(Received for publication, January 18, 1996; and in revised form, February 7, 1996)
From the
The murine Nkcc2/Slc12a1 gene encodes a
bumetanide-sensitive Na-K-Cl cotransporter that is expressed
exclusively in the kidney in the thick ascending limb of the loop of
Henle. Nuclear run-off assays demonstrated that kidney-specific
expression of Nkcc2 was due, at least in part, to
kidney-specific gene transcription. To begin study of the gene
promoter, a genomic clone that contained 13.5 kilobases of the
5`-flanking region of Nkcc2 was isolated. A single
transcription initiation site was located 1330 base pairs (bp) upstream
of the start codon. The sequence of the proximal 5`-flanking region
contained typical eukaryotic promoter elements including a TATA box,
two CCAAT boxes, and an initiator. A
(G-A)(C-T)
microsatellite and consensus
binding sites for hepatocyte nuclear factor 1, cAMP-response element
binding protein, CCAAT/enhancer-binding proteins, and basic
helix-loop-helix proteins, were also identified. To functionally
express the promoter, 2255 bp of the proximal 5`-flanking region was
ligated to a luciferase reporter gene and transfected into thick
ascending limb (TAL) cells, a stable cell line derived from
microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells
exhibited furosemide-sensitive Na-K(NH
)-Cl
cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater
following transfection into TAL cells compared with transfection into
cells that did not express Nkcc2 (NIH 3T3 fibroblasts).
Deletion analysis revealed that promoter activity in TAL cells was
similar in constructs extending from the transcription initiation site
to -1529 to -469, whereas further deletion to -190
resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory
elements required for maximal promoter activity are located in a 280-bp
DNA segment that contains consensus binding sites for several
transcription factors expressed in the kidney.
Relatively little is known regarding molecular mechanisms of
tissue-specific gene expression in the kidney, particularly when
compared with other organs such as the liver. Recently, cDNAs encoding
several proteins that are produced exclusively in the kidney have been
cloned including Tamm-Horsfall protein (THP), ()aquaporin-2
water channel, V
vasopressin receptor, ClC-K1 chloride
channel, vacuolar H
-ATPase B1 subunit, cysteine
conjugate
-lyase, vitamin D
hydroxylase-associated
protein, and kidney-specific cadherin. Also, promoters of the THP,
aquaporin-2, and V
vasopressin receptor genes have been
cloned and characterized(1, 2, 3) . However,
to date, no enhancer elements or kidney-specific transcription factors
that are responsible for tissue-specific expression of these genes have
been identified.
We have examined the murine renal Na-K-Cl
cotransporter gene (Nkcc2) as a model for kidney-specific gene
expression. The Na-K-Cl cotransporters are a family of integral plasma
membrane proteins that mediate coupled transport of
Na, K
, and Cl
with
a stoichiometry of 1:1:2 and are characteristically sensitive to
inhibition by loop diuretics such as furosemide and
bumetanide(4) . cDNAs that encode two distinct isoforms of
bumetanide-sensitive Na-K-Cl cotransporters, termed NKCC1 and NKCC2,
have been cloned. NKCC1 (also called BSC2) has been cloned from the
shark rectal gland, mouse inner medullary collecting duct (mIMCD-3)
cells, and human colonic carcinoma (T84)
cells(5, 6, 7) . The Nkcc1 gene is
expressed as a 6.5-7.4-kb transcript in many epithelial and
nonepithelial tissues including colon, kidney, lung, stomach, brain,
skeletal muscle, heart, and salivary
gland(5, 6, 7) . In secretory epithelia, such
as the shark rectal gland, the Nkcc1 gene product is a 195-kDa
protein that is localized exclusively in the basolateral membrane and
is activated by phosphorylation(4) . Thus, NKCC1 represents the
secretory Na-K-Cl cotransporter that operates in series with apical
Cl
channels to achieve net Cl
secretion. In nonepithelial cells, NKCC1 may be involved in cell
volume regulation(4, 6) .
