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phosphate cotransporter gene1
Biochimica et Biophysica Acta 1396 Ž1998. 267–272
Short sequence paper
Characterization of the 5X flanking region of the human NPT-1 Naqrphosphate cotransporter gene 1 Yutaka Taketani, Ken-ichi Miyamoto ) , Mika Chikamori, Keiko Tanaka, Hironori Yamamoto, Sawako Tatsumi, Kyoko Morita, Eiji Takeda Department of Clinical Nutrition, School of Medicine, UniÕersity of Tokushima, Kuramoto-cho 3, Tokushima 770, Japan Received 18 July 1997; revised 1 December 1997; accepted 10 December 1997
Abstract To elucidate the expression and regulation of the human type I Naqrphosphate transporter gene Ž NPT-1., the 5X flanking region of the NPT-1 gene was cloned, and its nucleotide sequence and function were determined. A genomic clone that contained approximately 14.0 kb of the 5X-flanking region of the NPT-1 gene was isolated. A single transcription start site was located 104 base pairs Žbp. upstream of the 3X end of exon 1. In addition to the sequence of the 5X-flanking region contained a sequence weakly homologous to a TATA box at position y41 to y36 and many transcriptional regulatory elements. Transient expression revealed that a 45-bp region of proximal to exon 1, which contained TATA-like sequence, was sufficient for promoting luciferase expression in OK-cells derived from opossum kidney proximal tubule. q 1998 Elsevier Science B.V. Keywords: Sodium-dependent phosphate cotransporter; Gene promoter; Kidney; ŽHuman.
Regulation of phosphate ŽPi. reabsorption in the renal proximal tubule is accomplished mainly by modulation of apical NaqrPi cotransporter w1x. The metabolic and hormonal factors that influence apical NaqrPi cotransporter exert their effects primarily by changing Pi reabsorption in the proximal tubule w1,2x. Abbreviations: Pi, inorganic phosphate; RACE, rapid amplification of cDNA end; HNF-1, hepatic nuclear factor-1; HNF-5, hepatic nuclear factor-5; CrEBP, CCAATrenhancer binding protein; Pho4, transcriptional activator protein of the phosphate regulon in Saccharomyces cereÕisiae ) Corresponding author. Fax: q81-886-33-7094; E-mail: [email protected] 1 The nucleotide sequence reported in this paper has been submitted to the DDBJrEMBLrGenbank nucleotide sequence databases with the following accession number D83236.
In recent years, two mammalian proximal tubular brush border NaqrPi cotransporters Žtypes I and II. have been identified by expression cloning techniques w3x. Functional analysis studies have shown that the characterization of type II transporter correspond to the well-known properties of proximal tubular brush border membrane of Pi transport w3x. Indeed, expression of type II NaqrPi cotransporter is observed only in kidney, while type I is primarily expressed in kidney, liver, and brain w3x. However, an increased Naq-dependent Pi uptake was also observed after stable transfection of the type I transporter into MDCK cells w4x and in Xenopus oocyte expression analysis w5x. The immunoreactive protein of the type I has a exclusively proximal tubular brush border location w6,7x. In addition, type I NaqrPi
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cotransporters have been identified subsequently by homology-based screening procedures in mouse ŽNpt-1. w8x, rat ŽRNaPi-1. w9x, rabbit ŽNaPi-1. w5x and human ŽNPT-1. w10,11x kidney. Homologous protein was also identified in rat brain-specific NaqrPi cotransporter ŽBNP-1. w12,13x. Antisense hybrid depletion strategy and oocytes injection experiment provided that types I and II cotransporters might account for full NaqrPi cotransport activity w11x. Recent studies have shown that the content of type II NaqrPi cotransporter protein expressed in brush border membrane and the related mRNA were reduced in X-linked hypophosphatemic Ž Hyp . mice showing also reduced brush border membrane NaqrPi cotransport activity w3x. In contrast, type I NaqrPi transporter seems to be unaffected in Hyp mice w8x. Interestingly, the expression of type I is altered by fasting and streptozotocin-induced diabetic states w14x. Insulin has a significant effect on renal Pi transport activity and type I NaqrPi cotransporter expression w1,2x. Thus two types of NaqrPi cotransporters are regulated by different factors and each subtype may be involved in a specific cellular function. Recently, we have characterized the human type II NaqrPi cotransporter Ž NaPi-3 . chromosomal gene and analyzed its promoter function w15x. To further clarify the differences