1 Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390; and 2 Unité d'Expression Génétique et Maladies, Centre National de la Recherche Scientifique URA 1644, Département de Biologie du Développement, Institut Pasteur, 75724 Paris cedex 15, France
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ABSTRACT |
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Kidney-specific cadherin (Ksp-cadherin)
is a tissue-specific member of the cadherin family that is expressed
exclusively in the kidney and developing genitourinary tract. Recent
studies have shown that the proximal 250 bp of the Ksp-cadherin gene
promoter are sufficient to direct tissue-specific gene expression in
vivo and in vitro. The proximal 120 bp of the promoter are
evolutionarily conserved between mouse and human and contain a DNase I
hypersensitive site that is kidney cell specific. At position 55, the
promoter contains a consensus recognition site for hepatocyte nuclear
factor-1 (HNF-1). Mutations of the consensus HNF-1 site and downstream GC-boxes inhibit promoter activity in transfected cells. HNF-1
and
HNF-1
bind specifically to the
55 site, and both proteins transactivate the promoter directly. Expression of Ksp-cadherin is not
altered in the kidneys of HNF-1
-deficient mice. However, expression
of a gain-of-function HNF-1
mutant stimulates Ksp-cadherin promoter
activity in transfected cells, whereas expression of a
dominant-negative mutant inhibits activity. These studies identify Ksp-cadherin as the first kidney-specific promoter that has been shown
to be regulated by HNF-1
. Mutations of HNF-1
, as occur in humans
with inherited renal cysts and diabetes, may cause dysregulated Ksp-cadherin promoter activity.
transcription factor; kidney-specific gene regulation; mouse inner medullary collecting duct-3 cells; deoxyribonuclease hypersensitive sites; maturity-onset diabetes of the young
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INTRODUCTION |
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KIDNEY-SPECIFIC CADHERIN (Ksp-cadherin or cadherin 16) is a unique, tissue-specific member of the cadherin family of cell adhesion proteins. Cadherins are plasma membrane glycoproteins that mediate calcium-dependent, homotypic cell-cell interactions and play important roles in morphogenesis, cell migration, cell differentiation, and signal transduction (2, 37). Ksp-cadherin is a structurally distinct member of the family that has a highly restricted tissue distribution. In the adult rabbit, mouse, and human, Ksp-cadherin is expressed exclusively in the kidney (30, 31). Within the kidney, Ksp-cadherin is restricted to renal tubules and is not expressed in glomeruli, blood vessels, or interstitial cells. The protein is located in the basolateral membrane of epithelial cells in all segments of the nephron, including proximal tubules, loops of Henle, distal tubules, and renal collecting ducts (29). During embryonic development, Ksp-cadherin is expressed in the developing kidney and genitourinary (GU) tract (29, 33). In the embryonic kidney (metanephros), Ksp-cadherin is expressed in developing renal tubules beginning after the S-shaped body stage and in the branching ureteric bud, which is the anlage of the renal collecting ducts, pelvis, and ureter. Ksp-cadherin mRNA transcripts have also been detected in mesonephric tubules and in the two paired sex ducts, the Wolffian (mesonephric) duct and the Müllerian (paramesonephric) duct. However, the expression in the developing GU tract is transient, since there is no expression in the adult renal pelvis, ureter, or male and female genital tracts.
To explore the mechanism of tissue-specific and developmentally
regulated expression of Ksp-cadherin, our laboratory previously cloned
and characterized the gene promoter (34). The proximal 2,608 bp of the Ksp-cadherin promoter direct expression of a luciferase reporter gene in transfected renal epithelial cells but not in mesenchymal cells (34). Deletion analysis has shown that
truncation of the promoter from 2,608 to
250 bp does not
significantly decrease promoter activity in renal epithelial cells.
However, further truncation to
113 bp reduces promoter activity by
40%, and truncation to
31 bp completely abolishes activity.
Transgenic mice containing 3.3 kb of the Ksp-cadherin promoter linked
to a lacZ reporter gene express
-galactosidase
exclusively in the ureteric bud and renal collecting ducts, verifying
that the activity of the promoter is tissue specific in vivo
(15). Recent studies have shown that 1,268 bp of the
promoter recapitulate the complete expression pattern of the endogenous
Ksp-cadherin gene in the kidney and developing GU tract, and the
proximal 250 bp of the promoter are sufficient for tissue-specific
expression (28). This region of the promoter contains
potential recognition sites for the transcription factor hepatocyte
nuclear factor (HNF-1).
HNF-1 consists of two proteins, HNF-1 and HNF-1
, that have been
implicated in tissue-specific gene regulation in the liver, pancreas,
kidney, and intestine (16, 20, 27). HNF-1
and HNF-1
contain an atypical homeodomain, an NH2-terminal
dimerization domain, and a POU domain. They bind to DNA as homodimers
or heterodimers and recognize an identical consensus sequence
(19). HNF-1
is a transcriptional activator and contains
three distinct activation domains within its COOH terminus. This domain
is not conserved in HNF-1
, which has been reported to function as an
activator or a repressor or to have no transcriptional activity,
depending on the promoter and cell type (20, 27). Both
HNF-1
and HNF-1
are highly expressed in the developing kidney but
in different patterns. HNF-1
is primarily expressed in proximal
tubules, whereas HNF-1
is expressed in all nephron segments and the
renal collecting system and sex ducts. The current study was undertaken
to investigate whether HNF-1
and/or HNF-1
regulate the
Ksp-cadherin gene promoter. We show that both proteins can bind
specifically to the promoter and directly stimulate its activity. In
addition, the activity of the Ksp-cadherin promoter is altered by the
expression of HNF-1
mutants that are similar to those found in
humans with inherited renal cysts and diabetes.
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MATERIALS AND METHODS |
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Materials.