A second isoform of the Na-K-Cl cotransporter, designated NKCC2 (also called BSC1), is encoded by a distinct gene (Nkcc2, symbol Slc12a1) that is located on a different chromosome(8) . cDNAs encoding NKCC2 have been cloned from the kidney in rabbit, rat, and mouse(9, 10, 11) . The NKCC2 protein is predicted to contain 12 transmembrane segments, and the amino acid sequence of murine NKCC2 is 64% identical to murine NKCC1. Northern blot analysis of adult tissues revealed that Nkcc2, in contrast to Nkcc1, is expressed as a 5.0-kb transcript that is only detected in the kidney (9, 10, 11) . Moreover, within the kidney, expression of Nkcc2 is restricted to the thick ascending limb of the loop of Henle (TALH)(10, 11) . Recently, the rat NKCC2 (BSC1) protein was immunolocalized to the apical membrane of the cortical and medullary TALH(12) . Thus, NKCC2 represents the apical Na-K-Cl cotransporter that mediates active reabsorption of NaCl in the TALH and is the clinically important site of action of loop diuretics. Studies using in situ hybridization of mouse embryos revealed that Nkcc2 transcripts are expressed in the metanephros but are absent from all other nonrenal tissues, verifying that Nkcc2 is kidney-specific in both adult and developing animals(11) . In the developing metanephros, Nkcc2 transcripts are absent from the nephrogenic zone, which contains uninduced mesenchyme, ureteric buds, and early epithelial structures (renal vesicles, S-shaped bodies), but are highly expressed in more mature nephrons, specifically in distal limbs of developing loops of Henle(11) . Northern blot analysis of embryonic mouse kidneys confirmed that Nkcc2 is induced at 14.5 days postcoitus, which corresponds to stages of nephrogenesis after development of the first S-shaped bodies. Taken together, these results indicate that Nkcc2 is a kidney-specific gene that is a marker for differentiation of the loop of Henle during kidney development.
The present study was undertaken to begin examining the molecular basis for kidney-specific expression of the murine Nkcc2 gene. The specific aims were to determine whether kidney-specific expression of Nkcc2 had a transcriptional basis, to clone and sequence the promoter of the Nkcc2 gene, and to evaluate whether the activity of the Nkcc2 promoter was kidney cell-specific.
Figure 5:
Sequence of the 5` end of the murine Nkcc2 gene. Panel A, partial restriction map of
genomic clone LMH8. Positions of restriction sites for SacI, XbaI, and SalI are indicated by the vertical bars. The bent arrow indicates the
transcription initiation site. The shaded bar indicates the
cloned 5`-flanking region. Panel B, proximal 5`-flanking
sequence (uppercase) and transcribed sequences (lowercase) of the mouse Nkcc2 gene obtained from
genomic clone
LMH8. Nucleotide positions are numbered to the right with respect to the transcription initiation site (bent arrow) at +1. Boxes enclose consensus
sequences for known regulatory elements. Single underlined nucleotides indicate restriction sites used for deletion analysis. Double underlined nucleotides indicate the GA microsatellite. Dotted underlined sequence was identical to cDNA sequence.
Nucleotides indicated in boldface are invariant in splice
donor and acceptor sites, and slashes indicate the splice
sites. Only a portion of the sequence of the first intron is shown. The straight arrow indicates oligonucleotide used for primer
extension analysis. CREB, cAMP-response element binding
protein; GAF, interferon-
activation factor; ISGF-3, interferon-
-stimulated gene factor 3; ER, estrogen receptor.
Na-K-Cl cotransport activity in TAL cells
was measured as furosemide-sensitive NH influx. TAL cells were grown to confluence on plastic coverslips
and then incubated at 37 °C in 140 mM NaCl, 25 mM HEPES (pH 7.4), 5 mM KCl, 1 mM MgCl
,
1 mM NaH
PO
, and 1 mM CaCl
. All solutions were gassed with 100%
O
, and experiments were performed in the nominal absence of
HCO
. Intracellular pH (pH
)
was measured using the permeant, pH-sensitive fluorescent dye
2-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM)
as described previously(19) . The cells on coverslips were
preloaded with BCECF-AM for 15 min, washed with the HEPES-buffered
solution, and then mounted in a microfluorometer. After a steady
base-line fluorescence ratio was obtained, 10 mM NH
Cl was added to the medium (isosmotically replacing
NaCl) in the presence or absence of 1 mM furosemide. pH
was calculated from the ratio of emission fluorescence at 535 nm
obtained at alternating excitation wavelengths of 495 and 440 nm, and
calibration was performed at the completion of each experiment using
the nigericin-high potassium technique.