in the functional role between type I and type II NaqrPi cotransporters, the 5X flanking region of the human type I NPT-1 gene have been characterized in this study. Cloning of 5X-flanking region of the human NPT-1 gene. We established the 5X end of NPT-1 mRNA sequence reported initially by Chong et al. w10x using the 5X RACE PCR technique with the 5X RACE ready cDNA library as previously described w15x. Two sequential polymerase chain reaction Ž PCR. amplification reactions were employed to identify the 5X end of the NPT-1 mRNA and to generate DNA fragment useful for initial sequencing. Sequence determination of the PCR products of these sequential reactions established the 5X end of the NPT-1. The cloning and DNA sequence analysis of the resulting product suggested that the original cDNA sequence reported by Chong et al. w10x was incomplete at the 5X end by 147 bases. We utilized the 5X RACE-derived DNA fragment to map the exon 1. However, we could not find any clones that contains the 5X end of NPT-1 cDNA. To further isolate clones containing exon 1, we pre-
Fig. 1. Cloning of the 5X flanking region of the NPT-1 gene. Ža. Sequences of Sau 3AI cassette w23x and used primers. Žb. Amplification of a probe containing a new position of the 5X flanking region of the NPT-1 gene from BamHI-digested genomic DNA in Sau 3AI cassettes Žopen box. by two round of PCR with the indicated primers ŽC1, C2, NPT-1-R, and NPT-1-R3.. Žc. A restriction map of the 5X flanking region of the NPT-1 gene containing exon 1 and intron 1. Nucleotides are numbered with the major transcription initiation site as q1. Restriction enzymes: H, HindIII; E, EcoRI.
pared a DNA probe by PCR with the Sau 3AI cassette cloning system ŽFig. 1a,b.. An ; 2-kb DNA fragment containing the 5X flanking region and intron 1 of the NPT-1 gene was thus amplified by PCR, labeled with 32 P, and used to screen a lEMBL-3 human genomic DNA library. A single clone, lNPT11, was obtained and was digested with HindIII or EcoRI, and the resulting fragments were subcloned into plasmid pBluescript II SKŽq. for sequencing. The restriction enzyme map of the 5X flanking region of the NPT-1 gene is shown in Fig. 1c. Identification of the transcription start site. The transcription initiation site of NPT-1 mRNA was determined by primer extension analysis and S1 nuclease mapping. A 32 P-labeled antisense oligonucleotide derived from NPT-1 cDNA was hybridized to total RNA from human kidney cortex and then extended by reverse transcriptase. By comparing with a
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major transcription site was identified as a guanosine residue 109 or a adenosine residue 104 bases upstream of the 3X end of the exon 1 Ž Fig. 3. . In addition, S1 nuclease protection assay was performed to identify the transcription initiation sites. In a experiment with 148 bp probe, a major protected fragment of 104 bp was observed in RNA from human kidney cortex ŽFig. 2b.. These results confirmed that the
Fig. 2. Primer extension analysis and SI nuclease mapping. Ža. Primer extension analysis was carried out essentially as described w15,24x. An oligonucleotide NPT-1-R3 was labeled at its 5X terminus with w g- 32 PxATP and T4 polynucleotide kinase. The labeled primer Ž100,000 counts per minute. was then hybridized at 428C for 1 h to total RNA Ž10 m g. isolated from human kidney cortex Žhuman kidney. or yeast t-RNA Žyeast t-RNA. in a reaction mixture Ž20 m l. containing 10 mM Tris–HCl ŽpH 8.3., 250 mM KCl, and 1 mM EDTA. Reverse transcription and electrophoresis were then performed as described w15,24x. The major products of primer extension are indicated by arrows. The products length was estimated with reference to molecular weight marker ŽM.. Žb. S1 nuclease mapping was carried out essentially as described w25x. Probe was prepared by synthesis with Klenow fragment of DNA polymerase I, w a- 32 PxdCTP, NPT-1-R2 primer Ž5X-CTGATTCGGGACAAAAAACAAGTG, corresponding to nucleotides q84 to q104. and HindIII digested NPT-1 DNA fragment containing exon 1 as template. The probes were hybridized with 50 m g of total RNA from human kidney cortex Žhuman kidney RNA. or yeast t-RNA Žyeast t-RNA. in a hybridization buffer Ž20 m l. containing 80% formamide, 40 mM PIPES ŽpH 6.4., 400 mM NaCl, and 1 mM EDTA. S1 nuclease reaction and electrophoresis were then performed as described w25x. The digest is indicated by arrow and analyzed the size with reference to sequence ladders obtained with the same primer using for probe preparation.
