Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1beta

Yun Bai1, Marco Pontoglio2, Thomas Hiesberger1, Angus M. Sinclair1, and Peter Igarashi1

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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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-1alpha and HNF-1beta 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-1alpha -deficient mice. However, expression of a gain-of-function HNF-1beta 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-1beta . Mutations of HNF-1beta , 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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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 beta -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-1alpha and HNF-1beta , that have been implicated in tissue-specific gene regulation in the liver, pancreas, kidney, and intestine (16, 20, 27). HNF-1alpha and HNF-1beta 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-1alpha is a transcriptional activator and contains three distinct activation domains within its COOH terminus. This domain is not conserved in HNF-1beta , 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-1alpha and HNF-1beta are highly expressed in the developing kidney but in different patterns. HNF-1alpha is primarily expressed in proximal tubules, whereas HNF-1beta is expressed in all nephron segments and the renal collecting system and sex ducts. The current study was undertaken to investigate whether HNF-1alpha and/or HNF-1beta 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-1beta mutants that are similar to those found in humans with inherited renal cysts and diabetes.


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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, pSV2beta gal, luciferin, and reporter lysis buffer were from Promega (Madison, WI). Galacton-Star beta -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-1alpha -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-1alpha (-/-) mice and their age-matched heterozygous HNF-1alpha (+/-) and wild-type HNF-1alpha (+/+) 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-HNF1alpha ) containing the mouse HNF-1alpha cDNA and a plasmid (pBJ5-HNF1beta ) containing the mouse HNF-1beta cDNA (B splice variant) were generous gifts from Dr. Gerald Crabtree (Stanford University). The 3.1-kb EcoRI fragment from pBJ5-HNF1beta was cloned into the EcoRI site of pBluescript II KS(+), creating the plasmid pBS-KS-HNF1beta . The HNF-1alpha coding region was amplified from pBS-KS-HNF1alpha with primers 5'-ACCATGGTTTCTAAGCTGAGCCAGCTG and 5'-GGGTTTAAACTTACTGGGAAGAGGAGGCCATCTGG and cloned into pcDNA3.1/V5/His-TOPO (Invitrogen), creating the expression plasmid pcDNA3-HNF1alpha . The HNF-1beta coding region was amplified from pBS-KS-HNF1beta with primers 5'-ACCATGGTGTCCAAGCTCACGTCGCTC and 5'-CGGGTTTAAACTCACCAGGCTTGCAGTGGACACTGT and cloned into pcDNA3.1/V5/His-TOPO, creating the expression plasmid pcDNA3-HNF1beta . The coding regions of both plasmids were sequenced completely.

An expression plasmid encoding a truncated form of mouse HNF-1beta lacking 236 amino acids at the COOH terminus (pcDNA3-HNF1beta Delta C) was produced by PCR amplification from pBS-KS-HNF1beta using primers 5'-ACCATGGTGTCCAAGCTCACGTCGCTC and 5'-CTGGTTGGAGCTATAGGCATCCAT and cloning into pcDNA3.1/V5/His-TOPO. A stop codon was introduced between the V5 and His epitope tags by PCR amplification with primers 5'-GTTTAAACGATATCGTTAACGCGTAGAATCGAGACCGAGGAG and 5'-ACCATGGTGTCCAAGCTCACGTCGCTC. The PCR product was digested with EcoRV, and the 100-bp fragment was cloned into the EcoRV site of the plasmid. An expression plasmid encoding a mutant form of mouse HNF-1beta corresponding to the human A263fsinsGG mutation (pcDNA3-A263fsinsGG) was produced by PCR amplification using primers 5'-ACCATGGTGTCCAAGCTCACGTCGCTC and 5'-CTAACCGGCCTCCCTCTCTTCCTTGC and cloning into pcDNA3.1/V5/His-TOPO.

A reporter plasmid containing the proximal Ksp-cadherin promoter was derived from pKsp(2608F)-Luc (34) by digestion with AvrII and NheI, removal of the 924- and 1,571-bp fragments, and recircularization. The resulting plasmid [pKsp(113)-Luc] contained nucleotides -113 to +74 of the Ksp-cadherin promoter linked to a luciferase reporter gene.

