Renal Tubule-specific Transcription and Chromosomal Localization of Rat Thiazide-sensitive Na-Cl Cotransporter Gene*

Yoshihiro TaniyamaDagger, Kazunori SatoDagger, Akira Sugawara, Akira Uruno, Yukio Ikeda, Masataka Kudo, Sadayoshi Ito, and Kazuhisa Takeuchi§

From the Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan

Received for publication, February 21, 2001, and in revised form, April 18, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The molecular mechanism underlying the renal expression localization of the thiazide-sensitive Na-Cl cotransporter (TSC) gene was studied. The TSC gene was localized to chromosome 19p12-14. In cultured cells, tissue-specific transcription activity of the 5'-flanking region of the rat rTSC gene (5'FL/rTSC) was demonstrated, and the major promoter region was located between position -580 and -141. To further examine the tissue-specific transcription, transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene were generated. Immunohistochemical analysis clearly showed that LacZ gene expression was co-localized to distal convoluted tubules (DCT) with TSC, indicating that the 5'FL/rTSC regulates the renal tubule-specific TSC expression. Because a transcription factor, HFH-3 (hepatocyte nuclear factor-3/folk head homologue-3), had also been localized to DCT, a possible role of the putative cis-acting element (HFH-3/rTSC, -400/-387 position) for HFH-3 binding in the tissue-specific transcription was examined. Deletion and mutation analyses suggested that transcription of the HFH-3/rTSC was actually responsive to HFH-3, and electrophoretic mobility shift assay showed a direct binding of in vitro synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the rTSC gene is localized to rat chromosome 19p12-24. The transcription regulatory region of the TSC gene confers DCT-specific gene expression. DCT-specific transcription factor HFH-3 may be involved in the renal tubule-specific transcription of TSC gene.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Thiazide-sensitive Na-Cl cotransporter (TSC)1 is an important molecule for reabsorption of NaCl in the kidney and a target of thiazide diuretics. TSC cDNAs were cloned, and the TSC function was characterized (1-3). Mutations possibly leading to loss-of-function in the human TSC gene have been shown to cause Gitelman's syndrome, which is characterized by dehydration, hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria (4, 5). Recently, TSC-deficient mice were generated, and their phenotype was shown to bear a good resemblance to Gitelman's syndrome (6). Thus, TSC plays an important role not only in NaCl metabolism but in acid-base balance and in the metabolism of other electrolytes such as potassium, magnesium, and calcium.

TSC mRNA expression has been shown to be localized to the distal convoluted tubule (DCT) in the kidney by in situ hybridization histochemistry (7, 8) as well as reverse transcription and polymerase chain reaction (RT-PCR) with microdissected nephron segments (9). Immunohistochemistry using a specific antibody against TSC has also shown that immunoreactive TSC is localized to DCT (10, 11). However, no study to uncover the molecular mechanism underlying the renal tubule-specific TSC expression has yet been performed in any species.

Recently, a transcription factor, hepatocyte nuclear factor-3/folk head homologue-3 (HFH-3), was isolated and partly characterized (12). HFH-3 belongs to an HFH/winged helix transcription factor family and is identical to FREAC-6 (13). Members of the family have been shown to be involved in tissue- or cell-specific gene expression, as well as cellular differentiation during embryonic development (14, 15). Interestingly, HFH-3 mRNA was reported to be localized to the epithelium of the DCT, where TSC is also localized. We therefore hypothesized that HFH-3 might be involved in the TSC gene expression.

In the present study, we studied the tissue-specific expression of the TSC gene in terms of gene transcription by an in vivo as well as in vitro experiments. The structure of the 5'-flanking region of the rat TSC gene (5'FL/rTSC) was revealed, and the rTSC gene was mapped to chromosome 19p12-14 by FISH. The transcription function was shown to be tissue-specific in cultured cells. Moreover, we generated transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene to examine the tissue-specific transcription of 5'FL/rTSC. Immunohistochemical analysis clearly demonstrated that LacZ expression was localized to DCT in the transgenic rat kidney, indicating that the 5'FL/rTSC regulates the DCT-specific TSC localization in vivo. Furthermore, we identified a functional HFH-3 binding site in the major promoter region of 5'FL/rTSC, which was actually bound by in vitro synthesized HFH-3. HFH-3 may be involved at least in part in the DCT-specific expression of TSC.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Southern Blot Analysis-- To use as a probe in Southern blot analysis of rTSC gene, an rTSC cDNA fragment was first cloned by a PCR-based method with a rat kidney cDNA library (rat kidney Marathon-ready cDNA, CLONTECH) as a template using a pair of primers (sense, 5'-CCC GGA GCC ATA ATG GCA GAG CTA CCC G-3' (from position 1 to 28 in previously reported rTSC cDNA (2)); antisense, 5'-CTT CTC CAG CTG GAG AGC GTG AGT TCC G-3' (from position 3,070 to 3,097 in rTSC transcript)) under the following PCR condition: 95 °C, 30 s and 70 °C, 5 min for 25 cycles. The PCR product was then subcloned into pCR-TOPO (Invitrogen) by a TA cloning method. Sequencing was performed to confirm the complete sequence identity with the previously reported rTSC cDNA (2). The BalI digested fragment of the rTSC cDNA (892 bp) was labeled with [alpha -32P]dCTP by a random primer method (BcaBEST labeling kit, TaKaRa) and used as a probe. Rat genomic DNA was extracted from liver as described previously (16). Genomic DNA was then digested with restriction enzymes BamHI, EcoRI, or HindIII. Digested DNA was then resolved on a 0.9% agarose gel and transferred to a nylon membrane (HyBond N+, Amersham Pharmacia Biotech). Hybridization was performed in Rapid-Hyb Buffer (Amersham Pharmacia Biotech) at 65 °C for 2 h with a radiolabeled probe (25 ng). The membrane was then incubated with 2× SSC, 0.1% SDS at room temperature for 20 min, 0.1× SSC, 0.1% SDS at 65 °C for 15 min, and 0.1× SSC, 0.1% SDS at 65 °C for 15 min. The blot was exposed to an x-ray film (Kodak) for 72 h.