molecular weight marker, the length of the two products was 200 and 195 nucleotides including the primer with additional restriction site Ž Fig. 2a. . Thus, the
Fig. 3. Nucleotide sequence of the 5X flanking region to exon 1 of the NPT-1 gene. Nucleotides are numbered with the major transcription initiation site, which is shown by arrow, as q1. Lower case letters show intron sequences, and bold lower case letters show consensus sequence of exon–intron junction known as GT–AG rule. Upper case letters show the sequences of exon or 5X flanking region. Various cis-regulatory elements are indicated as follows: CrEBP, CCAATrenhancer-binding protein binding site; AP-1, AP-1 binding site; AP-2, AP-2 binding site; HNF-1, hepatic nuclear factor-1 binding site; HNF-5, hepatic nuclear factor-5 binding site; GRE, glucocorticoid responsive element; TATA-like, a sequence weakly homologous to TATA box. The arrow head indicates the 5X end nucleotide derived from sequencing a major 5X RACE product. The underlined sequence indicates the translation initiation site.
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major initiation site was the adenosine residue located at 104 bases upstream of the 3X end of exon 1. Characterization of the NPT-1 gene promoter region. The nucleotide sequences of exon 1 and its 5X flanking region are shown in Fig. 3. Although the proximal ; 1.5 kb of 5X flanking of the NPT-1 gene does not contain typical TATA or GC boxes, a TTTTAA sequence, which shows weak homology to a TFIID binding site, is apparent at y41 to y36 bp relative to the transcription initiation site ŽFig. 3.. This sequence may play the role of a TATA box. Several well-characterized cis-acting elements including two AP-1 at positions y1151 and y418, two AP-2 sites at positions y886 and y846, a CCAATrenhancer binding protein binding site ŽCrEBP sites. at positions y637. In addition, two HNF-1 and two HNF-5 sequences at position y1287, y1091, y1405, y1219 were detected in the promoter. Furthermore, we determined the sequences of exon–intron junctions of 5X and 3X end of intron 1 and 5X and of intron 2. These sequences conserved the GT–AG rule w16x Ž Fig. 3. .
Promoter actiÕity of the 5X flanking region of the NPT-1 gene. Reporter constructs were made in which the 5X upstream sequences of the human NPT-1 gene was fused to a luciferase reporter gene. When transfected into OK-cells derived from opossum kidney proximal tubule, a construct containing sequences y1419 to q104 from the pNPT1.4 was able to detect expression of luciferase activity which on average was 1.5-fold compared with those in the vector alone. Progressive deletion to positions y670 and y45 resulted in an increase in activity, reaching a maximum 300% of the pNPT1.4 construct. In contrast, the pNPT1.4rev was decreased ; 60% of the maximum activity. These results suggest the presence of multiple negative regulatory elements localized between y1419 and y45 in the NPT-1 gene promoter. In addition, deletion of the proximal sequence y45 to q104 ŽpNPTD 0.04. completely abolished the induction of luciferase activity. The reverse orientation of the promoter Žy45 to q104. relative to the reporter did not produce luciferase activity Ždata not shown.. Thus, a 45-bp region of proximal to exon 1,
Fig. 4. Functional analysis of the human NPT-1 gene promoter. Various truncated promoter fragments were prepared by PCR and digestion with appropriate restriction enzymes indicated in top of figure, and was ligated into a pGL-3 luciferase reporter plasmid. These reporter plasmids were named as indicated. Numbers indicated distance in base pairs from the transcription initiation site. Restriction enzyme cleavage sites ŽS, SacI; H, HindIII. and putative regulatory elements were also shown at the top of figure. The b-galactosidase expression vector pCMV-b was used as an internal control. OK-cells were provided from American Type Cell Collection ŽCRL1840. and cultured at 378C under 5% CO 2 in F-12rDMEM Ž1:1. containing 10% fetal bovine serum. Transfection of OK-cells was performed by Lipofectamine with 0.5 m g of the NPT-1 promoter-luciferase reporter vector and 0.5 m g of pCMV-b per 5 = 10 5 cells. After transfection, the cells were incubated under standard conditions for 48 h. Cells were then harvested in cell lysis buffer w24x and the lysate assayed for luciferase activity. b-galactosidase activity, and protein concentration w24x. Luciferase activity was corrected for differences in transfection efficiency between experiments by normalizing to b-galactosidase activity values. Data are means" S.E.M. of triplicate determinations. These results were representative of three separate experiments.