Luciferase activity was measured in cell lysates using methods
similar to those described previously(22) . Cells were washed
twice with Ca-, Mg
-free
phosphate-buffered saline and then lysed by incubation for 20 min at
room temperature in 900 µl of reporter lysis buffer (Promega).
Lysed cells were scraped into microcentrifuge tubes and freeze-thawed
once using a dry ice-ethanol bath. After brief vortexing and
centrifugation at 14,000
g to remove cell debris, 20
µl of supernatant was aliquoted into 75
12-mm plastic tubes
(Sarstedt). The reaction was initiated by rapid addition of 100 µl
of luciferase assay reagent (Promega) containing 20 mM of
Tricine (pH 7.8), 1.07 mM (MgCO
)
Mg(OH)
5H
0,
2.67 mM MgSO
, 0.1 mM EDTA, 33.3 mM DTT, 270 µM coenzyme A, 530 µM ATP, and
470 µM luciferin. Light output was integrated over 10 s at
room temperature using an Optocomp I photon-counting luminometer (MGM
Instruments). Data were normalized for growth hormone concentration,
which was measured in the conditioned medium using a solid phase
radioimmunoassay according to the manufacturer's directions
(Nichols Institute Diagnostics). Luciferase measurements were performed
in triplicate, and measurements of growth hormone were performed in
duplicate. Standard curves were generated in each experiment to insure
that results were within the linear range of the assay.
Figure 1:
Measurement of the rate of
transcription of the Nkcc2 gene using nuclear run-off assays.
Nuclei were isolated from liver (left) and renal medulla (right), and nuclear run-off assays were performed as
described under ``Experimental Procedures.'' Radiolabeled
transcripts (5 10
cpm) were hybridized to 5 µg
of filter-immobilized cDNAs encoding NKCC2 (top), GAPDH (middle), or empty pBluescript (bottom). Three
independent experiments gave identical results, and a representative
autoradiogram is shown. Exposure in a PhosphorImager was for 7
days.
Figure 2:
Measurement of apical
Na-K(NH)-Cl cotransport activity in murine
TAL cells. Intracellular pH was measured in confluent monolayers of TAL
cells as described under ``Experimental Procedures.'' The arrow indicates replacement with medium containing 10 mM NH
Cl (isosmotically substituted for NaCl). The left trace (A) is in the absence of 1 mM furosemide, while the right trace (B) is in the
presence of 1 mM furosemide (solid line). Results of
a representative experiment are shown.
To determine whether TAL cells expressed the Nkcc2 gene, Northern blot analysis was performed. As shown in Fig. 3, TAL cells expressed an Nkcc2 transcript that was identical in size (5.0 kb) to the transcript expressed in native kidney. In contrast, no Nkcc2 transcripts were detected in NIH 3T3 fibroblasts (Fig. 3), whereas transcripts encoding GAPDH were present in both cell types (not shown). It is important to note, however, that the amount of RNA subjected to analysis was 10-fold greater in TAL cells compared with native kidney. Moreover, since TALH comprises a minority population of tubules in the kidney, the level of expression of Nkcc2 in TAL cells was considerably less than in native thick ascending limb cells. Nevertheless, experiments shown in Fig. 6and Fig. 7indicate that the level of expression was adequate for studies of the Nkcc2 promoter when a sufficiently sensitive reporter gene assay was used.
Figure 3:
Northern blot analysis of expression of Nkcc2 in TAL cells. Poly(A) RNA from TAL
cells (15 µg), NIH 3T3 fibroblasts (15 µg), and mouse kidney
(1.5 µg) was hybridized with a
P-labeled antisense Nkcc2 riboprobe. The autoradiogram was exposed overnight.