FISH-- Chromosome mapping of the rTSC gene was carried out by FISH based on a previously reported method (17). At first, three different DNA fragments of the rTSC gene were amplified by PCR with three primer pairs (sense-1, 5'-CCC GGA GCC ATA ATG GCA GAG CTA CCC G-3' (from position 1 to 28 in previously reported rTSC cDNA (2)), and antisense-1, 5'-GCC ACA CCC ACG GCA TTG GCA AAG GCG A-3' (from position 693 to 720 in rTSC transcript); sense-2, 5'-CCT CCT CAT CCC TTA TCT CCT GCA CCG C-3' (from position 2,570 to 2,599 in rTSC transcript), and antisense-2, 5'-CTC GTC CTT GAG CCG TCA TTG AGA CGG-3' (from position 2,799 to 2,826 in rTSC transcript); and sense-3, '-CCG TCT CAA TGA CGG CTT CAA GGA CGAG-3' (from position 2,799 to 2,826 in rTSC transcript), and antisense-3, 5'-CTT CTC CAG CTG GAG AGC GTG AGT TCC G-3' (from position 3,070 to 3,097 in rTSC transcript)) under the following PCR condition: 98 °C, 10 s; 68 °C, 8 min, for 30 cycles, and final extension at 72 °C for 10 min. The PCR products were labeled with digoxigenin and used as probes in FISH.

Cloning of the 5'-Flanking Region of rTSC Gene (5'FL/rTSC)-- The 5'FL/rTSC was cloned by a PCR-based method using the Genome Walker Kit (CLONTECH) for rat gene cloning. Briefly, the first PCR was carried out based on the manufacturer's instruction with the gene-specific primer (GSP)-1, 5'-ATG GGT CAA ATG GCT GGG CTG GCT ATT G-3' (designed to hybridize with the region from position 120 to 147 in rTSC cDNA (2)), and the adapter primer (AP)-1, 5'-GTA ATA CGA CTC ACT ATA GGG C-3' (supplied in the kit). Nested PCR was then performed with the GSP-2, 5'-TGC ACA GAG CAT CGC CTG GCA TCT CTG T-3' (designed to anneal to the region from position 31 to 58 in rTSC cDNA (2)), and the AP-2, 5'-ACT ATA GGG CAC GCG TGG T-3', using the first PCR product as a template. The resultant PCR product was subcloned into pCR-TOPO (Invitrogen), and sequenced in both directions. Putative transcription factor binding sites in 5'FL/rTSC were identified by the TRANSFAC 3.4 data base using Transcription Element Search Software (TESS) (18).

Primer Extension Method-- Primer extension was performed using the primer extension system-AMV reverse transcriptase (Promega) by a modification of the previously reported method (19). Total RNA was extracted from rat renal cortex by the guanidinium thiocyanate-cesium chloride centrifugation method (16). An oligonucleotide (5'-ACT GCA CAG AGC ATC GCC TGG CAT CTC TGT CAC GGG TAG C-3') designed to anneal to the region from position 21 to 60 in rTSC cDNA (2) was labeled with IRD700 (Aloka) at the 5'-end and used as a primer. Annealing was carried out with 1 pmol of the primer and 25 µg of total RNA at 50 °C for 8 h, and the extension reaction was performed at 42 °C for 2 h. The extended product was resolved on a 0.8% polyacrylamide gel. To determine the length of the DNA product, the sequence ladder of pBluescript SK(+) (Invitrogen) was also resolved in the same gel. The fluorescent DNA was analyzed by automated DNA analyzer (LI-COR dNA analyzer 4200).

Rapid Amplification of the 5'-end of cDNA (5'-RACE)-- 5'-RACE was performed with rat kidney Marathon-ready cDNA (CLONTECH). PCR was carried out according to the manufacturer's instruction with the adapter primer, 5'-GCC ACA CCC ACG GCA TTG GCA AAG GCG A-3' (supplied in the kit), and an rTSC-specific primer, 5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3' (from position 693 to 720 in rTSC transcript), under the following conditions: 95 °C, 30 s; 68 °C, 2 min, for 30 cycles. The PCR product was then subcloned into pCR-TOPO (Invitrogen) by a TA cloning method. The 5'-end of the rTSC cDNA was determined by sequencing.

