technologies). Protected fragments were analyzed with 8%
PAGE followed by autoradiography with X-OMAT film (Kodak, Rochester, NY).
Sequencing.
All manual sequencing was carried out with the T7Sequencing
kit (Amersham Pharmacia Biotech) and deoxyadenosine
5'-[-35S]thiotriphosphate following the
manufacturer's protocol. Automated sequencing was carried out with the
ThermoSequenase fluorescent-labeled primer cycle-sequencing kit with
7-deaza-dGTP (Amersham Pharmacia Biotech) and the M13Rev and
M13Fwd(-38) primers (LI-COR, Lincoln, NE) following the manufacturer's
protocol. Reactions were run on the LI-COR DNA sequencer model 4000L,
and data were collected with the software LI-COR BaseImagIR data
collection version 4.
-rENaC promoter sequence analysis.
To locate consensus transcription factor binding sites,
-rENaC
5'-flanking DNA was analyzed with FINDPATTERNS (Wisconsin sequence
analysis package) against the TFSITES database and the MatInspector
version 2.2 search engine (28)
(http://transfac. gbf.de/cgi-bin/matSearch/matsearch2.pl).
Reporter constructs.
Cloned genomic DNA fragments containing portions of the putative
-rENaC promoter were inserted upstream of the secreted alkaline phosphatase (SEAP) gene in the promoterless expression vector pSEAP2-Basic (Clontech, Palo Alto, CA). A combination of naturally occurring restriction sites and fragments generated by PCR were used to
assemble reporter constructs (see Fig. 4A).
Transient transfection.
MDCK, MLE-15, and Caco-2 cells were seeded on Costar six-well plates
(Corning, Corning, NY) and transfected with LipofectAMINE (Life
Technologies). SEAP2 reporter constructs were cotransfected with a Rous
sarcoma virus (RSV) promoter-driven -galactosidase (
gal)
expression vector (RSV
gal; an internal control for transfection efficiency). Transfection, harvesting, and SEAP and
gal assays were
carried out as previously described (27). For assays with the pGL3 reporters, the pGL3 construct was cotransfected with the
internal control plasmid pRL-TK (Promega). Firefly and
Renilla luciferase activities were measured with the
dual-luciferase reporter assay kit (Promega).
DNA mobility shift assay.
A variety of double-strand DNA fragments derived from the 424- to
311-bp region of the
-rENaC promoter was used to attempt to
identify the nuclear proteins binding to this region. These included a
34-bp oligonucleotide (nt
343 to
310; designated GSAL3) and an
18-bp double-strand DNA fragment (nt
347 to
330; designated neg
)
with a putative AP-1 consensus sequence. These probes were created by
annealing complementary in vitro synthesized oligonucleotides. Standard
protocols for mobility shift DNA-binding (gel shift) assays were
followed, with nuclear extracts prepared from MDCK, MLE-15, and Caco2
cells (3). Binding reaction mixtures contained 10 mM
Tris · HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 10%
glycerol, 2 µg of poly(dI-dC), 20 µg of BSA, 0.05% Nonidet P-40, 8 µg of nuclear protein extract, and 20,000 counts/min of 32P end-labeled probe. Three antibodies were used in the
gel shift assays: 1) c-Jun/AP-1 (D) × TransCruz,
2) p-c-Jun (KM-1) × TransCruz, and 3) Oct-1
(C-21) × TransCruz (Santa Cruz Biotechnology, Santa Cruz,
CA). Binding reactions were incubated for 30 min at room temperature
followed by electrophoresis for 4 h at 80 V on 5% acrylamide
nondenaturing gels in 1.0× Tris-borate-EDTA. Dried gels were exposed
to X-OMAT film overnight.
Statistical analysis. Reporter gene activities in transiently transfected cells are presented as mean ± SE from n = 3 or 6 wells as indicated in Figs. 4-6, and the significance between the means of different groups was calculated with an analysis of variance (Instat software by GraphPad, San Diego, CA). A probability (P) of <0.05 was considered significant. All transfections were performed at least twice with triplicate wells.