Positions of molecular weight standards (in kb) are shown on the left. The arrow indicates the 5-kb Nkcc2 transcript.
Figure 6: Functional expression of the Nkcc2 promoter in TAL cells and NIH 3T3 fibroblasts. NIH 3T3 fibroblasts (columns 1-3) or TAL cells (columns 4-6) were co-transfected with 4 µg of pXGH5 and 4 µg of pGL3-Basic (columns 1 and 4), 4 µg pGL3-Control (columns 2 and 5), or 4 µg pGL3B-NKCC2 (columns 3 and 6). 72 h after transfection, cells were lysed and assayed for luciferase activity and growth hormone. Normalized light output is shown. Data are mean (±S.E.) of four separate experiments. n.s., not significantly different from column 2 (p = 0.26, t test). *, significantly greater than column 3 (p < 0.001, t test).
Figure 7: Deletion analysis of the proximal 5`-flanking region of the Nkcc2 gene. Panel A, recombinant plasmids containing nested deletions of the proximal 5`-flanking region of Nkcc2 (in pGL3-Basic) were generated by site-directed mutagenesis and restriction digestion. The bent arrows indicate transcription initiation sites, and the closed bars indicate the putative HNF-1 site at nucleotide position -211. Plasmids were transfected into TAL cells, and luciferase activity was measured in cell lysates after 72 h. To control for transfection efficiency, cells were co-transfected with pXGH5, and growth hormone was measured in the conditioned medium. Panel B, shaded bars indicate normalized light output relative to the plasmid containing 2.3 kb of proximal 5`-flanking region. Data are mean (±S.E.) of six independent experiments.
Figure 4:
Mapping of Nkcc2 transcription
initiation sites by primer extension analysis. An antisense
oligonucleotide complementary to the 5`-untranslated region of the
mouse Nkcc2 cDNA (nucleotides -107 to -131
numbered with respect to the translation start codon, Fig. 5B) was end-labeled, annealed to 10 µg of
poly(A) RNA from mouse kidney (left lane),
NIH 3T3 fibroblasts (middle lane), or TAL cells (right
lane) and then elongated with reverse transcriptase. Products were
analyzed on 6% polyacrylamide sequencing gels, and a representative
autoradiogram is shown. Lanes between samples were intentionally left
blank to preclude the possibility of sample spillover. The arrow indicates the major primer extension products. Positions of
molecular size standards (in bp) are indicated on the left.
To confirm the location of
the transcription initiation site using an independent method and to
obtain the sequence of the 5` end of the transcript, ligation-anchored
PCR was performed. Amplification using a gene-specific primer that
annealed 241 bp 3` to the translation start codon produced cDNAs that
were 471 bp in length, which confirmed that the authentic 5` end of the Nkcc2 transcript was located 230 bp 5` to the start codon (not
shown). The ligation-anchored PCR product was cloned and sequenced and
compared with the sequence of LMH8. The nucleotide sequences were
identical in the region of overlap (indicated by dotted underlined
nucleotides in Fig. 5B), verifying the identity of
the genomic clone. However, beginning at nucleotide +35, the
sequence of
LMH8 contained an additional 1101 bp of sequence that
was not present in the cDNA. This sequence was flanked by consensus
splice donor (MAG
GTRAGT) and acceptor (Y
NYAG
G)
sites(15) . These results indicated that the first exon of Nkcc2 was 34 bp in length and noncoding. The first intron was
1101 bp, and the second exon contained the translation start codon. The
5` end of the transcript that was identified by ligation-anchored PCR
and primer extension analysis corresponded to a single transcription
initiation site in the gene that was located 1330 bp upstream to the
start codon. The genomic clone
LMH8 contained an additional 13.5
kb of 5`-flanking sequence that was upstream to this transcription
initiation site (Fig. 5A).