Cell Culture-- HEK293 (a human embryonic kidney epitheloid cell line), A10 (a rat vascular smooth muscle cell line), and HepG2 (a human hepatocyte cell line) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

RT-PCR-- RT-PCR was performed to confirm endogenous expression of TSC mRNA in HEK293, HepG2, or A10 cells using a One-step RNA PCR kit (TaKaRa) according to the manufacturer's instruction. Total RNA was extracted from these cells by RNeasy kit (Qiagen). Endogenous hTSC mRNA in HEK293 or HepG2 cells were detected with a pair of primers: sense, 5'-AGG CAG GCA TCG TCC TGA CCT G-3' (from position 519 to 540 in hTSC cDNA (3)); antisense, 5'-GCA GGG TCC TTG AGG TCA CCA G-3' (from position 1,107 to 1,128 in hTSC transcript). rTSC mRNA in A10 cells was also detected by RT-PCR with a pair of primers: sense, 5'-AAT GGC AAG GTC AAG TCG G-3' (from position 608 to 626 in rTSC cDNA (2)); antisense, 5'-GAT GCG GAT GTC ATT GAT GG-3' (from position 793 to 812 in rTSC transcript). Human mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also detected with a pair of primers: sense, 5'-CCA TGG AGA AGG CTG GGG-3' (from position 371 to 388 in human (h) GAPDH cDNA); antisense, 5'-CAA AGT TGT CAT GGA TGA CC-3' (from position 546 to 565 of hGAPDH transcript (20)). rGAPDH mRNA from rat renal cortex was detected by RT-PCR with a pair of primers: sense, 5'-TCC CTC AAG ATT GTC AGC AA-3' (from position 451 to 470 in rGAPDH cDNA (21)); antisense, 5'-AGA TCC ACA ACG GAT ACA TT-3' (from position 739 to 758 in rGAPDH transcript).

Construction of a Human HFH-3 Expression Vector-- The protein coding region of hHFH-3 cDNA was cloned by a PCR-based method with human kidney Marathon-ready cDNA using a pair of primers (sense, 5'-AGT TCC CCA GCA TCG GCC AGG AGC-3' (from position 46 to 70 in hHFH-3 cDNA (12)); antisense, 5'-TCT GGC ATG TCC ACC TGG CTC AGG-3' (from position 1,148 to 1,171 in hHFH-3 transcript)) under the following PCR condition: 95 °C, 30 s; 68 °C, 2 min, for 30 cycles. An expression vector of hHFH-3 (pcHFH-3) was synthesized by subcloning the hHFH-3 cDNA into an expression vector, pcDNA1/Amp (Invitrogen). Sequencing was performed to confirm the complete sequence identity to the one reported previously (12).

Construction of Luciferase Reporter Gene Vectors-- To examine the transcription activity of 5'FL/rTSC, a chimeric firefly luciferase expression vector containing 5'FL/rTSC (rTSC/Luc) was constructed. 5'FL/rTSC was subcloned into promoter-less firefly luciferase expression vector, PicaGene Basic Vector (Nippon Gene). Deletion mutants of rTSC/Luc were then created with exonuclease III/Mung bean nuclease as described previously (22, 23). A double strand DNA containing the putative HFH-3 binding site located from position -400 to -387 in 5'FL/rTSC (HFH-3/rTSC) was made by annealing oligonucleotides 5'-AGC TAC AGG CTT CCT TTT TGT TAT ATG CAT GTC CT-3' (sense) and 5'-TCG AAG GAC ATG CAT ATA ACA AAA AGG AAG CCT GT-3' (antisense), in which underlining indicates the putative HFH-3 binding site (consensus sequence DBD TRT TTR YDT D (12)) located from position -400 to -387 in 5'FL/rTSC. The product was then subcloned upstream of a thymidine kinase gene promoter in a luciferase expression vector (HFH-3/rTSC-tk-Luc). An expression vector carrying the mutated HFH-3 binding site (mHFH-3/rTSC-tk-Luc) was also synthesized with oligonucleotides 5'-AGC TAC AGG CTT CCT TGG GGT TAT ATG CAT GTC CT-3' (sense) and 5'-TCG AAG GAC ATG CAT ATA ACC CCA AGG AAG CCT GT-3' (antisense), in which italic capital letters indicate mutated nucleotides and underlining indicates the putative HFH-3 binding site. The same mutation was also introduced by QuikChange site-directed mutagenesis kit (Stratagene) in the -580 rTSC/Luc with oligonucleotides 5'-AAG ACA GGC TTC CTT GGG GTT ATA TGC ATG TCC-3' (sense) and 5'-GGA CAT GCA TAT AAC CCC AAG GAA GCC TGT CTT-3' (antisense), in which italic capital letters indicate mutated nucleotides and underlining indicates the putative HFH-3 binding site. The resultant mutated construct was designated -580 (mHFH-3) rTSC/Luc.

Transient Transfection and Luciferase Assay-- HEK293, A10, and HepG2 cells were cultured in 6-well culture plates before transfection. Transfection was performed with 2 µg/well of luciferase expression vector as well as 0.5 µg/well control beta -galactosidase expression vector (pSV-beta -galactosidase control vector, Promega) using DOTAP liposomal transfection reagent (Roche). After the transfection, culture medium was replaced with Dulbecco's modified Eagle's medium containing 1% charcoal/resin-stripped fetal bovine serum. 48 h after transfection, luciferase expression was determined by measuring its enzymatic activity using a luciferase assay system (Promega). Transfection efficiency was normalized by beta -galactosidase expression (determined by its enzymatic activity) as reported previously (23). To examine the effect of HFH-3 on the transcription activity of 5'FL/rTSC, pcHFH-3 or mock DNA was co-transfected with -580 rTSC/Luc, -580 (mHFH-3) rTSC/Luc, HFH-3/rTSC-tk-Luc, or mHFH-3/rTSC-tk-/Luc in HepG2 cells, which have been shown to lack HFH-3 expression (12).