Restriction endonucleases and DNA-modifying enzymes were from New
England Biolabs (Beverly, MA) or Roche Molecular Biochemicals (Indianapolis, IN). pGL3 plasmids, pSV2gal, luciferin, and reporter lysis buffer were from Promega (Madison, WI). Galacton-Star
-galactosidase substrate was obtained from Tropix (Bedford, MA).
Plasmid prep kits and Effectene transfection reagent were from Qiagen
(Valencia, CA). Oligonucleotides were synthesized by Integrated DNA
Technologies (Coralville, IA) and were purified by polyacrylamide gel
electrophoresis. The QuikChange site-directed mutagenesis system was
from Stratagene (La Jolla, CA). Antibodies were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Tissue culture media were from
Life Technologies (Gaithersburg, MD). Other reagents were of molecular
biological grade from Sigma (St. Louis, MO), Promega, or Roche
Molecular Biochemicals.
Animals.
HNF-1-deficient mice were produced by replacing the first exon with
a LacZ cassette, as described previously (24). Studies were performed using 2- to 3-wk-old HNF-1
(
/
) mice and their age-matched heterozygous HNF-1
(+/
) and wild-type HNF-1
(+/+) littermates. Mice were genotyped by PCR analysis of tail DNA, as
described previously (24). The genetic background of the animals was either C57BL/6 × 129/Sv or 129Sv/DBA2/C57BL/6.
Plasmids.
A plasmid (pBS-KS-HNF1) containing the mouse HNF-1
cDNA and a
plasmid (pBJ5-HNF1
) containing the mouse HNF-1
cDNA (B splice variant) were generous gifts from Dr. Gerald Crabtree (Stanford University). The 3.1-kb EcoRI fragment from pBJ5-HNF1
was
cloned into the EcoRI site of pBluescript II KS(+), creating
the plasmid pBS-KS-HNF1
. The HNF-1
coding region was amplified
from pBS-KS-HNF1
with primers 5'-ACCATGGTTTCTAAGCTGAGCCAGCTG and
5'-GGGTTTAAACTTACTGGGAAGAGGAGGCCATCTGG and cloned into
pcDNA3.1/V5/His-TOPO (Invitrogen), creating the expression plasmid
pcDNA3-HNF1
. The HNF-1
coding region was amplified from
pBS-KS-HNF1
with primers 5'-ACCATGGTGTCCAAGCTCACGTCGCTC and
5'-CGGGTTTAAACTCACCAGGCTTGCAGTGGACACTGT and cloned into
pcDNA3.1/V5/His-TOPO, creating the expression plasmid pcDNA3-HNF1
.
The coding regions of both plasmids were sequenced completely.
Site-directed mutagenesis.
Site-directed mutagenesis was performed using QuikChange kits
(Stratagene) according to the manufacturer's directions. Complementary sense and antisense oligonucleotides containing the desired mutations (Table 1) were synthesized and purified
by PAGE. The mutagenic primers were annealed to the luciferase reporter
plasmid and extended using PfuTurbo DNA polymerase. Eighteen
cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and elongation at 68°C for 10 min were performed. The parental
DNA template was then digested with DpnI, and the mutated
plasmids were transformed into Escherichia coli. The
presence of the desired mutation was verified by DNA sequencing. DNA
for transfections was purified by alkaline lysis maxipreps and
anion-exchange chromatography (Qiagen). Mutant reporter plasmids were
produced containing the mutations listed in Table 1, except
mutation 6.
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Cell culture and transfection. Mouse inner medullary collecting duct cells (mIMCD-3) were a generous gift from Dr. Stephen Gullans (Harvard Medical School) and were grown in DMEM supplemented with 10% FBS (26). HeLa cells were obtained from American Type Culture Collection (Manassas, VA) and were maintained in Iscove's modified Dulbecco's media supplemented with 2% FBS and 5% newborn calf serum. C3H/10T1/2 cells were obtained from ATCC and were maintained in basal Eagle medium supplemented with 10% FBS.
Plasmids were transiently transfected in cultured cells using Effectene (Qiagen). Cells were plated in six-well dishes at a density of 1.2-3 × 105 cells/well. After overnight growth to ~60% confluence, the cells were washed one time with PBS and then transfected with 0.6-3 µg plasmid. Plasmid DNA and enhancer (2 times the amount of DNA, vol/wt) were combined in 100 µl of buffer and incubated for 2-5 min. Effectene reagent (10 µl) was added, and the mixture was incubated for 5-10 min at room temperature to permit complex formation. Growth medium (0.6 ml) was added, and the mixture was added drop-wise on the cells. In each experiment, the total amount of transfected DNA was maintained constant by the addition of empty expression plasmid. To control for differences in transfection efficiency, cells were cotransfected with a constant amount of plasmid pSV2-Reporter gene assays.
Luciferase activity was measured in cell lysates using methods similar
to those described previously (34). Cells were washed with
PBS and then lysed by incubation for 20 min at room temperature in 150 µl Reporter Lysis Buffer (Promega). Lysates were frozen one time at
70°C, thawed, and then centrifuged at 14,000 g.
Supernatant (20 µl) was added to 100 µl of Luciferase Assay Reagent
(Promega). After a 1-s delay, light output was measured for 10 s
using an Optocomp I luminometer (MGM Instruments, Hamden, CT).
Luciferase activity was normalized to
-galactosidase activity, which
was measured by adding 20 µl of cell lysate to 300 µl of reaction buffer containing Galacton-Star (Tropix), 100 mM sodium phosphate (pH
7.5), 1 mM MgCl2, and 5% Sapphire-II (Tropix). After
incubation for 60 min at room temperature, light output was measured
for 5 s using a luminometer. In some experiments (see Fig.