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RESULTS |
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Transcription start site analysis.
To define the promoter of -rENaC, the 5'-end of the mRNA needed to
be precisely identified. This was done with RLM-RACE, a modification of
the 5'-RACE procedure designed to amplify cDNA only from full-length,
capped mRNA (Fig. 1). When RLM-RACE was performed on total RNA from rat kidneys, a single product of ~500 bp
was detected. This product was dependent on cleavage of the pyrophosphate linkage between the cap structure and the mRNA (Fig. 1A, compare lanes 1 and 2),
demonstrating its specificity to the 5'-end of the decapped mRNA. After
subcloning of the amplification mixture, 30 isolates were analyzed and
sequenced. Twenty-two subclones contained sequences homologous to
-rENaC, all of which were contiguous.
-rENaC sequences extended
to 56 nt upstream of the published cDNA sequence (GenBank accession no.
X77932). Multiple end points were detected, suggesting that multiple
transcription start sites are used within a 50-bp region (Fig.
1B). The most commonly found end point was an A residue at
position +42. The sequence surrounding this position closely matches
the consensus initiator sequence commonly found in TATA-less promoters
(19).
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Promoter sequence and cis-element consensus sequences.
Isolation of the -clone
27.1.1 and its subsequent subcloning and
Southern blotting with the exon I probe revealed a 2-kb XbaI
fragment that contained the first exon. Sequencing of this fragment
showed that it contained 1.2 kb of the 5'-flanking sequence of the
-rENaC gene. No TATA box was found, which is consistent with the
observed multiple start sites and consensus initiator sequence.
Although mRNA induction studies in the colon (12, 35)
showed that
-rENaC mRNA levels were increased by
glucocorticosteroids, no consensus GRE was found. Many other
cis-element consensus sites were observed as indicated in
Fig. 3. The sequence contained consensus AP-1 sites, PEA3 elements, Oct-1 elements, a nuclear factor-
B element, and numerous Sp1 sites.
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Characterization of -rENaC promoter activity.
The longest
-rENaC promoter constructs, which terminate 5' at
1,221 bp, and the shortest, which terminate 5' at
311 bp (Fig.
4A), were tested for
transcriptional activity in transfected cells.
-1221a and
-311a
both terminate 3' at the BstEII site at +22 bp, whereas
-1221 and
-311 extend in the 3' direction to +100 bp, covering
nearly all of exon I. It was observed that
-1221 produced
approximately threefold more activity than
-1221a and that
-311
produced three- to fivefold more activity than
-311a (Fig.
4B). This phenomenon was observed consistently in each of
the three epithelial cell lines, MDCK, MLE-15, and Caco-2. The negative
control, the empty pSEAP2-Basic vector, showed minimal activity. From
this point on, all constructs were created with the 3' terminus at +100
bp.
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Effect of -rENaC (
424- to
311-bp) element on heterologous
promoters.
The 114-bp fragment derived from the
-rENaC
424- to
311-bp
flanking DNA containing the putative negative element was tested in two heterologous promoters (Fig.
5A) in MLE-15 cells. This fragment was inserted in front of an
-rENaC promoter fragment,
548, and in front of a TK promoter. The
-rENaC (
424- to
311-bp) element suppressed activity of the
-rENaC promoter by
~25% (Fig. 5C). In contrast, the TK promoter activity was
enhanced by the
-rENaC (
424- to
311-bp) element (Fig.
5D).
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Role of AP-1 consensus element within the -rENaC (
424- to
311-bp) element.
As indicated in Fig. 3, the
424- to
311-bp region contained
putative consensus AP-1 and Oct-1 sites, both of which have previously
been reported to be able to function as repressors or activators of
transcription depending on context (18, 33, 36). Using
mobility shift experiments, we were able to demonstrate that the AP-1
element in the 114-bp
-rENaC sequence was capable of specifically
binding Jun proteins in vitro (Fig.