Electrophoretic Mobility Shift Assay (EMSA)-- An expression construct to synthesize a fusion protein of the HFH-3 DNA binding domain and glutathione S-transferase (GST) (12) was a generous gift from Dr. Robert H. Costa (University of Illinois at Chicago). GST-HFH-3 fusion protein was isolated from Escherichia coli cultures and purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech) according to the manufacturer's instruction. The purity of GST-HFH-3 was confirmed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (data not shown). The double strand DNA of HFH-3/rTSC was radiolabeled and used as a probe. EMSA was performed with the probe and in vitro synthesized GST-HFH-3 as described previously (23, 24). A double strand DNA containing the consensus HFH-3 binding was created by annealing with oligonucleotides 5'-AGC TGC ACG TTC GTT GTT TAT GTA CCG AGC G-3' (sense) and 5'-TCG ACG CTC GGT ACA TAA ACA ACG AAC GTG C-3' (antisense), in which underlining indicates the consensus HFH-3 binding sequence, and was used as a competitor. mHFH-3/rTSC oligonucleotides (containing the mutated HFH-3 binding site) were also used as competitor.

Generation of Transgenic Rats Bearing 5'FL/rTSC-LacZ-- The transgene used herein consisted of the 2,110-bp rTSC promoter fused to the LacZ gene. A fragment encoding the rTSC promoter region (-2,110 to +45) was excised from rTSC/Luc with KpnI and subcloned to the KpnI site of pSV-beta -galactosidase control vector (Promega). The KpnI site of this plasmid was located 87-bp upstream of the LacZ coding region, and the frame for the intact LacZ gene expression was confirmed by sequencing. The transgene fragment excised by digestion with SalI was purified using the QIAquick gel extraction kit (Qiagen).

Transgenic rats were generated using pronuclear microinjection as the standard procedure. The microinjection was performed by YS New Technology Laboratory (Tochigi, Japan). Briefly, the transgene fragment was microinjected to the pronuclei of fertilized single-cell oocytes obtained from Harlan Sprague-Dawley rats. Embryos that survived microinjection were transferred into the oviduct of pseudo-pregnant Wistar rats. Transgenic rats were selected with amplification of LacZ by PCR from genomic DNA extracted from tails. The number of integrated copies of the transgene was estimated by Southern blotting with the transgene fragment as a probe.

Immunohistochemistry-- The transgenic and nontransgenic rats (16-week) were anesthetized with diethyl ether, and perfused with saline through cannulae inserted into the left ventricles. The kidneys were removed and fixed for 24 h with 20% formaldehyde and mounted onto paraffin blocks. Rabbit anti-beta -galactosidase polyclonal antibody (Biogenesis) was used at a dilution of 1:200. Rabbit anti-TSC antibody, a gift from Dr. Steven C. Hebert (11), was used at a dilution of 1:250. Staining was performed by the avidin-biotinylated peroxidase complex method using an RTU Vectastain universal avidin-biotin complex kit (ABC, Vector) and a VIP substrate kit (Vector). Sections were counter-stained with hematoxylin.

Data Analysis-- p < 0.05 by Student's t test was considered statistically significant.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Southern Blot Analysis and Chromosome Mapping of rTSC Gene-- A single band was observed in either the BamHI or HindIII digest of rat genomic DNA (~6.5 and ~4.2 kilobase pairs in length, respectively), suggesting that rTSC is a single copy gene (Fig. 1). Additionally, two bands in the EcoRI digest suggested the presence of EcoRI site in the intron between TSC exons complementary to the probe. To determine the chromosomal localization of rTSC gene, we next performed FISH using three different DNA fragments of rTSC gene as probes. After hybridization, one hundred metaphases were analyzed by recording the number and position of fluorescent spots on chromosomes. Twin fluorescent spots were identified in eighty three metaphases at chromosome 19, and single fluorescent spot at the same chromosome in twenty metaphases. A typical metaphase showing the presence of twin fluorescent spots at chromosome 19 is shown in Fig. 2A. The position of fluorescent spots were shown in Fig. 2B, and these spots were clustered at 19p12-14 (p < 0.01).


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Fig. 1.   Southern blot analysis of the rTSC gene. Rat genomic DNA was digested with BamHI, EcoRI, or HindIII and hybridized with the radiolabeled BalI fragment of rTSC cDNA. A single band is observed in the BamHI or HindIII digested sample.


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Fig. 2.   Chromosomal localization of the rTSC gene. One hundred myeloblasts in the metaphase were screened by FISH with three different labeled DNA fragments of rTSC gene. A, the typical metaphase representing the twin spots at chromosome 19p12-14 locus in the FISH. B, a diagram illustration of the position of fluorescence spots. Twin spots were detected at chromosome 19 in 83 of 100 metaphases, and a single spot was also identified at the same chromosome region in 12 metaphases. These spots are shown to be clustered at 19p12-14 (p < 0.01).