7C) in which the expression of HNF-1
mutants affected
-galactosidase activity, luciferase activity was normalized to
protein concentration. Protein concentration was measured using the
Pierce Coomassie Plus Protein Assay (Rockford, IL) with BSA as the standard.
In vitro translation.
In vitro translation was performed using TNT Quick-coupled
transcription/translation kits (Promega) according to the
manufacturer's directions. pBluescript plasmid (1 µg) containing the
full-length cDNAs encoding HNF-1 or HNF-1
(pBS-KS-HNF1
and
pBS-KS-HNF1
, respectively) was added to TNT Quick master mix in a
total volume of 50 µl. Transcription was performed using T7 RNA
polymerase, and translation was performed using rabbit reticulocyte
lysates. Reactions were incubated at 30°C for 60-90 min, and 5 µl of the reaction product were used for electrophoretic
mobility shift assays (EMSA). Unprogrammed lysates were used as
negative controls.
Preparation of nuclear extracts from cultured cells.
Nuclear extracts were prepared from mIMCD-3 cells using the method of
Andrews and Faller (1). Confluent cells were grown in
100-mm dishes (~5 × 106 cells) and then were
scraped into 1.5 ml PBS and pelleted. Cells were lysed by incubation
for 15 min in ice-cold medium containing 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Nuclei were pelleted by
centrifugation and then extracted for 20 min at 4°C with 20 mM
HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF. After centrifugation for 45 min at 14,000 g, the supernatant was
diluted 1:1 with buffer containing 5 mM HEPES-KOH (pH 7.9), 20%
glycerol, 1 mM DTT, and 0.2 mM PMSF and was stored at 70°C.
EMSA.
EMSA were performed as described previously (34).
Double-stranded oligonucleotides containing the sequences of the
promoter from 46 to
13 bp
(5'-CTAGGCGGGGCCACGGCTGCTCCTGTGGGCCCCG) or from
79 to
50 bp
(5'-GGCTCTCCAAAGTCAATAAGTAACTTGGGG) were end labeled with
32P using T4 polynucleotide kinase. Binding reactions (20 µl) contained 25 mM NaCl, 10 mM Tris · Cl (pH 7.5), 1 mM
MgCl2, 5 mM EDTA, 5% glycerol, 1 mM DTT, 1 × 106 counts/min labeled probe, and 2-4 µg nuclear
extracts or 5 µl programmed lysates. In some reactions, a 5- to
100-fold molar excess of unlabeled oligonucleotide was included as a
specific competitor. All binding reactions contained 200 ng
poly(dI · dC) or poly(dA · dC) as nonspecific
competitor. After incubation at 4°C or room temperature for 20 min,
samples were loaded on 4% polyacrylamide gels and electrophoresed at 8 V/cm for 2 h in 0.5× Tris-borate-EDTA. Gels were dried,
and the radiolabeled bands were detected by autoradiography using Kodak
BioMax MS film.
Northern blot analysis.
Total RNA was isolated from the kidneys of HNF-1 mutant homozygous,
heterozygous, and wild-type mice and subjected to Northern blot
analysis as described previously (24, 25). Northern blots were hybridized with a 700-bp mouse Ksp-cadherin cDNA and an
11
-hydroxysteroid dehydrogenase type 1 cDNA as a control.
Autoradiography and quantification of band intensities were performed
using a PhosphorImager.
DNase I hypersensitive site mapping.
DNase I hypersensitive site mapping was performed according to Boyes
and Felsenfeld (7). mIMCD-3 or 10T1/2 cells (1 × 108 cells) were trypsinized, washed two times in PBS, and
then resuspended in 1 ml of 0.34 M sucrose, 10 mM HEPES (pH 8.0), 60 mM
KCl, 2 mM EDTA, 1.5 mM DTT, 0.5 mM spermine, and 0.15 mM spermidine
(buffer A) containing 0.5% IGEPAL CA-630. All buffers were
ice-cold unless otherwise indicated. Nuclei were immediately pelleted
at 2,060 g for 5 min at 4°C and then washed one time in 5 ml buffer A without IGEPAL CA-630. Nuclei were resuspended
in 1 ml of 10 mM HEPES (pH 8.0), 20 mM KCl, 3 mM MgCl2, and
6 mM CaCl2 (buffer B), and 100-µl aliquots
(1 × 107 nuclei) were added to tubes containing
increasing concentrations of DNase I (Sigma) diluted in 100 µl
buffer B. The concentrations of DNase I ranged from 0.2 to
10 µg, and one sample was kept as a no DNase I control. Nuclei
containing DNase I were placed in a 37°C water bath for 5 min, and
then the reaction was stopped by adding 200 µl of 0.1 M Tris (pH
7.5), 20 mM EDTA, 1% SDS, and 0.5 M NaCl (buffer C).
Buffer C was also added to the no DNase I control. Samples
were treated with RNase A for 1 h at 37°C and then digested with
10 µg proteinase K overnight at 55°C. Genomic DNA was purified by
phenol/chloroform extraction and ethanol precipitation. DNase I
"fadeout" was confirmed by agarose gel electrophoresis and staining
with ethidium bromide. DNA (10 µg) was digested with HindIII, electrophoresed on a 0.8% agarose gel, transferred
to a nylon membrane, and hybridized with 5'- or 3'-probes near the HindIII sites. The probes used were a 443-bp
HindIII/StuI restriction fragment containing
nucleotides 1,265 to
822 bp (5'-probe) and a 713-bp fragment
containing nucleotides +2,005 to +2,718 bp (3'-probe) that was
generated by PCR with primers 5'-CCTATCAGGAGACTCAAACACGGC and
5'-AATGTGGAAGCGAAGGTCGG. PCR reaction conditions were 40 cycles of
94°C for 30 s, 57°C for 30 s, and 72°C for 1 min using
genomic DNA from mIMCD-3 cells as the template.