6A, lanes 1-13). Using antibodies directed against
Oct-1, we have not been able to demonstrate Oct-1 binding to the
putative Oct-1 consensus sequence within the
-rENaC (
424- to
311-bp) element; the major DNA-protein complexes formed with a probe
(GSAL3) encompassing both consensus sites appear to involve Jun but not
Oct-1 proteins (Fig. 6A, lanes
14-21). However, mutation of the AP-1 consensus element within the context of the
-424 promoter did not restore transcriptional activity in SEAP reporter constructs (Fig.
6B). Effectiveness of the mutation within the AP-1 consensus
element was assessed with further gel shifts; the mutated sequence
failed to compete with the wild-type sequence for binding to nuclear protein (Fig. 6C).
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Effect of deletion of Sp1 consensus elements.
The activity of TATA-less promoters is frequently dependent on Sp1
sites in the proximal promoter region. The -rENaC 5'-flanking sequence displays two clusters of Sp1 sites, at
211 to
170 and
51
to
1 bp. These sequences were deleted, singly and in combination, in
the context of the
-311 promoter and inserted into the pGL3-Basic luciferase reporter (Fig. 7A).
Transient transfections of these constructs in all three epithelial
cell lines showed that the proximal Sp1 cluster was essential for
promoter activity, whereas deletion of the distal cluster had no effect
(Fig. 7B).
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DISCUSSION |
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When RLM-RACE was used to amplify the 5'-end of -rENaC cDNAs,
only one major product was detected by gel electrophoresis. However,
sequencing of independent subclones revealed multiple start sites over
a 50-bp region, with a favored position within a consensus initiator
sequence at +42 bp. All RACE-derived sequences were contiguous with
each other, the reported cDNA sequence, and our 5'-flanking genomic
sequence (Fig. 3), with no indication of alternative splicing or
additional upstream exons. To confirm the functional existence of
multiple start sites, we performed RPA using RNA from rat tissues and
cultured rat FDLE cells. The longest protected fragment in RPA agreed
with the most upstream transcription start site predicted by RLM-RACE.
Two other clusters of protected fragments were also noted, again
indicating multiple start sites with favored positions at +21 and +42
bp. Although RPA would suggest that the +21-bp position is favored over
the +42 bp most commonly found in RLM-RACE, the sizes of these
fragments overall agree well between the two methods.
Our studies indicate that the -rENaC promoter fits the criteria of a
TATA-less promoter. In the 5'-flanking DNA that we obtained, we did not
detect a TATA box. A GC-rich area upstream from +1 bp contained
multiple Sp1 sites characteristic of a TATA-less promoter
(19). Such genes typically initiate transcription at multiple sites over a region of 20-200 bp. We detected multiple start sites over a 50-bp region (Figs. 1 and 2). Our results in Fig.
4B, in which promoter constructs ending at +22 bp exhibited significantly less activity than those extending to +100 bp, suggest that these multiple start sites make an important contribution to the
overall promoter activity.
The promoters of each of the homologous -,
-, and
-ENaC genes
share a certain degree of similarity. They all lack a TATA box but
contain multiple consensus PEA3 and Sp1 elements (2, 10, 27,
38). For example, although the three subunits are all TATA-less,
the
-subunit gene resembles the
-rENaC gene in that it also
contains a GC-rich start site containing multiple Sp1 consensus sites
(2, 38). The importance of the Sp1 sites at the
transcription start site of the human
-ENaC were investigated by
Auerbach et al. (2). Consistent with our studies
of
-ENaC (Fig. 7), transcriptional activity from a minimal human
-ENaC promoter was dependent on these sites. In
-ENaC promoters,
the Sp1 consensus elements are found considerably further upstream; their functionality has not yet been confirmed (27, 32).
It is also of interest to note that Sp1/Sp3 sites have been
demonstrated to be important for both basal and hyperoxia-induced
activity in the promoter of the
1-subunit of Na-K-ATPase
(40, 41). Transepithelial ion transport requires both
apical Na channels (ENaC) and basolateral Na pumps (Na-K-ATPase), and
thus some mechanism to coordinately regulate expression of both these
types of ion transporters may exist. Like Na-K-ATPase, ENaC mRNA
expression has also been shown to be regulated by oxygen concentration
(29), although we have not explored the role of the
-rENaC Sp1 consensus sites in this phenomenon.