Structure of 5'FL/rTSC-- A 2.1-kilobase pair fragment of 5'FL/rTSC was isolated and sequenced (DDBJ/EMBL/GenBankTM data bases with accession number AB024534). 5'FL/rTSC (Fig. 3) contains several putative consensus transcription factor recognition sequences such as: a TATA box at 42 bases upstream (-42) of the transcription initiation site; three SRY binding sites (SRY) at position -1,931, -1,728, and -189; five Pit-1 binding sites (Pit-1) at positions -1,486, -1,328, -1,219, -990, and -809; a CCAAT/enhancer-binding protein (C/EBP) family binding site at position -463 and three C/EBPbeta binding sites (C/EBPbeta ) at positions -1,461, -1,118, and -1,011; two SP-1 binding sites (SP-1) at positions -153 and -131; two glucocorticoid-responsive elements (GRE) at positions -1,091 and -579; cAMP-responsive element (CRE) at position -1,021; an AP-2 binding site (AP-2) at position -788; a c-MYC binding site (c-myc) at position -141; and an HFH-3 binding site at position -393.


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Fig. 3.   Nucleic acids sequence of the 5'flanking region of the rTSC gene. An asterisk and position +1 indicate the transcription initiation site. An underline indicates the protein coding sequence. Double underlines indicate the following putative cis-acting elements: TATA, TATA box; SRY, SRY biding site; Pit-1, Pit-1 binding site; C/EBP, CAAT/enhancer-binding protein (C/EBP) binding site; SP-1, SP-1 binding site; HFH-3, HFH-3 binding site; GRE, glucocorticoid-responsive element; CRE, cAMP-responsive element; AP-2, AP-2 binding site; c-MYC, c-MYC binding site. The sequence has been deposited in the DDBJ/EMBL/GenBankTM data bases with accession number AB024534.

Transcription Initiation Site of rTSC Gene-- The transcription initiation site of the rTSC gene was determined by both the primer extension method and 5'-RACE. Total RNA extracted from rat renal cortex was reverse transcribed using the 5'-end labeled DNA primer (designed to be a complement to the region from position 21 to 60 of rTSC cDNA). As shown in Fig. 4A, the longest extended product had 67 bases in length, indicating that the transcription initiation site of the rTSC gene is located 8 bases upstream of the 5'-end of the cloned cDNA (2) (Fig. 4C). Moreover, we performed 5'-RACE to confirm further the transcription initiation site. A PCR product 720 bp in length was subcloned, and the 5'-end was determined by sequencing. As shown in Fig. 4B, the 5'-end was completely identical to the transcription initiation site determined by the primer extension method (Fig. 4C).


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Fig. 4.   Determination of the transcription initiation site of rTSC gene. A, a blot obtained by the primer extension method. An asterisk indicates the longest extended product, 67 bases in length. B, sequence of the 5'end of rTSC cDNA obtained by the 5'RACE. The arrow region indicates the primer sequence, and an asterisk indicates the transcription initiation site. C, the sequence around the transcription initiation site (asterisk) in the rTSC gene. The arrow region is the primer sequence used in the primer extension, and the underline indicates the protein coding region.

Transcription Activity of 5'FL/rTSC-- Luciferase reporter gene assay was performed to examine the transcription activity of 5'FL/rTSC. HEK293 cells (in which endogenous TSC mRNA expression was detected) were transfected with either luciferase expression vector alone (control) or rTSC/Luc. Luciferase expression with rTSC/Luc was significantly higher than with control (Fig. 5A; 26.2 ± 1.8-fold compared with control) in transfected HEK293 cells, suggesting that the 5'FL/rTSC is transcriptionally active. We next conducted the deletion analysis of 5'FL/rTSC. As shown in Fig. 5B, transfection of the luciferase expression vector containing the full-length (~2.1 kilobase pairs) 5'FL/rTSC (-2,093 rTSC/Luc) showed the highest luciferase expression, and the transcription activity of 5'FL/rTSC was dependent on the fragment length. Luciferase activity was high in cells transfected with the rTSC/Luc containing deletion fragments from position -2,093 to -580. However, further deletion of the promoter to position -141 resulted in a marked decrease in luciferase activity.


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Fig. 5.   Transcription activity of 5'FL/rTSC. A, luciferase expression in HEK293 cells transfected with either rTSC/Luc or a control vector (lacking the 5'FL/rTSC). The vertical line indicates the relative luciferase activity after normalization of transfection efficiency by beta -galactosidase activity (n = 6). Bars represent the mean ± S.E. (n = 6; *, p < 0.01). B, transcription activities of deleted fragments of 5'FL/rTSC in HEK293 cells. Deleted fragment lengths are shown on the left, and bars on the right represent the mean ± S.E. of relative luciferase activity (n = 6; *, p < 0.01 compared with -580 rTSC/Luc).

Tissue-specific Transcription of rTSC Gene-- Endogenous expression of TSC mRNA was examined in HEK293, HepG2, or A10 cells by the RT-PCR method. An expected length of PCR product (609 bp) was detected with the total RNA from HEK293 cells (Fig. 6A), and its sequence identity to rTSC was confirmed, whereas no PCR products for rTSC were detected in the other cells. As a positive control, GAPDH mRNA was detected in either HEK293, HepG2, or A10 cells by RT-PCR (Fig. 6A). The PCR product of rTSC mRNA was also detected with the total RNA from renal cortex by the same RT-PCR protocol. TSC was thus indicated to be expressed in HEK293 cells but not in either HepG2 or A10 cells. We therefore used these cell lines to examine the tissue-specific transcription activity of 5'FL/rTSC. As shown in Fig. 6B, the transcription activity of 5'FL/rTSC estimated by luciferase expression was pronounced in HEK293 cells, whereas it was modest in both HepG2 and A10 cells. It was thus suggested that the 5'FL/rTSC is able to regulate the tissue-specific gene transcription.