The proximal 5`-flanking region contained consensus recognition sequences for several transcription factors that are known to be involved in tissue-specific gene expression. At positions -261 and -290 there were the sequences TTGTGCAAT and TGAAGCAAT that exactly matched the consensus binding site (TKNNGYAAK) (15) for CCAAT/enhancer binding proteins (C/EBP), which mediate tissue-specific expression in liver and adipocytes. Two other sequences at positions -704 and -1184 (TTAGGAAAT and TGGGGAAAT) matched the consensus C/EBP site at 8/9 nucleotides. At positions -169, -294, -1070, and -1081 there were the sequences CAGTTG, CAACTG, CACTTG, and CAACTG, which contained the E-box motif (CANNTG) that was first identified in the enhancers of B-cell-specific genes(29) . At position -211, there was the sequence GATTAATGATTTACT that matched 13/15 nucleotides with a consensus binding site for hepatocyte nuclear factor 1 (HNF-1), a transcription factor that is involved in tissue-specific expression in the liver and other organs(30) .
Several consensus binding sites were identified for transcription
factors that function as effector molecules in signal transduction
pathways. These included the sequence TGACGTAG at nucleotide
-1111, which exactly matched the consensus recognition site
(TGACGYMR) (31) for cAMP-response element binding protein. Also
present were consensus binding sites for NF-B (GGGRHTYYCC) (15) at -138; interferon-
activation factor
(TTNCNNNAA) (31) at -93, -245, and -714;
interferon-
-stimulated gene factor 3 (YAGTTTCWYTTTYCC) (15) at -359; activator protein-1 (TGASTMA) (16) at -111, -838, -971, -1039, and
-1118; and activator protein-2 (CCCMNSSS) (16) at
-432, -477, and -1180. Although three half-sites for
estrogen receptor (AGGTCA) (16) were identified at -561,
-640, and -726, no consensus binding sites for
glucocorticoid or mineralocorticoid receptors were found.
The sequence of the Nkcc2 promoter was aligned with the sequences of cloned promoters from other kidney-specific genes including human aquaporin-2 (2) and rat V2 vasopressin receptor(3) , but no significant regions of homology were identified (data not shown). Of particular interest was the alignment with the gene encoding THP, which is expressed in the same nephron segment (TALH) as Nkcc2. The human, bovine, and rat THP promoters have been cloned, sequenced, and functionally expressed in vitro(1) . The rat THP gene also contains a consensus HNF-1 binding site that is located 280 bp 5` to the transcription initiation site within a phylogenetically conserved region. However, no other extended regions of sequence similarity between the THP and Nkcc2 genes were found (not shown).
Previous studies using Northern blot analysis and in situ hybridization indicated that expression of transcripts encoding
the apical Na-K-Cl cotransporter (NKCC2) was restricted to the kidney
in both adult and developing
mammals(9, 10, 11) . In the present study,
nuclear run-off assays demonstrated that kidney-specific expression was
due, at least in part, to kidney-specific transcription of the Nkcc2 gene. The cis-acting regulatory elements that
govern initiation of transcription may be interspersed throughout the
gene. However, in most cases the proximal promoter region contains
sufficient information for efficient and tissue-specific gene
transcription, although distal enhancers may be required to achieve
maximal levels of expression. For example, regulatory elements located
within the first 300 bp 5` to the transcription initiation site are
sufficient to mediate tissue-specific expression of the albumin,
-globin, and growth hormone genes in hepatocytes, erythroid cells,
and somatotrophs, respectively(32) . These proximal regulatory
elements represent binding sites for tissue-specific or tissue-enriched
proteins that activate gene transcription. Accordingly, we first
focused on elements within the proximal 5`-flanking region that may be
responsible for kidney-specific transcription of Nkcc2.
We
isolated a 20.1-kb genomic clone that contained 13.5 kb of the proximal
5`-flanking region of the Nkcc2 gene. The sequence of the
proximal 5`-flanking region contained a TATA box and two CCAAT boxes,
which are typical eukaryotic promoter elements in tissue-specific genes
but are frequently absent from genes that are expressed ubiquitously.