Statistical analysis. Results of reporter gene assays are expressed as the means ± SE of three to nine independent transfections assayed in duplicate. The statistical significance of differences between the means was evaluated using Student's t-test or one-way ANOVA as appropriate. Scheffé's test was used for post hoc comparisons. Differences were considered to be significant at P < 0.05.
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RESULTS |
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The proximal Ksp-cadherin gene promoter is evolutionarily conserved
between mouse and human.
The DNA elements that are essential for regulation of gene expression
are often conserved between different species. To identify evolutionarily conserved regions within the Ksp-cadherin gene promoter,
the sequence of the mouse promoter was compared with the sequence of
the human genome in the NCBI database. A BLASTN search was performed
using 1,268 bp of the mouse promoter, since this region is sufficient
to recapitulate the complete expression pattern of the gene in
transgenic mice (28). A draft (phase 1)
sequence of the human Ksp-cadherin gene was identified in the High
Throughput Genomic Sequences database (accession no. AC009084). The
human sequence was contained within a 222-kb bacterial artificial chromosome (BAC) derived from chromosome 16, which was consistent with
the chromosomal localization of the Ksp-cadherin gene
(CDH16), as previously determined by fluorescence in situ
hybridization (31). An alignment of the sequence of the
BAC with the sequence of the human Ksp-cadherin cDNA revealed that the
BAC contained the entire Ksp-cadherin gene on a single 56-kb contig.
The gene consisted of 18 exons distributed over 10.7 kb (Fig.
1A). All of the exon-intron
boundaries conformed to the GT/AG rule (data not shown). The contig
also contained 39 kb of sequence 5' to the human Ksp-cadherin gene,
which included the gene promoter. The sequence of the human promoter
was independently verified using adapter-ligation PCR and was 98%
identical to the sequence derived from the BAC (data not shown).
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The proximal Ksp-cadherin promoter contains a DNase I
hypersensitive site that is cell type specific.
The binding of transcription factors to specific sites in DNA is
accompanied by local disruptions of chromatin that render DNA in the
region more susceptible to digestion by exogenous nucleases (13). Sites that are relatively sensitive to digestion by
deoxyribonuclease (so-called DNase hypersensitive sites), particularly
those that are cell type specific, represent potential binding sites
for transcription factors involved in tissue-specific expression. To identify DNase hypersensitive sites in the Ksp-cadherin gene, nuclei
were harvested from mIMCD-3 renal epithelial cells, which express
endogenous Ksp-cadherin, and were incubated with graded concentrations
of DNase I. Genomic DNA was then isolated, and a 4.2-kb
HindIII restriction fragment containing the first six exons
of the gene and 1.3 kb of the 5'-flanking region was visualized by
indirect end labeling. Figure
2A shows the genomic structure of the 4.2-kb fragment and the 3'-probe that was used for indirect end
labeling. Figure 2B shows that incubation with increasing concentrations of DNase I resulted in gradual disappearance of the
4.2-kb parental band and the appearance of a 3-kb subband. The size of
the subband corresponded to a DNase I hypersensitive site located
~100 bp 5' to the transcription initiation site. The position of the
hypersensitive site was confirmed by hybridization to a 5'-probe (data
not shown). No additional sites were observed within the 1.3-kb region
of the 5'-flanking sequence. As a negative control, nuclei were
harvested from 10T1/2 cells, which do not express Ksp-cadherin, and
were treated identically. Figure 2C shows that incubation
with increasing concentrations of DNase I resulted in gradual
disappearance of the parental band but no appearance of a subband.
These results demonstrate that the proximal 100 bp of the Ksp-cadherin
promoter contain a DNase I hypersensitive site that is cell type
specific, the appearance of which correlates with gene transcription.
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Mutations of the consensus HNF-1 site and GC-rich region inhibit
Ksp-cadherin promoter activity in mIMCD-3 cells.
Previous analysis of the Ksp-cadherin promoter indicated that the 82-bp
region of the promoter from nucleotides 113 to
31 was essential for
promoter activity in transfected renal epithelial cells and that
mutations of the region from nucleotides
60 to
71 produced varying
degrees of inhibition of promoter activity (34). To
confirm and extend these results, we performed additional mutational
analysis of the proximal promoter. A series of 3-bp transversion
mutations were introduced into the minimal Ksp-cadherin promoter
downstream of the four mutations (m1-m4) characterized previously (Table 1). Mutation 13 disrupted highly conserved nucleotides in the putative HNF-1 site at nucleotide
55, and mutations 5-10 disrupted the GC-rich region extending
from nucleotides
15 to
43. Luciferase reporter plasmids containing
the mutant and wild-type promoter sequences were transiently
transfected into mIMCD-3 cells, and luciferase activity was measured 48 h later (Fig. 3A). As previously shown, mutations
2-4, which disrupted both the consensus HNF-1 site and the
overlapping HNF-3 site, inhibited promoter activity by 70-80%.
Mutation 1, which disrupted the consensus HNF-3 site, but
not the HNF-1 site, produced only a slight inhibition that was not
statistically significant. Mutation 13, which disrupted only
the consensus HNF-1 site, inhibited promoter activity by 80%.
Mutations of the downstream GC-rich region (m5, m7, m8, and
m9) also inhibited promoter activity, although
mutation 10 had no significant effect. These results
indicated that mutations of the consensus HNF-1 binding site and some
of the GC boxes inhibited Ksp-cadherin promoter activity in transfected
renal epithelial cells.
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HNF-1 and HNF-1
bind specifically to the Ksp-cadherin
promoter.
The mouse Ksp-cadherin gene promoter contained a consensus HNF-1
binding site at nucleotide position
55 of the noncoding strand. The
sequence of the promoter (5'-AGTTACTTATTGACT-3') matched the consensus
sequence
(5'-RGTTAATNATTAACM-3')
(19) at 12 of 15 positions and was identical at 9 positions that are highly conserved in known HNF-1 sites (underlined).