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Fig. 6.   Tissue-specific transcription activity of 5'FL/rTSC. A, endogenous mRNA expression of TSC in cultured cells, HEK293 (human renal tubular epithelial cell line), HepG2 (human hepatocarcinoma cell line), and A10 (rat aortic smooth muscle cell line). PCR was performed with (+) or without (-) RT. B, transcription activity of 5'FL/rTSC in these cells. Bars represent the mean ± S.E. (n = 6; *, p < 0.01 compared with the levels in HEK293 cells).

Establishment of Transgenic Rats Bearing 5'FL/TSC-LacZ-- 227 microinjected oocytes were transplanted into eight Wistar rats. 58 rats were born, seven of which had the transgene. The copy number of the integrated transgene was estimated as three to six copies by Southern blotting. Every transgenic rat grew normally in appearance. The transgene was inherited in the Mendelian fashion.

Expression Co-localization of LacZ with TSC-- The transgenic rat harboring six copies of the transgene was subjected to immunohistochemistry. In the cortical region of the transgenic rat kidney, immunoreactive beta -galactosidase was observed at cortical distal tubules (Fig. 7A), whereas no stain was observed in the control rat (Fig. 7B). Using the sequential sections from the transgenic rat kidney, we observed co-localization of immunoreactive beta -galactosidase (arrows, Fig. 7, C and E) and TSC (arrowheads, panels D and F). No immunostaining was observed in the medullar region (Fig. 7G).


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Fig. 7.   Expression of beta -galactosidase in transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene. In the cortical region of transgenic rat kidney, immunoreactive beta -galactosidase is observed at distal tubules (A), whereas no stains are observed in a control rat (B). In the sequential sections from the transgenic rat, co-localization of immunoreactive beta -galactosidase (arrows, C and E) and TSC (arrowheads, D and F) is observed at the cortical distal tubules. In the medullar region, no immunoreactive beta -galactosidase (G) is detected.

A Role of HFH-3 on Transcription Activity of 5'FL/rTSC-- To examine the effect of HFH-3 overexpression on the transcription activity of 5'FL/rTSC, hHFH-3 cDNA was isolated, and an expression vector carrying the cDNA (pcHFH-3) was synthesized. In vitro translated HFH-3 with this construct was resolved in SDS- polyacrylamide gel electrophoresis, and we identified a new band with the expected size corresponding to HFH-3 protein (data not shown). As shown in Fig. 8, luciferase expression in HepG2 cells transfected with -2,093 rTSC/Luc was significantly higher by cotransfection with pcHFH-3 than with mock (2.28 ± 0.14-fold expression compared with mock; p < 0.01). This enhancement of transcription with pcHFH-3 was also observed in transfection with -1,139 rTSC/Luc and -580 rTSC/Luc, whereas it was not observed with -141 rTSC/Luc. Thus, the HFH-3-responsive element was suggested to be present between position -580 and -141, and we identified an element homologous to HFH-3 binding consensus sequence (DBD TRT TTR YDT D) at position -393 (TCC TTT TTG TTA TA).


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Fig. 8.   Effect of HFH-3 overexpression on the transcription activity of 5'FL/rTSC. HFH-3 was overexpressed with pcHFH-3 in HepG2 cells (lacking TSC or HFH-3 mRNA expression) transfected with deletion mutants of 5'FL/rTSC. Deleted fragment lengths are shown on the left, and the bars on the right represent the mean ± S.E. of relative luciferase activity (n = 6; *, p <0.01). Hatched or open bars represent the data from cells transfected with pcHFH-3 (HFH-3 (+)) or with a vector lacking HFH-3 cDNA (Control), respectively.

To evaluate the transcription responsiveness of HFH-3 to putative HFH-3 binding site, we mutated this site and examined the effect of HFH-3 overexpression on its transcription activity. In HEK293 cells expressing HFH-3 mRNA (data not shown), transcription activity of -580 (mHFH-3) rTSC/Luc was reduced markedly (Fig. 9, open column) compared with that of -580 rTSC/Luc (Fig. 9, hatched column).


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Fig. 9.   Loss of transcription activity by a mutation of HFH-3/rTSC. Transcription activity of HFH-3/rTSC was examined in HEK293 cells (expressing HFH-3 mRNA) using -580 (mHFH-3)/rTSC in which the HFH-3/rTSC was mutated. Bars represent the mean ± S.E. (n = 12; *, p < 0.01).

Functional Characterization of HFH-3 Binding Site in 5'FL/rTSC-- We next characterized the HFH-3 binding site in 5'FL/rTSC (HFH-3/rTSC) by a luciferase reporter gene assay using a heterologous promoter. In HepG2 cells (which lacks HFH-3 mRNA expression (data not shown)) transfected with HFH-3/rTSC-tk-Luc, luciferase expression was increased according to the amount of transfected pcHFH-3 (Fig. 10, hatched column). The overexpression of HFH-3 induced 2.78 ± 0.09-fold increases in luciferase expression at the maximum compared with control (mock transfection) (p < 0.01), whereas no increment in luciferase expression was observed in cells transfected with mHFH-3/rTSC-tk-Luc (Fig. 10, open column).