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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Table 1.   Sequences of mutations used in this study

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-beta Gal or pCMV-beta Gal encoding E. coli beta -galactosidase. Transfected cells were grown in standard culture medium for 48 h and then were assayed for luciferase and beta -galactosidase activity.

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 beta -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-1beta mutants affected beta -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-1alpha or HNF-1beta (pBS-KS-HNF1alpha and pBS-KS-HNF1beta , 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-1alpha 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 11beta -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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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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Fig. 1.   Evolutionary conservation of the kidney-specific cadherin (Ksp-cadherin) promoter. A: genomic structure of the human Ksp-cadherin gene obtained from the sequence of a bacterial artificial chromosome (BAC) clone. Filled boxes indicate exons. B: alignment of the sequences of the Ksp-cadherin promoter from the mouse (M) and human (H). Sequence alignment was performed using the GeneStream program LALIGN with gap penalties of -16 and -4. Colons, positions of identity; -, gaps introduced to optimize the alignment; arrow, transcription initiation site; horizontal bars, consensus transcription factor binding sites. [The hepatocyte nuclear factor (HNF)-1 site shown is the reverse complement of the consensus.] Nucleotide positions are numbered at right relative to the transcription initiation site at +1. C: plot of the percent identity between the mouse and human Ksp-cadherin promoters. Horizontal bar indicates 80% identity.

Figure 1B shows the alignment of the proximal promoters of the mouse and human Ksp-cadherin genes, and Fig. 1C shows a plot of the percent identity over the region. Overall, the mouse and human promoters were 62% identical (excluding gaps). However, two regions were >80% identical: a 38-bp sequence beginning at nucleotide -762 containing three to four tandem repeats (data not shown) and the 120-bp region immediately upstream of the transcription start site. The proximal 120 bp of the promoter were even more highly conserved than the 5'-untranslated region of the mRNA transcript. The consensus binding site for HNF-1 at nucleotide -55, the consensus HNF-3 site at nucleotide -62, and the GC-rich region from nucleotides -15 to -43 were highly conserved in the mouse and human promoters, highlighting the potential importance of these elements for promoter activity.

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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Fig. 2.   DNase I hypersensitive site mapping of the 5'-end of the Ksp-cadherin gene. A: genomic structure of a 4.2-kb HindIII restriction fragment containing exons 1-6 (gray boxes) and 1.3 kb of 5'-flanking region of the Ksp-cadherin gene. Bracketed line indicates the 3'-probe used for end labeling. B: Southern blot analysis of genomic DNA from mouse inner medullary collecting duct (mIMCD)-3 cells after digestion with graded concentrations of DNase I. Filled arrowhead, parental 4.2-kb band; open arrowhead, 3-kb subband, which corresponds to a hypersensitive at the position shown by the vertical arrow in A. C: Southern blot analysis of genomic DNA from 10T1/2 cells, which do not express endogenous Ksp-cadherin. Filled arrowhead, parental 4.2-kb band. No subbands were detected in 10T1/2 cells.

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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Fig. 3.   Mutational analysis of the Ksp-cadherin promoter. A: luciferase reporter plasmids (0.6 µg) containing the wild-type (wt) or mutated (m1-m13) Ksp-cadherin promoter (nucleotides -113 to +74) were transiently transfected into mIMCD-3 cells, and luciferase activity was measured after 48 h. To control for transfection efficiency, cells were cotransfected with 10 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Data are means ± SE of 9 independent transfections. The mutations that were used are listed in Table 1. *P < 0.05 compared with wild-type promoter. B: luciferase reporter plasmids (0.6 µg) containing the wild-type or mutated Ksp-cadherin promoter were transiently transfected into mIMCD-3 cells (filled) or HeLa cells (open bars). Cells were cotransfected with 5 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Data are means ± SE of 6 independent transfections. *P < 0.05 compared with wild-type promoter.

Figure 3B compares the effects of mutations of the Ksp-cadherin promoter in transfected mIMCD-3 cells, which express endogenous HNF-1, and HeLa cells that do not contain HNF-1. Transfection of the wild-type promoter-reporter plasmid in mIMCD-3 cells produced high levels of luciferase activity, and mutations of the consensus HNF-1 binding site (m3 and m13) inhibited activity by 75-80% (filled bars). In contrast, the wild-type Ksp-cadherin promoter had low activity in HeLa cells, and mutations 3 and 13 had no significant effect (open bars). These results indicated that the effects of the mutations were specific.