The sequences of the mouse and human promoters were identical in this
region except for a single nucleotide substitution at a redundant
position (N). The putative HNF-1 binding site overlapped with the HNF-3
site at nucleotide
62 and was contained within the 82-bp region that had previously been shown to be important for promoter activity in
transfected renal epithelial cells. Moreover, mutations of the sequence
that altered highly conserved nucleotides (m3 and m13) inhibited promoter activity in mIMCD-3 cells by 80%
(Fig. 3).
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HNF-1 and HNF-1
transactivate the Ksp-cadherin promoter
directly.
To test whether HNF-1
or HNF-1
could transactivate the
Ksp-cadherin promoter, reporter gene assays were performed in
transiently transfected HeLa cells, which do not express endogenous
HNF-1. HeLa cells were cotransfected with expression plasmids encoding HNF-1
or HNF-1
and a luciferase reporter plasmid containing the
Ksp-cadherin promoter. Figure
5A shows
that expression of HNF-1
stimulated luciferase activity by 11-fold
(open bars), whereas expression of HNF-1
produced a 3-fold
stimulation (filled bars). HNF-1
has previously been shown to
be a stronger transcriptional activator than HNF-1
. At higher
concentrations of expression plasmid, luciferase activity began to
decrease, consistent with the phenomenon of squelching. Expression of
HNF-1
together with HNF-1
resulted in stimulation of activity
that was comparable to HNF-1
by itself (data not shown).
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Expression of Ksp-cadherin in HNF-1 mutant mice.
Transgenic mice in which the HNF-1
(Tcf1) gene has been
disrupted by homologous recombination exhibit renal abnormalities and
decreased insulin secretion. The renal abnormalities, which include
glycosuria, amino aciduria, and phosphaturia, resemble human Fanconi
syndrome (24). The expression of the renal
Na+/glucose cotransporter is inhibited in the kidneys from
homozygous mutant mice and may be the mechanism for renal glycosuria
(25). To examine the expression of Ksp-cadherin in the
kidneys of HNF-1
mutant mice, Northern blot analysis was performed.
As shown in Fig. 6, the expression of
Ksp-cadherin mRNA normalized to the expression of 11
-hydroxysteroid
dehydrogenase type 1 was similar in HNF-1
mutant homozygotes,
heterozygotes, and wild-type mice. These results indicated that
HNF-1
was not essential for the expression of the Ksp-cadherin gene
in vivo.
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Mutations of HNF-1 alter the activity of the Ksp-cadherin
promoter.
Because HNF-1
homozygous null mutations are embryonically lethal
before the time of kidney organogenesis (3, 10), it was
not possible to examine Ksp-cadherin gene expression in HNF-1
mutant
mice. As an alternative approach, we examined the effect of mutations
of HNF-1
on transactivation of the Ksp-cadherin promoter.
The P328L329fsinsCCTCT mutation of the human HNF-1
gene produces a dominant gain-of-function mutant that contains an
intact DNA-binding domain, a deletion of 230 amino acids from the COOH
terminus, and a replacement with 29 amino acids from the frame-shifted
open reading frame (5, 35). To test the effects of a
similar mutation of mouse HNF-1
on Ksp-cadherin promoter activity,
mIMCD-3 cells were cotransfected with expression plasmids encoding
either wild-type HNF-1
or an HNF-1
mutant containing a
COOH-terminal deletion similar to the one in the human
P328L329fsinsCCTCT mutant (HNF-1
C). Figure
7A shows that expression of
wild-type HNF-1
had no effect on promoter activity, presumably
because the cells endogenously express HNF-1
. In contrast, transfection with the COOH-terminal deletion mutant produced a concentration-dependent stimulation of promoter activity, consistent with a gain-of-function mutation. Next, we tested the effect of a
dominant-negative mutation of HNF-1
on Ksp-cadherin promoter activity. The A263fsinsGG mutation of the human HNF-1
gene
produces a protein that can dimerize with wild-type HNF-1
but is
unable to bind DNA and therefore functions as a dominant-negative
mutant (32). Figure 7B shows that a mouse
A263fsinsGG mutant inhibited the activity of the Ksp-cadherin
promoter by 40% when transfected in mIMCD-3 cells that endogenously
express wild-type HNF-1
. The A263fsinsGG mutant had no
significant effect on the activity of the promoter containing mutations
of the HNF-1 binding site (m3 and m13),
indicating that the inhibition was specific. Figure 7C shows
the results of coexpression of HNF-1
and the A263fsinsGG dominant-negative mutant in HeLa cells. Expression of wild-type HNF-1
by itself stimulated promoter activity fourfold, whereas coexpression of the A263fsinsGG mutant inhibited activation by 60%, consistent with a dominant-negative mutation. Taken together, these results support the role of endogenous HNF-1
in
Ksp-cadherin promoter activity.
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DISCUSSION |
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HNF-1 and HNF-1
were first identified in the liver, where
they regulate the promoters of liver-specific genes such as albumin and
1-antitrypsin (11). However, HNF-1
and
HNF-1
are also highly expressed in other epithelial tissues,
including the kidney (6). The expression of HNF-1
and
HNF-1
in the kidney overlaps with the expression of Ksp-cadherin.
Like Ksp-cadherin, HNF-1
and HNF-1
are expressed exclusively in
tubular epithelial cells in the kidney and are not expressed in
glomeruli or in nonepithelial cells. HNF-1
is expressed in all
segments of the nephron and in renal collecting tubules, whereas
HNF-1
is primarily expressed in proximal tubules (12,
24). HNF-1
is also expressed in the ureteric bud, mesonephric
(Wolffian) duct, and Müllerian duct, which are all sites of
Ksp-cadherin gene expression in the developing embryo (9).