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Fig. 10.   Transactivation of HFH-3/rTSC by HFH-3. Luciferase constructs containing the truncated wild-type HFH-3/rTSC (HFH-3/rTSC-tk-Luc) or mutated HFH-3/rTSC (mHFH-3/rTSC-tk-Luc) fused upstream of tk promoter was transfected into HepG2 cells (lacking TSC expression), and HFH-3 was overexpressed by co-transfection with pcHFH-3. The luciferase expression with HFH-3/rTSC-tk-Luc is stimulated in proportion to the amounts of transfected pcHFH-3 DNA, whereas it is kept unchanged in cells transfected with HFH-3/rTSC-tk-Luc. Bars represent the mean ± S.E. (n = 10; *, p < 0.01).

Direct Binding of HFH-3 to HFH-3/rTSC in EMSA-- EMSA was performed to examine a direct binding between HFH-3 and HFH-3/rTSC. As shown in Fig. 11, in vitro synthesized HFH-3 protein reacted with the radiolabeled HFH-3/rTSC and formed a protein-DNA complex (Lane 1). Using unlabeled HFH-3/rTSC as a cold competitor, the formation of protein-DNA complex was significantly inhibited (lane 2). Moreover, the complex disappeared with an excess of oligonucleotides containing the HFH-3 binding consensus sequence as a cold competitor (lane 3). However, the protein-DNA complex was kept unchanged with an excess of oligonucleotides of mHFH-3/rTSC as a competitor (lane 4). These results indicate that HFH-3/rTSC is actually bound by HFH-3 and the binding is sequence-specific.


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Fig. 11.   Protein-DNA interaction between HFH-3 and HFH-3/rTSC. EMSA was performed with the in vitro synthesized GST-HFH-3 fusion protein (GST-HFH-3) and 32P-labeled HFH-3/rTSC fragment (32P-HFH-3-TSC) as a probe. Formation of a protein-DNA complex is observed (lane 1). The complex formation is significantly inhibited with an excess of unlabeled HFH-3/rTSC fragment (T) (lane 2) as well as oligonucleotides containing consensus HFH-3 binding site (C) (lane 3). On the other hand, no inhibition of the complex formation is observed with an excess of oligonucleotides containing the mutated (m) HFH-3/rTSC (lane 4). Lane 5, radiolabeled probe alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rTSC gene was shown to be localized to 19p12-14 by FISH. In either human or mouse, the TSC gene has also been mapped to chromosome 16q13 (3, 4, 25) or 8 (26), respectively. These results suggest the segmental chromosome similarity in their regions.

Genes of the Na-K-Cl cotransporter (NKCC2) (2), sodium phosphate cotransporter (NPT2) (27), chloride channel CLC-K1 (28), and kidney-specific cadherin (Ksp-cadherin) (29) have been shown to be expressed specifically in the kidney. The transcription regulatory regions of those genes have already been cloned (30-32), and the tissue-specific transcription mechanisms have been partly examined. Particularly, the kidney-specific CLC-K1 gene transcription has recently been shown to depend on an interaction between a transcription factor, MAZ, and a transcription repressor, KKLF (33). In the present study, we analyzed the transcriptional function of the rTSC gene in terms of tissue-specific gene expression.

The 5'FL/rTSC contains two putative glucocorticoid-responsive elements (Fig. 3). It has been reported that glucocorticoids and mineralocorticoids stimulate the thiazide-sensitive sodium transport at DCT, inducing an increase in the number of thiazide diuretic binding sites (34, 35) or in TSC protein expression (36). The glucocorticoid-responsive elements may possibly be involved in the steroid-induced TSC expression at the gene transcription level. A putative cAMP-responsive element was also identified, implying that TSC transcription could be regulated by intracellular cAMP formation by an agent such as calcitonin, which has been shown to increase the number of renal thiazide diuretic binding sites (37). Moreover, a couple of putative C/EBPbeta binding sites were identified. Because C/EBPbeta is expressed in kidney and induced by interleukin-6 mediating an inflammatory response (38), TSC gene transcription may possibly be affected by inflammation via interleukin-6.

Luciferase reporter gene analysis was performed with the chimeric reporter expression vector containing 5'FL/rTSC (rTSC/Luc). When rTSC/Luc was transfected into HEK293 cells (which express TSC mRNA), marked luciferase expression was observed (Fig. 5A). Additionally, the transcription activity is dependent upon the length of 5'FL/rTSC, indicating that the 5'FL/rTSC has a significant transcription function. Deletion analysis also showed a marked decrease (75%) in the transcription activity between position -580 and -141 (Fig. 5B), suggesting that a major promoter is located between these positions.

Tissue-specific transcription of TSC gene was focused in the present study. As shown in Fig. 6A, transcription activity of 5'FL/rTSC was pronounced in HEK293 cells expressing TSC mRNA, whereas it was suppressed in either HepG2 or A10 cells lacking TSC mRNA expression (Fig. 6), suggesting that the 5'FL/rTSC plays a crucial role in the tissue-specific TSC gene transcription. To further study the tissue-specific transcription of TSC in kidney, we generated the transgenic rats with the 5'FL/rTSC fused upstream of the LacZ gene as a transgene. As shown in Fig. 7, immunoreactive beta -galactosidase was present at the same tubular region as TSC, probably at DCT in the renal cortical region, and neither was detected in the other renal tissues. Thus, this in vivo gene transcription model has clearly demonstrated that DCT-specific TSC gene expression is dependent on the 5'FL/rTSC, and it is suggested that the region contains some important elements responsible for the DCT-specific transcription.