Although the recognition sequences of novel transcription factors that
mediate kidney-specific gene expression remain unknown, consensus
binding sites were identified for several transcription factors that
regulate tissue-specific expression in other organs and are also
present in the kidney. Four potential binding sites for members of the
C/EBP family were identified. C/EBP, C/EBP
(NF-IL6, LAP), and
albumin D site-binding protein are basic leucine zipper proteins that
activate a variety of tissue-specific genes, particularly during
terminal differentiation in liver and adipocytes (33) . Recent
studies suggest that tissue-specific expression of genes encoding
aldolase B and cytosolic phosphoenolpyruvate carboxykinase in the renal
proximal tubule may also involve C/EBP(34, 35) .
Although C/EBP family members have been detected in the renal cortex
and medulla (34, 36) , the levels of expression are
very low compared with liver, and the resolution of existing studies is
insufficient to ascertain whether these transcription factors are
present in the loop of Henle. Four E-boxes were identified in the Nkcc2 promoter that could represent binding sites for basic
helix-loop-helix (bHLH) proteins. Tissue-specific bHLH proteins (e.g. MyoD, MASH1) are important for gene expression in
muscle, neurons, lymphocytes, and pancreas(29) . In general,
tissue-specific (class B) bHLH proteins bind DNA preferentially as
heterodimers with ubiquitous (class A) bHLH proteins (e.g. E12, E47). Although the E2A gene products, E12 and E47,
are expressed in the kidney(37) , no class B proteins that are
potential binding partners and kidney-specific have been identified.
Inhibitory HLH proteins (e.g. Id-1) that bind to class B
proteins but lack a basic domain and cannot bind DNA are also produced
in the kidney. The down-regulation of Id-1 expression that occurs
during nephron development has been taken as indirect evidence for the
existence of class B proteins that function during terminal
differentiation in the kidney(38) .
Of potential importance
was the presence of a consensus binding site for HNF-1 at -211.
The HNF-1 family consists of two distinct proteins, HNF-1 (also
called HNF-1 or LF-B1) and HNF-1
(also called vHNF-1 or LF-B3),
which are transcription factors that regulate liver-specific expression
of genes such as
-antitrypsin, albumin, and
fibrinogen(30) . HNF-1
and HNF-1
are diverged
homeodomain proteins that bind to DNA as dimers and recognize an
inverted palindrome that contains the consensus sequence
RGTTAATNATTAACM. In addition to liver, HNF-1
and HNF-1
are
also highly expressed in certain extrahepatic tissues, including the
kidney. Recent studies using transgenic mice indicate that
tissue-specific expression of the cytosolic phosphoenolpyruvate
carboxykinase gene in the renal proximal tubule requires a promoter
element (P2) that contains a consensus HNF-1 binding site(39) .
In the developing mouse kidney, expression of HNF-1
is
stage-dependent and immediately precedes expression of Nkcc2;
HNF-1
is absent from uninduced mesenchyme and early nephrogenic
structures (ureteric buds, renal vesicles, comma-shaped bodies) but is
induced at 14.5 days postcoitus in late S-shaped bodies and remains
highly expressed in differentiated renal tubules, including the loops
of Henle(40) . The presence of a consensus HNF-1 binding site
in the Nkcc2 promoter and the overlapping patterns of
expression of Nkcc2 and HNF-1
in the developing kidney
raise the possibility that Nkcc2 may be a target for
transcriptional activation by HNF-1
. Consistent with this
hypothesis, deletion of a 280-bp DNA segment containing the putative
HNF-1 site caused a 76% reduction in promoter activity (see below).
Recent preliminary studies suggest that HNF-1
can bind to the site
at -211 and transactivate promoter activity. (
)
Several consensus binding sites were identified for
transcription factors that function as effector molecules in signal
transduction pathways. These included the cAMP-response element binding
protein. Although apical Na-K-Cl cotransport activity in the murine
TALH is stimulated by arginine vasopressin via a pathway that
presumably involves cAMP(41) , it is currently not known
whether this effect entails changes in Nkcc2 gene expression.
Thus, the importance of the binding site for cAMP-response element
binding protein remains uncertain. As well, the Nkcc2 promoter
contained consensus recognition sites for NF-B, which mediates the
transcriptional response to cytokines; interferon-
activation
factor and interferon-
stimulated gene factor 3, which consist of
oligomers of STAT proteins that mediate the response to interferons;
AP-1, which consists of heterodimers of Fos and Jun proteins; and AP-2,
which is inducible by protein kinase C. However, no information is
currently available regarding regulation of Nkcc2 gene
expression by any of these signaling pathways.