HNF-1alpha and HNF-1beta 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).

To determine whether HNF-1alpha and/or HNF-1beta could bind to the -55 site of the Ksp-cadherin promoter, EMSA was performed using programmed reticulocyte lysates and a radiolabeled oligonucleotide containing the -55 site. Figure 4A shows that binding activity was present in lysates programmed with HNF-1alpha (lane 3) or HNF-1beta (lane 9) but was not present in unprogrammed lysates (lane 2). Binding was specific since it could be competed with increasing concentrations of unlabeled wild-type oligonucleotide (lanes 4, 6, 10, and 12) but not with equivalent amounts of an oligonucleotide containing a mutation (m3) of the consensus HNF-1 site (lanes 5, 7, 11, and 13). A consensus HNF-1 binding site from the beta -fibrinogen promoter also competed for binding, although less effectively than the wild-type sequence (lanes 8 and 14).


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Fig. 4.   Binding of HNF-1alpha and HNF-1beta to the Ksp-cadherin promoter. A: electrophoretic mobility shift assay (EMSA) was performed using a radiolabeled oligonucleotide containing nucleotides -79 to -50 of the Ksp-cadherin promoter and reticulocyte lysates programmed with HNF-1alpha (lanes 3-8), HNF-1beta (lanes 9-14), unprogrammed lysates (un; lane 2), or no protein (lane 1). Binding reactions were performed in the presence of unlabeled wild-type (wt) oligonucleotide (lanes 4, 6, 10, and 12), unlabeled mutated (m3) oligonucleotide (lanes 5, 7, 11, and 13), unlabeled oligonucleotide containing a consensus HNF-1alpha binding site from the rat beta -fibrinogen gene (lanes 8 and 14), or no competitor (Comp) (lanes 3 and 9). B: EMSA was performed using the -79/-50 oligonucleotide and nuclear extracts from untransfected mIMCD-3 cells (lanes 2-10) or no protein (lane 1). Binding reactions were performed in the presence of an antibody (Ab) directed against HNF-1alpha (lanes 3 and 7), HNF-1beta (lanes 4 and 8), irrelevant antibody (lanes 5 and 9), or 100-fold excess unlabeled wild-type oligonucleotide (lane 10). C: EMSA was performed using the -79/-50 oligonucleotide and reticulocyte lysates programmed with HNF-1alpha (lanes 2-6), HNF-1beta (lanes 7-11), or unprogrammed lysates (lane 1). Binding reactions were performed in the presence of no competitor (lanes 2 and 7), 5-fold (lanes 3 and 8) or 50-fold (lanes 5 and 10) excess unlabeled wild-type oligonucleotide, or 5-fold (lanes 4 and 9) or 50-fold (lanes 6 and 11) excess unlabeled mutated (m13) oligonucleotide. D: EMSA was performed using the -79/-50 oligonucleotide and nuclear extracts from untransfected mIMCD-3 cells (lanes 2-6) or no protein (lane 1). Binding reactions were performed in the presence of no competitor (lane 2), 5-fold (lane 3) or 50-fold (lane 5) excess unlabeled wild-type oligonucleotide, or 5-fold (lane 4) or 50-fold (lane 6) excess unlabeled mutated (m13) oligonucleotide. Arrows, retarded complexes; S.S., supershift; F, unbound oligonucleotide.

Northern blot analysis showed that mIMCD-3 cells endogenously expressed HNF-1beta but did not express HNF-1alpha , consistent with the pattern of expression of these genes in the renal collecting duct from which this cell line was derived (data not shown). To determine whether mIMCD-3 cells contained endogenous HNF-1beta that bound to the -55 site, EMSA was performed using nuclear extracts. Figure 4B, lanes 2 and 6, shows that nuclear extracts from mIMCD-3 cells contained proteins that bound to an oligonucleotide containing the -55 site in a concentration-dependent manner. Binding was specific, since it could be competed with a 100-fold excess of unlabeled oligonucleotide (lane 10). To verify the presence of HNF-1beta in the DNA-protein complex, binding was performed in the presence of specific antibodies against HNF-1alpha or HNF-1beta . Addition of an antibody against HNF-1beta resulted in a supershift, indicating that HNF-1beta was present in the DNA-protein complex (lanes 4 and 8). In contrast, no supershift was observed after addition of an equal amount of irrelevant antibody (lanes 5 and 9). Moreover, no supershift was observed after addition of an antibody directed against HNF-1alpha (lanes 3 and 7), consistent with the absence of HNF-1alpha in mIMCD-3 cells. Control experiments verified that the HNF-1alpha antibody could supershift the complex formed by HNF-1alpha in programmed reticulocyte lysates (data not shown).