During kidney development, HNF-1
and HNF-1
are expressed before
Ksp-cadherin (3, 17, 22). HNF-1
appears at embryonic
day 12.5 and is expressed in the branching ureteric bud,
comma-shaped bodies, S-shaped bodies, and developing renal tubules.
HNF-1
appears at embryonic day 14.5 and is expressed in
late S-shaped bodies and maturing proximal tubules. Thus the patterns
of expression, particularly of HNF-1
, are consistent with a role in
regulating Ksp-cadherin gene expression.
Previous studies identified an 82-bp region within the Ksp-cadherin
promoter that is essential for activity in transfected renal epithelial
cells. In the present study, we show that this region contains a
cell-specific DNase-hypersensitive site that represents a potential
binding site for a tissue-specific transcription factor. This region
contains a consensus HNF-1 site at nucleotide 55 that is highly
conserved between mice and humans. Mutations that disrupt the consensus
HNF-1 binding site inhibit promoter activity in transiently transfected
renal epithelial cells. Studies using EMSA show that HNF-1
and
HNF-1
bind specifically to the
55 site of the Ksp-cadherin
promoter. Cotransfection experiments show that HNF-1
and HNF-1
both transactivate the promoter. HNF-1
and HNF-1
are very likely
to activate the promoter directly rather than indirectly, because two
independent mutations of the consensus HNF-1 site that prevent HNF-1
binding also prevent transactivation. Expression of a dominant
gain-of-function mutant of HNF-1
stimulates Ksp-cadherin promoter
activity, whereas expression of a dominant-negative mutant inhibits
promoter activity. Taken together, these results demonstrate that
HNF-1
and HNF-1
can directly bind to the Ksp-cadherin gene
promoter and regulate its activity. To our knowledge, Ksp-cadherin is
the first kidney-specific promoter that has been shown to be regulated
by HNF-1
.
Recent studies suggest that HNF-1 plays an important role in gene
regulation in the renal proximal tubule. The promoter of the cytosolic
phosphoenolpyruvate carboxykinase (PEPCK) gene contains an
HNF-1 binding site that is essential for its expression in renal
proximal tubules in transgenic mice (23). Studies in
cultured cells have shown that HNF-1
can also regulate the promoters
of the facilitative glucose transporter (GLUT2) and dipeptidyl
peptidase IV, genes that are expressed in renal proximal tubules and in other tissues (8, 14). Knockout mice that lack HNF-1
develop renal Fanconi syndrome with glucosuria (24), and
humans with heterozygous mutations of HNF-1
have a diminished renal
threshold for glucose (21). The impairment in urinary
glucose reabsorption in HNF-1
mutant mice is because of decreased
expression of SGLT2, which encodes an Na+/glucose
cotransporter that is expressed in renal proximal tubules and the
intestine (25). These results suggest that HNF-1
regulates tissue-specific gene expression in the proximal tubule.
Although HNF-1
can bind to the Ksp-cadherin promoter, the expression
of Ksp-cadherin transcripts is not altered in HNF-1
mutant mice, suggesting that HNF-1
is not essential for Ksp-cadherin gene expression. It is possible that expression of HNF-1
compensates for
the absence of HNF-1
. The majority of genes with
cis-acting elements that bind to HNF-1
, including SGLT1,
GLUT2, and PEPCK, are not affected by the lack of HNF-1
, probably
because of the abundant expression of HNF-1
in the kidney.
HNF-1 can also bind to the Ksp-cadherin promoter and stimulate its
activity. HNF-1
has previously been shown to regulate the
cAMP-dependent transcription of the urokinase-type plasminogen activator gene in LLC-PK1 renal epithelial cells
(18). During kidney development, the expression of
HNF-1
coincides with renal tubulogenesis (9, 17, 22).
Like Ksp-cadherin, HNF-1
is expressed in all segments of the nephron
and in renal collecting tubules, mesonephric tubules, mesonephric duct,
ureteric bud, and Müllerian duct. Ksp-cadherin is expressed in
mIMCD-3 cells that endogenously express HNF-1
but do not express
HNF-1
. Taken together with the observation that mutations of
HNF-1
affect Ksp-cadherin promoter activity in transfected cells,
these results suggest that HNF-1
may be the principal member of the
HNF-1 family that regulates the Ksp-cadherin promoter in vivo. However,
the function of HNF-1
in the kidney remains unclear because HNF-1
mutant embryos do not survive past embryonic day 7.5, which
is before the period of kidney organogenesis and Ksp-cadherin
expression (3, 10). Studies using conditional gene
targeting will be needed to determine whether HNF-1
is required for
Ksp-cadherin gene expression in the kidney and GU tract.
Mutations of the human HNF-1 gene (TCF2) are responsible
for the autosomal dominant disorder maturity-onset diabetes mellitus of
the young, type 5 (MODY5). In addition to impaired insulin secretion,
some patients with MODY5 develop renal cysts and abnormalities of the
female genital tract, including rudimentary uterus and vaginal aplasia
(4, 36). Mutations of HNF-1
that involve the
homeodomain and/or POU domain interfere with DNA binding and produce
loss-of-function or dominant-negative effects (32). In the present study, a mouse HNF-1
mutant (A263fsinsGG)
lacking the third helix of the homeodomain inhibited the activation of the Ksp-cadherin promoter produced by wild-type HNF-1
, consistent with a dominant-negative mutation. In contrast, the P328L329fsdelCCTCT mutation of human HNF-1
produces a dominant gain-of-function mutant.
This mutation consists of a deletion of a CCTCT pentanucleotide at
codon 328 that is predicted to encode a truncated protein containing an
intact homeodomain and POU domain in which 230 amino acids have
been deleted from the COOH terminus and replaced with 29 amino acids
from the frame-shifted reading frame (5). Previous studies
have shown that expression of the P328L329fsdelCCTCT mutant in HeLa
cells produces greater activation of a synthetic promoter containing
four HNF-1 binding sites than expression of wild-type HNF-1
(35).