HFH-3 belongs to the HFH/winged helix transcription factor family (12), and its family members are involved in tissue-specific gene expression and cell differentiation during embryonic development (14, 15). HFH-3 mRNA is localized to DCT in kidney (12), but the target gene of HFH-3 has not yet been identified. We hypothesized that HFH-3 might be involved in the renal tubule-specific gene transcription of TSC, because a putative HFH-3 binding site was identified in the 5'FL/rTSC. We first examined the effect of HFH-3 overexpression on the transcription activity of 5'FL/rTSC in the transfected HEK293 cells. As shown in Fig. 7, the stimulation was significantly observed with -2,093, -1,139, and -580 rTSC/Luc but not with -141 rTSC/Luc. Consistent with this result, there is a putative HFH-3 binding site (HFH-3/rTSC) at the -393 position (Fig. 3), 5'-TCC TTT TTG TTA T-3', which bears a homology to the previously reported HFH-3 binding sequence (DBD TRT TTR YDT D) (12) by 10 of 13 nucleotides (77%). To examine the transcription activity of HFH-3/rTSC, the HFH-3/rTSC in -580 rTSC/Luc was mutated, and its transcription function was examined in HEK293 cells (expressing both TSC and HFH-3 mRNAs). By the mutation, we observed a marked loss of transcription function. It is therefore suggested that the HFH-3/rTSC plays an important role in TSC gene transcription in HEK293 cells. We next focused on the transcriptional function of HFH-3/rTSC by examining a functional interaction between HFH-3 and HFH-3/rTSC in HepG2 cells. HFH-3 overexpression activated the transcription of HFH-3/rTSC-tk-Luc in a dose-dependent manner (Fig. 8), whereas the transcription stimulation was not observed in transfected cells with the mHFH-3/rTSC. These results indicate that HFH-3 can stimulate the transcription through an interaction with HFH-3/rTSC. However, the magnitude of the transcription stimulation (~2-3-fold that of mock) was not comparable with the transcription activity of -2,029 rTSC/Luc in HEK293 cells. Some other elements in the 5'FL/rTSC may also be involved in the gene transcription to enhance the HFH-3 transactivation.

EMSA was performed to examine an interaction between HFH-3 and HFH-3/rTSC. In EMSA, a protein-DNA complex was formed with the radiolabeled HFH-3/rTSC and in vitro synthesized HFH-3. The complex formation was inhibited with an excess of the HFH-3 consensus sequence oligonucleotides but not with an excess of mHFH-3/rTSC. HFH-3 therefore can actually bind the HFH-3/rTSC and may lead to transactivation of TSC gene. Putative target genes for HFH-3 have been suggested to be the mineralocorticoid receptor, Na/H exchanger NHE3, Na/K-ATPase alpha 2 subunit, and Na/K-ATPase beta 2 subunit (12), although any interaction with HFH-3 has not been proven even in these the transcription activity of the rTSC gene genes. We have demonstrated here that HFH-3 can stimulate through the HFH-3/rTSC via an interaction with HFH-3. This is the first report in which a target gene for HFH-3 has been identified.

In conclusion, we have localized the rTSC gene to chromosome 19p12-14 and characterized its promoter region. The promoter determines the DCT-specific gene expression of rTSC, and DCT-specific HFH-3 may be involved at least in part in renal tubule-specific rTSC gene transcription.

    ACKNOWLEDGEMENTS

We thank Dr. K. Nakagawara (Nihon Gene Research Laboratories) for help with FISH; Dr. Ichiro Miyoshi for assistance in creating and breeding transgenic rats (Institute of Animal Experiments, Tohoku University Graduate School of Medicine); Yasuteru Kondo (Tohoku University School of Medicine) for laboratory assistance; Dr. R.H. Costa (Department of Biochemistry, University of Illinois at Chicago) for gift of a construct to synthesize the GST-HFH-3 fusion protein; Dr. S. C. Hebert (Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University) for supply of anti-TSC antibody; Dr. N. Takahashi (Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill) for critical reading of this manuscript; and Dr. Yoshiaki Kondo (Department of Pediatrics, Tohoku University Graduate School of Medicine) for valuable discussion.

    FOOTNOTES

* This study was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture, Japan and by the Takeda Foundation for Metabolic Disorder Research, Japan.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB024534.

Dagger These authors were equal contributors to this study.

§ To whom correspondence should be addressed: Molecular Biology Unit, Division of Nephrology, Endocrinology, and Vascular Medicine, Dept. of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Tel.: 81-22-717-7163; Fax: 81-22-717-7168; E-mail: kazut2i@mail.cc.tohoku.ac.jp.

Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M101614200

    ABBREVIATIONS

The abbreviations used are: TSC, thiazide-sensitive Na-Cl cotransporter; h, human; r, rat; HFH-3, hepatocyte nuclear factor-3/folk head homologue-3; DCT, distal convoluted tubule; RT-PCR, reverse transcription-polymerase chain reaction; FISH, fluorescence in situ hybridization; EMSA, electrophoretic mobility shift assay; RACE, rapid amplification of cDNA ends; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; tk, thymidine kinase; Luc, luciferase; GST, glutathione S-transferase; bp, base pair(s); C/EBP, CCAAT/enhancer-binding protein.

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ABSTRACT
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RESULTS
DISCUSSION
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