To determine whether an independent mutation of the consensus HNF-1 site also disrupted protein binding, we studied the effects of mutation 13 (Table 1). Competitive EMSA was performed using lysates programmed with HNF-1alpha or HNF-1beta (Fig. 4C) or nuclear extracts from mIMCD-3 cells (Fig. 4D). In each case, there was specific binding to an oligonucleotide containing the -55 site, which could be competed with excess unlabeled wild-type oligonucleotide. However, like mutation 3, an excess of unlabeled oligonucleotide containing mutation 13 was unable to compete, indicating that mutation 13 disrupted the HNF-1 binding site. Taken together, these results demonstrate that disruption of conserved nucleotides within the consensus recognition site prevented HNF-1 binding.

HNF-1alpha and HNF-1beta transactivate the Ksp-cadherin promoter directly. To test whether HNF-1alpha or HNF-1beta 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-1alpha or HNF-1beta and a luciferase reporter plasmid containing the Ksp-cadherin promoter. Figure 5A shows that expression of HNF-1alpha stimulated luciferase activity by 11-fold (open bars), whereas expression of HNF-1beta produced a 3-fold stimulation (filled bars). HNF-1alpha has previously been shown to be a stronger transcriptional activator than HNF-1beta . At higher concentrations of expression plasmid, luciferase activity began to decrease, consistent with the phenomenon of squelching. Expression of HNF-1alpha together with HNF-1beta resulted in stimulation of activity that was comparable to HNF-1alpha by itself (data not shown).


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Fig. 5.   Transactivation of the Ksp-cadherin promoter by HNF-1alpha and HNF-1beta . A: HeLa cells were cotransfected with 0.6 µg pKsp(113)-Luc and the indicated amounts of pcDNA3-HNF1alpha (open bars) or pcDNA3-HNF1beta (filled bars). Cells were cotransfected with 5 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Normalized luciferase activity is shown relative to cells transfected with 0.5 µg empty pcDNA3 expression plasmid. Data are means ± SE of 9 independent transfections. *P < 0.05 compared with cells transfected with empty pcDNA3. B: HeLa cells were cotransfected with 0.6 µg pcDNA3-HNF1alpha (black and gray bars) or pcDNA3-HNF1beta (open and hatched bars) and 0.6 µg luciferase reporter plasmid containing the wild-type Ksp-cadherin promoter (filled and open bars) or mutated (m3) promoter (gray and hatched bars). Cells were also transfected with 5 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Data are means ± SE of 9 independent transfections. *P < 0.002 compared with wild-type promoter. C: effects of mutations of the Ksp-cadherin promoter on transactivation by HNF-1alpha . HeLa cells were cotransfected with 0.1 µg pcDNA3-HNF1alpha (filled bars) or empty pcDNA3 (open bars) and 0.6 µg luciferase reporter plasmid containing the wild-type or mutated (m1-m7) Ksp-cadherin promoter. Cells were also transfected with 10 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Data are means ± SE of 3 independent transfections. *P < 0.05 compared with wild-type promoter.

To distinguish whether activation of the Ksp-cadherin promoter by HNF-1 was direct or indirect, HeLa cells were cotransfected with HNF-1 expression plasmids and a mutated reporter plasmid containing the same mutation of the -55 site (m3) that inhibited HNF-1 binding. Figure 5B shows that cotransfection of HNF-1beta and the wild-type promoter resulted in a two- to threefold stimulation of luciferase activity (open bars), whereas cotransfection of HNF-1alpha and the wild-type promoter resulted in a six- to eightfold stimulation (filled bars), similar to the results shown in Fig. 5A. The stimulation of luciferase activity by HNF-1alpha and HNF-1beta was completely abolished by cotransfection of a reporter plasmid containing the mutated promoter (gray and hatched bars). These results establish that both HNF-1alpha and HNF-1beta can stimulate the Ksp-cadherin promoter and that an intact HNF-1 binding site at position -55 is required for transactivation.