In the present study, we extend these results by showing that a mouse
HNF-1 mutant lacking 236 amino acids at the COOH terminus functions
as a dominant activator of the Ksp-cadherin gene promoter. Deletion of
the COOH-terminal domain by itself is sufficient to produce
constitutive activation, indicating that the 29 amino acids that are
introduced by the frame-shifted open reading frame are not required.
The mechanism by which deletion of the COOH-terminal domain of HNF-1
produces constitutive transcriptional activation is not known.
Mammalian one-hybrid assays unexpectedly showed that the deleted region
of the COOH terminus did not contain a transrepression domain (data not
shown). Rather, a GAL4 fusion protein containing the COOH terminus
strongly stimulated the activity of a GAL4-responsive promoter,
demonstrating that the COOH terminus of mouse HNF-1
, like HNF-1
,
contains a transactivation domain. The reason deletion of the
transactivation domain produces a stronger activator is not known but
is likely to involve complex interactions of the COOH terminus with the
rest of the HNF-1
protein.
Humans who are heterozygous for mutations of the HNF-1 gene
(TCF2) exhibit renal cystic diseases, including cystic
dysplasia and the hypoplastic form of glomerulocystic disease. However, the target genes that are responsible for these phenotypes are not
known. The observation that HNF-1
regulates the activity of the
Ksp-cadherin promoter raises the possibility that dysregulated expression of Ksp-cadherin, a putative cell adhesion molecule of
urogenital epithelia, may contribute to developmental abnormalities of
the kidney and GU tract in humans with HNF-1
mutations. Further studies will be required to determine whether the expression of Ksp-cadherin is affected in humans with MODY5 and kidney cysts.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Crabtree for providing HNF-1 and HNF-1
plasmids, A. Kaup for expert technical assistance, B. McNally for critically reviewing the manuscript, and D. Copeland and K. Trueman for
assistance with the preparation of the manuscript. We thank one of the
reviewers for suggesting the control experiments shown in Figs.
3B and 7B.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-42921.
Address for correspondence: P. Igarashi, Div. of Nephrology, UT Southwestern, 5323 Harry Hines Blvd., MC 8856, Dallas, TX 75390-8856 (E-mail: peter.igarashi{at}utsouthwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 7, 2002;10.1152/ajprenal.00128.2002
Received 3 April 2002; accepted in final form 6 May 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Andrews, NC,
and
Faller DV.
A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.
Nucleic Acids Res
19:
2499,
1991[ISI][Medline].
2.
Angst, BD,
Marcozzi C,
and
Magee AI.
The cadherin superfamily: diversity in form and function.
J Cell Sci
114:
629-641,
2001
3.
Barbacci, E,
Reber M,
Ott MO,
Breillat C,
Huetz F,
and
Cereghini S.
Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification.
Development
126:
4795-4805,
1999
4.
Bingham, C,
Bulman MP,
Ellard S,
Allen LIS,
Lipkin GW,
van't Hoff WG,
Woolf AS,
Rizzoni G,
Novelli G,
Nicholls AJ,
and
Hattersley AT.
Mutations in the hepatocyte nuclear factor-1 gene are associated with familial hypoplastic glomerulocystic kidney disease.
Am J Hum Genet
68:
219-224,
2001[ISI][Medline].
5.
Bingham, C,
Ellard S,
Allen L,
Bulman M,
Shepherd M,
Frayling T,
Berry PJ,
Clark PM,
Lindner T,
Bell GI,
Ryffel GU,
Nicholls AJ,
and
Hattersley AT.
Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1.
Kidney Int
57:
898-907,
2000[ISI][Medline].
6.
Blumenfeld, M,
Maury M,
Chouard T,
Yaniv M,
and
Condamine H.
Hepatic nuclear factor 1 (HNF1) shows a wider distribution than products of its known target genes in developing mouse.
Development
113:
589-599,
1991[Abstract].
7.
Boyes, J,
and
Felsenfeld G.
Tissue-specific factors additively increase the probability of the all-or-none formation of a hypertensive site.
EMBO J
15:
2496-2507,
1996[Abstract].
8.
Cha, JY,
Kim H,
Kim KS,
Hur MW,
and
Ahn Y.
Identification of transacting factors responsible for the tissue-specific expression of human glucose transporter type 2 isoform gene.
J Biol Chem
275:
18358-18365,
2000
9.
Coffinier, C,
Barra J,
Babinet C,
and
Yaniv M.
Expression of the vHNF1/HNF1 homeoprotein gene during mouse organogenesis.
Mech Dev
89:
211-213,
1999[ISI][Medline].
10.
Coffinier, C,
Thepot D,
Babinet C,
Yaniv M,
and
Barra J.
Essential role for the homeoprotein vHNF1/HNF1 in visceral endoderm differentiation.
Development
126:
4785-4794,
1999
11.
de Simone, V,
and
Cortese R.
Transcription factors and liver-specific genes.
Biochim Biophys Acta
1132:
119-126,
1992[ISI][Medline].
12.
de Simone, V,
de Magistris L,
Lazzaro D,
Gerstner J,
Monaci P,
Nicosia A,
and
Cortese R.
LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia.
EMBO J
10:
1435-1443,
1991[Abstract].
13.
Elgin, SCR
DNase I-hypersensitive sites of chromatin.
Cell
27:
413-415,
1981[ISI][Medline].
14.
Erickson, RH,
Lai RS,
and
Kim YS.
Role of hepatocyte nuclear factor 1 and 1
in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells.
Biochem Biophys Res Commun
270:
235-239,
2000[ISI][Medline].
15.