Second, we detected a negative regulatory element between positions
311 and
424 bp in the
-rENaC promoter. Similarly, Auerbach et
al. (2) have shown a negative regulatory element between positions
1248 and
1525 of the human
-ENaC gene. Analysis of the
- and
-ENaC "negative" sequences indicates that they share a
number of consensus transcription factor binding sites, including AP-1
and Oct-1 sites. Inserting the
-rENaC
424- to
311-bp element into heterologous promoters suggested that it was context specific; it
was successful in suppressing an
-rENaC minimal promoter, but it
enhanced the activity of a TK promoter. This could be consistent with
Oct-1 and/or AP-1 playing a role because both have been reported to be
able to function as repressors or enhancers depending on context
(18, 33, 36). One factor that may have contributed to this
could be the fact that
-rENaC, like
-rENaC, has a TATA-less promoter, whereas the TK promoter possesses a TATA box. We have noted a
significant decrease in transcriptional activity mediated by an
upstream region of the rat
-ENaC promoter (26, 27). However, the magnitude of the effect is much less than that observed here for the
-rENaC negative element. No such negative element has
been reported in studies of the human
-ENaC promoter (10, 27,
32).
A final point of comparison lies in the steroid inducibility of the
ENaC subunits. Previous studies have shown the -ENaC promoters of
both human and rat to contain functionally active GREs fitting a
classic consensus sequence (10, 27, 32), whereas neither
human nor rat
-ENaC promoters contain a consensus GRE or respond to
dexamethasone in such experiments (37, 38). No
consensus GREs were found in the 1,221 bp of the 5'-flanking DNA of the
-rENaC gene we examined, although one may exist further upstream.
Overall, the data described here for the -rENaC promoter (Sp1 sites
surrounding the transcription start, presence of an upstream negative
element, and absence of classic consensus GREs) suggest that the
- and
-subunits may be coordinately regulated. In contrast, although the
-subunit promoter is also a TATA-less promoter, its Sp1
sites are located further upstream, it does not appear to contain a
strong negative element upstream, and it does contain classic,
consensus GREs. We suggest that the
- and
-subunit genes are more
similar to each other than they are to the
-subunit gene and may
share a common mechanism of steroid induction in the colon, distinct
from that operational on
-ENaC in kidneys and lungs. In this light,
it is of interest to note the observation that the
- and
-ENaC
genes are tightly linked in both humans and rodents; Voilley et al.
(39) found that the human
- and
-subunit genes were
found within the same 400-kb genomic fragment. This tight linkage
indicates that these genes are physically close enough to theoretically
physically share regulatory DNA elements. A more extensive
characterization of this region, to finely map regulatory elements
relative to each of the subunit genes, will be necessary to prove such
a hypothesis. The work in our present report, representing the first
description of the
-rENaC promoter region, forms a starting point
for such studies.
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ACKNOWLEDGEMENTS |
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For advice and technical assistance, we thank Brent Steer, Bijan
Rafii, Christopher Fladd, Yanxia Wen, and Vicky Hannam. We also
acknowledge Dr. B. Rossier for rat epithelial sodium channel -subunit cDNA, Dr. J. Whitsett for the MLE-15 cell line, and Dr. V. Giguere for the Rous sarcoma virus
-galactosidase construct and the
thymidine kinase promoter.
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FOOTNOTES |
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This work was supported by the Canadian Institutes of Health Research Group in Lung Development (Project 8).
Address for reprint requests and other correspondence: G. Otulakowski, Lung Biology Research, Hospital for Sick Children Research Institute, 555 University Ave., Toronto, Ontario M5G 1X8, Canada (E-mail: gotulak{at}sickkids.on.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 March 2001; accepted in final form 15 August 2001.
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