To extend these results, additional mutational analysis was performed. Figure 5C shows the results of cotransfection of HeLa cells with wild-type or mutant reporter plasmids and an HNF-1alpha expression plasmid (filled bars) or an equivalent amount of empty pcDNA3.1 (open bars). The mutations had no significant effect on the basal promoter activity in HeLa cells (open bars). However, mutations 2, 3, and 13 strongly inhibited or abolished transactivation by HNF-1alpha . These mutations disrupted highly conserved nucleotides in the consensus HNF-1 binding site. In contrast, mutations 1 and 7, which do not alter the consensus HNF-1 site, had no significant effect on transactivation. Mutation 4, which affects a sequence that is not as highly conserved, produced an intermediate effect.

Expression of Ksp-cadherin in HNF-1alpha mutant mice. Transgenic mice in which the HNF-1alpha (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-1alpha mutant mice, Northern blot analysis was performed. As shown in Fig. 6, the expression of Ksp-cadherin mRNA normalized to the expression of 11beta -hydroxysteroid dehydrogenase type 1 was similar in HNF-1alpha mutant homozygotes, heterozygotes, and wild-type mice. These results indicated that HNF-1alpha was not essential for the expression of the Ksp-cadherin gene in vivo.


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Fig. 6.   Expression of Ksp-cadherin in HNF-1alpha mutant mice. Top: Northern blot analysis on total RNA (15 µg) from the kidneys of animals at age 12-15 days. Four samples for each genotype are shown {from left to right: wild-type [HNF1(+/+)], heterozygous [HNF1(+/-)], and mutant homozygous [HNF1(-/-)]}. KSP, Ksp-cadherin; 11beta HSD1, 11beta -hydroxysteroid dehydrogenase type 1. Bottom: histogram showing mean values and SE of the Ksp-cadherin signal normalized to 11beta HSD1.

Mutations of HNF-1beta alter the activity of the Ksp-cadherin promoter. Because HNF-1beta 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-1beta mutant mice. As an alternative approach, we examined the effect of mutations of HNF-1beta on transactivation of the Ksp-cadherin promoter. The P328L329fsinsCCTCT mutation of the human HNF-1beta 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-1beta on Ksp-cadherin promoter activity, mIMCD-3 cells were cotransfected with expression plasmids encoding either wild-type HNF-1beta or an HNF-1beta mutant containing a COOH-terminal deletion similar to the one in the human P328L329fsinsCCTCT mutant (HNF-1beta Delta C). Figure 7A shows that expression of wild-type HNF-1beta had no effect on promoter activity, presumably because the cells endogenously express HNF-1beta . 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-1beta on Ksp-cadherin promoter activity. The A263fsinsGG mutation of the human HNF-1beta gene produces a protein that can dimerize with wild-type HNF-1beta 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-1beta . 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-1beta and the A263fsinsGG dominant-negative mutant in HeLa cells. Expression of wild-type HNF-1beta 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-1beta in Ksp-cadherin promoter activity.