Interestingly, the
5`-flanking region of murine Nkcc2 contained a simple sequence
repeat consisting of (G-A)(C-T)
.
Microsatellites have occasionally been found in the 5`-flanking regions
of vertebrate genes, where their function, if any, remains unknown.
Because they are also found in introns, 3`-flanking region, and
intergenic regions, the presence of a microsatellite in the proximal
5`-flanking sequence of Nkcc2 may be coincidental. However, in Drosophila, GA repeats are present in the promoters of the
heat shock protein 26 and heat shock protein 70 genes, where they
comprise a DNase I hypersensitive site that is essential for inducible
promoter activity. Moreover, a Drosophila protein, called GAGA
factor, has been identified that binds to the GA sequence and appears
to alter chromatin structure during gene activation(42) . In
mammals, a corresponding transcription factor that binds to
double-stranded sequences containing GA repeats has not yet been
identified. However, polypyrimidine tracts can also form triple helical
H-DNA via Hogness base pairing, and a protein that binds to the
resulting single-stranded DNA of the CT repeat has been identified in
several mammalian species(43) . Taken together, these results
raise the possibility that the GA-rich sequence may be important for
altering chromatin structure during transcription of the Nkcc2 gene. Alternatively, since GA repeats are also genetically
unstable, the sequence may contribute to hypermutation of the
nonrepetitive flanking region.
To verify that the cloned 5`-flanking
region contained a functional gene promoter, transient transfection
experiments were performed. These studies utilized a stable cell line
(TAL cells) that was derived from the murine TALH. TAL cells exhibited
furosemide-sensitive intracellular acidification following exposure to
apical NH/NH
, expressed a 5-kb Nkcc2 transcript by Northern blot analysis, and produced a
primer extension product that was similar in size to the product from
native kidney. Taken together, these results demonstrated that TAL
cells expressed authentic NKCC2 and were, therefore, appropriate
recipient cells for studies of the promoter. Transfection experiments
showed that the cloned Nkcc2 promoter was highly active in
cells that expressed endogenous Nkcc2 (TAL cells) but was
relatively inactive in cells that did not express Nkcc2 (NIH
3T3 fibroblasts, HeLa cells, and S1 proximal tubule cells). These
results suggested that cell type-specific promoter activity may
contribute to kidney-specific and nephron segment-specific expression
of Nkcc2. Moreover, regulatory elements that were required for
cell-specific promoter activity were likely to be contained within the
cloned 3.5-kb fragment. However, these experiments do not exclude the
possibility that additional distal regulatory elements may be required
for high level, tissue-specific expression of Nkcc2 in vivo.
Further experiments utilizing transgenic mice will be required to
verify whether the cloned Nkcc2 promoter contains regulatory elements
that are sufficient to confer kidney-specific expression in an intact
organism. To begin identifying the locations of regulatory elements
that were required for promoter activity in TAL cells in
vitro, deletion analysis was performed. These studies indicated
that critical cis-acting regulatory elements were located
between 190 and 469 bp 5` to the transcription start site. This region
was interesting because it contained the putative HNF-1 site, GA
microsatellite, two consensus C/EBP binding sites, and an E-box,
further underscoring the potential importance of one or more of these
elements for expression of Nkcc2.
In conclusion, we have
demonstrated that kidney-specific expression of Nkcc2 is due,
at least in part, to kidney-specific gene transcription. We have
identified a stable murine cell line (TAL cells) that exhibits apical
Na-K(NH)-Cl cotransport activity and
endogenously expresses the 5-kb Nkcc2 transcript. We have
cloned and sequenced the murine Nkcc2 promoter and observed
that the promoter exhibits kidney cell-specific activity. Deletion
analysis has identified a 280-bp DNA segment that is required for
maximal promoter activity and contains consensus recognition sequences
for several transcription factors expressed in the kidney.
A preliminary account of this work has been published in abstract form (13) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U45313[GenBank].