Y. Taketani et al.r Biochimica et Biophysica Acta 1396 (1998) 267–272
which contained a TATA-like sequence, was sufficient for promoting luciferase expression in OK-cells ŽFig. 4.. In a previous study, we isolated a type I NaqrPi cotransporter Ž NPT-1. gene from a human kidney cDNA library w11x. Functional analysis in Xenopus oocytes showed NPT-1 to be a high affinity NaqrPi cotransporter which suggest that at least two high affinity NaqrPi cotransporter Ž NPT-1 and NaPi-3. are present in the human renal cortex w11x. The structure of human type II Ž NaPi-3 . chromosomal gene has been determined recently w15x. The organization of the genes was approximately 16-kb long and comprising 13 exons and promoter regions. There is a typical TATA box and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2, and SP-1 sites in the promoter. This region also contains three direct repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone superfamily, including vitamin D. Deletion analysis indicated that the upstream region Žfrom nt-2409 to nt-1259. in the NaPi-3 gene promoter is important in kidney specific gene expression w15x. However, we could not find out homologous region for specific gene expression in kidney between the NaPi-3 gene and the NPT-1 gene. As shown in Fig. 3, the NPT-1 gene promoter may belong to the family of promoter sequences that bind already known liver-enriched transactivators Ž i.e., CrEBP, HNF-1, HNF-5. w17,18x. Indeed, NPT-1 is abundant in the liver w11x. Moreover, the levels of HNF-1 mRNA are known to be reduced in diabetic rat w19x. Since insulin and glucose stimulate the expression of rat type I NaqrPi cotransporter gene w14x, changes in the amount of HNF-1 protein may affect the expression of the NPT-1 gene in a model animals with diabetes mellitus. More recently, Pontoglio et al. w20x reported that HNF-1 knockout mice manifested renal Fanconi syndrome characterized by phosphaturia, polyuria, glucosuria, and generalized aminoaciduria. This syndrome is the results from multiple transport dysfunctions mainly in the renal proximal tubules. Most of these transports rely on the Naq-dependent cotransporter w20x. The reduced number of Naqrglucose cotransporter molecule was detected in the mutant mice w20x. These observations suggest that HNF-1 may regulate the expression of
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apical Naq-dependent cotransporter genes in the kidney. Interestingly, the existence of overlaps between HNF-5 and a glucocorticoid binding site at a position y1231 to y1213 is observed in the NPT-1 gene promoter. This element is important in glucocorticoid receptor action in the rat tyrosine aminotransferase ŽTAT. gene promoter w21x. Rat TAT gene is expressed specially in liver where its transcription rate is increased by glucocorticoids w22x. These observations suggest that glucocorticoids may be important regulators of NPT-1 in the liver. Finally, these features of the NPT-1 gene promoter will lead to a better understanding of the mechanism of tissue-specific expression and the physiological regulation by the metabolic and hormonal factors. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Setsuro Fujii Memorial Foundation, the Uehara Memorial Foundation, and the Salt Science Research Foundation. References w1x H. Murer, J. Biber, in: D.W. Seldin, G. Giebisch ŽEds.., The Kidney: Physiology and Pathophysiology, 2nd edn., Raven Press, New York, 1992, pp. 2481–2509. w2x T.J. Berndt, F.G. Knox, in: D.W. Seldin, G. Giebisch ŽEds.., The Kidney: Physiology and Pathophysiology, 2nd edn., Raven Press, New York, 1992, pp. 2511–253. w3x H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 433 Ž1997. 379–389. w4x E.S. Quabius, H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 430 Ž1995. 132–136. w5x A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, H. Murer, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 9608– 9612. w6x M. Levi, S.A. Kempson, M. Lotscher, J. Biber, H. Murer, J. ¨ Membr. Biol. 154 Ž1996. 1–9. w7x J. Biber, M. Arar, B. Kaissling, H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 426 Ž1994. 5–11. w8x S.S. Chong, C.A. Kozak, L. Liu, K. Kristjansson, S.T. Dunn, J.E. Brourdeau, M.R. Hughes, Am. J. Physiol. 268 Ž1995. F1038–F1045. w9x H. Li, Z. Xie, Cell. Mol. Biol. Res. 41 Ž1995. 451–460. w10x S.S. Chong, K. Kristjansson, H.Y. Zoghbi, M.R. Hughes, Genomics 18 Ž1993. 335–339. w11x K. Miyamoto, S. Tatsumi, T. Sonoda, H. Yamamoto, H. Minami, Y. Taketani, E. Takeda, Biochem. J. 305 Ž1995. 81–85. w12x B. Ni, X.-W. Yan, J. Wang, S.M. Paul, J. Neurosci. 15 Ž1995. 5789–5799.