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Fig. 7.   Effects of mutations of HNF-1beta on Ksp-cadherin promoter activity. A: mIMCD-3 cells were cotransfected with 0.6 µg pKsp(113)-Luc and the indicated amounts of pcDNA3-HNF1beta (open bars) or pcDNA3-HNF1beta Delta C (filled bars). Cells were cotransfected with 5 ng pCMV-beta Gal, and luciferase activity was normalized to beta -galactosidase activity. Normalized luciferase activity is shown relative to cells transfected with 1 µg empty pcDNA3 expression plasmid. Data are means ± SE of 9 independent transfections. *P < 0.05 compared with cells transfected with empty pcDNA3. B: mIMCD-3 cells were cotransfected with luciferase reporter plasmids (0.6 µg) containing the wild-type or mutated (m3, m13) Ksp-cadherin promoter and either 0.2 µg pcDNA3-A263fsinsGG (open bars) or 0.2 µg empty pcDNA3 expression plasmid (filled bars). Luciferase activity was normalized to beta -galactosidase activity. Data are means ± SE of 6 independent transfections. *P < 0.05 compared with cells transfected with empty pcDNA3. C: HeLa cells were cotransfected with 0.6 µg pKsp(113)-Luc and 0.1 µg pcDNA3-HNF1beta and/or 0.1 µg pcDNA3-A263fsinsGG. Luciferase activity was normalized to protein concentration. Data are means ± SE of 6 independent transfections. *P < 0.05 compared with cells transfected with HNF-1beta alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HNF-1alpha and HNF-1beta were first identified in the liver, where they regulate the promoters of liver-specific genes such as albumin and alpha 1-antitrypsin (11). However, HNF-1alpha and HNF-1beta are also highly expressed in other epithelial tissues, including the kidney (6). The expression of HNF-1alpha and HNF-1beta in the kidney overlaps with the expression of Ksp-cadherin. Like Ksp-cadherin, HNF-1alpha and HNF-1beta are expressed exclusively in tubular epithelial cells in the kidney and are not expressed in glomeruli or in nonepithelial cells. HNF-1beta is expressed in all segments of the nephron and in renal collecting tubules, whereas HNF-1alpha is primarily expressed in proximal tubules (12, 24). HNF-1beta 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-1alpha and HNF-1beta are expressed before Ksp-cadherin (3, 17, 22). HNF-1beta 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-1alpha 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-1beta , 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-1alpha and HNF-1beta bind specifically to the -55 site of the Ksp-cadherin promoter. Cotransfection experiments show that HNF-1alpha and HNF-1beta both transactivate the promoter. HNF-1alpha and HNF-1beta 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-1beta stimulates Ksp-cadherin promoter activity, whereas expression of a dominant-negative mutant inhibits promoter activity. Taken together, these results demonstrate that HNF-1alpha and HNF-1beta 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-1beta .

Recent studies suggest that HNF-1alpha 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-1alpha 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-1alpha develop renal Fanconi syndrome with glucosuria (24), and humans with heterozygous mutations of HNF-1alpha have a diminished renal threshold for glucose (21). The impairment in urinary glucose reabsorption in HNF-1alpha 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-1alpha regulates tissue-specific gene expression in the proximal tubule. Although HNF-1alpha can bind to the Ksp-cadherin promoter, the expression of Ksp-cadherin transcripts is not altered in HNF-1alpha mutant mice, suggesting that HNF-1alpha is not essential for Ksp-cadherin gene expression. It is possible that expression of HNF-1beta compensates for the absence of HNF-1alpha . The majority of genes with cis-acting elements that bind to HNF-1alpha , including SGLT1, GLUT2, and PEPCK, are not affected by the lack of HNF-1alpha , probably because of the abundant expression of HNF-1beta in the kidney.

HNF-1beta can also bind to the Ksp-cadherin promoter and stimulate its activity. HNF-1beta 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-1beta coincides with renal tubulogenesis (9, 17, 22). Like Ksp-cadherin, HNF-1beta 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-1beta but do not express HNF-1alpha . Taken together with the observation that mutations of HNF-1beta affect Ksp-cadherin promoter activity in transfected cells, these results suggest that HNF-1beta may be the principal member of the HNF-1 family that regulates the Ksp-cadherin promoter in vivo. However, the function of HNF-1beta in the kidney remains unclear because HNF-1beta 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-1beta is required for Ksp-cadherin gene expression in the kidney and GU tract.

Mutations of the human HNF-1beta 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-1beta 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-1beta mutant (A263fsinsGG) lacking the third helix of the homeodomain inhibited the activation of the Ksp-cadherin promoter produced by wild-type HNF-1beta , consistent with a dominant-negative mutation. In contrast, the P328L329fsdelCCTCT mutation of human HNF-1beta 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-1beta (35).

In the present study, we extend these results by showing that a mouse HNF-1beta 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-1beta 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-1beta , like HNF-1alpha , 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-1beta protein.

Humans who are heterozygous for mutations of the HNF-1beta 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-1beta 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-1beta 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-1alpha and HNF-1beta 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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ABSTRACT
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RESULTS
DISCUSSION
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