Igarashi, P,
Shashikant CS,
Thomson RB,
White DA,
Liu-Chen S,
Ruddle FH,
and
Aronson PS.
Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice.
Am J Physiol Renal Physiol
277:
F599-F610,
1999
16.
Kuo, CJ,
Conley PB,
Hsieh CL,
Francke U,
and
Crabtree GR.
Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1.
Proc Natl Acad Sci USA
87:
9838-9842,
1990[Abstract].
17.
Lazzaro, D,
de Simone V,
de Magistris L,
Lehtonen E,
and
Cortese R.
LFB1 and LFB3 homeoproteins are sequentially expressed during kidney development.
Development
114:
469-479,
1992[Abstract].
18.
Marksitzer, R,
Stief A,
Menoud PA,
and
Nagamine Y.
Role of LFB3 in cell-specific cAMP induction of the urokinase-type plasminogen activator gene.
J Biol Chem
270:
21833-21838,
1995
19.
Mendel, DB,
and
Crabtree GR.
HNF-1, a member of a novel class of dimerizing homeodomain proteins.
J Biol Chem
266:
677-680,
1991
20.
Mendel, DB,
Hansen LP,
Graves MK,
Conley PB,
and
Crabtree GR.
HNF-1 and HNF-1
(vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro.
Genes Dev
5:
1042-1056,
1991[Abstract].
21.
Menzel, R,
Kaisaki PJ,
Rjasanowski I,
Heinke P,
Kerner W,
and
Menzel S.
A low renal threshold for glucose in diabetic patients with a mutation in the hepatocyte nuclear factor-1 (HNF-1
) gene.
Diabet Med
15:
816-820,
1998[ISI][Medline].
22.
Ott, MO,
Rey-Campos J,
Cereghini S,
and
Yaniv M.
vHNF1 is expressed in epithelial cells of distinct embryonic origin during development and precedes HNF1 expression.
Mech Dev
36:
47-58,
1991[ISI][Medline].
23.
Patel, YM,
Yun JS,
Liu J,
McGrane MM,
and
Hanson RW.
An analysis of regulatory elements in the phosphoenolpyruvate carboxykinase (GTP) gene which are responsible for its tissue-specific expression and metabolic control in transgenic mice.
J Biol Chem
269:
5619-5628,
1994
24.
Pontoglio, M,
Barra J,
Hadchouel M,
Doyen A,
Kress C,
Poggi Bach J,
Babinet C,
and
Yaniv M.
Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.
Cell
84:
575-585,
1996[ISI][Medline].
25.
Pontoglio, M,
Prie D,
Cheret C,
Doyen A,
Leroy C,
Froguel P,
Velho G,
Yaniv M,
and
Friedlander G.
HNF1 controls renal glucose reabsorption in mouse and man.
EMBO Rep
1:
359-365,
2000
26.
Rauchman, MI,
Nigam SK,
Delpire E,
and
Gullans SR.
An osmotically tolerant inner medullary collecting duct cell line derived from an SV40 transgenic mouse.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F416-F424,
1993
27.
Rey-Campos, J,
Chouard T,
Yaniv M,
and
Cereghini S.
vHNF1 is a homeoprotein that activates transcription and forms heterodimers with HNF1.
EMBO J
10:
1445-1457,
1991[Abstract].
28.
Shao, X,
Johnson JE,
Richardson JA,
Hiesberger T,
and
Igarashi P.
A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract.
J Am Soc Nephrol
13:
1824-1836,
2002
29.
Thomson, RB,
and
Aronson PS.
Immunolocalization of Ksp-cadherin in the adult and developing rabbit kidney.
Am J Physiol Renal Physiol
277:
F146-F156,
1999
30.
Thomson, RB,
Igarashi P,
Biemesderfer D,
Kim R,
Abu-Alfa A,
Soleimani M,
and
Aronson PS.
Isolation and cDNA cloning of Ksp-cadherin, a novel kidney-specific member of the cadherin multigene family.
J Biol Chem
270:
17594-17601,
1995
31.
Thomson, RB,
Ward DC,
Quaggin SE,
Igarashi P,
Muckler ZE,
and
Aronson PS.
cDNA cloning and chromosomal localization of the human and mouse isoforms of Ksp-cadherin.
Genomics
51:
445-451,
1998[ISI][Medline].
32.
Tomura, H,
Nishigori H,
Sho K,
Yamagata K,
Inoue I,
and
Takeda J.
Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1 mutations in familial type 2 diabetes mellitus.
J Biol Chem
274:
12975-12978,
1999
33.
Wertz, K,
and
Herrmann BG.
Kidney-specific cadherin (cdh16) is expressed in embryonic kidney, lung, and sex ducts.
Mech Dev
84:
185-188,
1999[ISI][Medline].
34.
Whyte, DA,
Li C,
Thomson RB,
Nix SL,
Zanjani R,
Karp SL,
Aronson PS,
and
Igarashi P.
Ksp-cadherin gene promoter. I. Characterization and renal epithelial-cell-specific activity.
Am J Physiol Renal Physiol
277:
F587-F598,
1999
35.
Wild, W,
Pogge von Strandmann E,
Nastos A,
Senkel S,
Lingott-Frieg A,
Bulman M,
Bingham C,
Ellard S,
Hattersley AT,
and
Ryffel GU.
The mutated human gene encoding hepatocyte nuclear factor 1 inhibits kidney formation in developing Xenopus embryos.
Proc Natl Acad Sci USA
97:
4695-4700,
2000
36.
Woolf, AS.
Diabetes, genes, and kidney development.
Kidney Int
57:
1202-1203,
2000[ISI][Medline].
37.
Yagi, T,
and
Takeichi M.
Cadherin superfamily genes: functions, genomic organization, and neurologic diversity.
Genes Dev
14:
1169-1180,
2000