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w13x B. Ni, P.R. Rosteck, N.S. Nadi, S.M. Paul, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 5607–5611. w14x H.L. Ping Ren, M. Onwochei, R.J. Ruch, Z. Xie, Am. J. Physiol. 271 Ž1996. E1021–1028. w15x Y. Taketani, K. Miyamoto, K. Tanaka, K. Katai, M. Chikamori, S. Tatsumi, H. Segawa, H. Yamamoto, K. Morita, E. Takeda, Biochem. J. 324 Ž1997. 927–934. w16x R. Breathnach, P. Chambon, Annu. Rev. Biochem. 50 Ž1981. 349–383. w17x V. De Simone, R. Cortese, Curr. Opin. Cell Biol. 3 Ž1991. 960–965. w18x E. Lai, J.E. Darnell, Trends Biochem. Sci. 16 Ž1991. 427– 430.
w19x G. Barrera-Hernandez, I.E. Wanke, N.C. Wong, J. Biol. Chem. 271 Ž1996. 9969–9975. w20x M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. Poggi Bach, C. Babinet, M. Yaniv, Cell 84 Ž1996. 575–585. w21x T. Grange, J. Roux, G. Rigaud, R. Pictet, Nucleic Acids Res. 19 Ž1990. 131–139. w22x D.K. Granner, J.L. Hargrove, Mol. Cell. Biochem. 53 Ž1983. 113–114. w23x J. Compton, Nature 350 Ž1991. 91–92. w24x T. Arakawa, M. Nakamura, T. Yoshimoto, S. Yamamoto, FEBS Lett. 363 Ž1995. 105–110. w25x A.J. Berk, P.A. Sharp, Cell 12 Ž1977. 721–732.
Short sequence paper
Characterization of the 5X flanking region of the human NPT-1 Naqrphosphate cotransporter gene 1 Yutaka Taketani, Ken-ichi Miyamoto ) , Mika Chikamori, Keiko Tanaka, Hironori Yamamoto, Sawako Tatsumi, Kyoko Morita, Eiji Takeda Department of Clinical Nutrition, School of Medicine, UniÕersity of Tokushima, Kuramoto-cho 3, Tokushima 770, Japan Received 18 July 1997; revised 1 December 1997; accepted 10 December 1997
Abstract To elucidate the expression and regulation of the human type I Naqrphosphate transporter gene Ž NPT-1., the 5X flanking region of the NPT-1 gene was cloned, and its nucleotide sequence and function were determined. A genomic clone that contained approximately 14.0 kb of the 5X-flanking region of the NPT-1 gene was isolated. A single transcription start site was located 104 base pairs Žbp. upstream of the 3X end of exon 1. In addition to the sequence of the 5X-flanking region contained a sequence weakly homologous to a TATA box at position y41 to y36 and many transcriptional regulatory elements. Transient expression revealed that a 45-bp region of proximal to exon 1, which contained TATA-like sequence, was sufficient for promoting luciferase expression in OK-cells derived from opossum kidney proximal tubule. q 1998 Elsevier Science B.V. Keywords: Sodium-dependent phosphate cotransporter; Gene promoter; Kidney; ŽHuman.
Regulation of phosphate ŽPi. reabsorption in the renal proximal tubule is accomplished mainly by modulation of apical NaqrPi cotransporter w1x. The metabolic and hormonal factors that influence apical NaqrPi cotransporter exert their effects primarily by changing Pi reabsorption in the proximal tubule w1,2x. Abbreviations: Pi, inorganic phosphate; RACE, rapid amplification of cDNA end; HNF-1, hepatic nuclear factor-1; HNF-5, hepatic nuclear factor-5; CrEBP, CCAATrenhancer binding protein; Pho4, transcriptional activator protein of the phosphate regulon in Saccharomyces cereÕisiae ) Corresponding author. Fax: q81-886-33-7094; E-mail: [email protected] 1 The nucleotide sequence reported in this paper has been submitted to the DDBJrEMBLrGenbank nucleotide sequence databases with the following accession number D83236.
In recent years, two mammalian proximal tubular brush border NaqrPi cotransporters Žtypes I and II. have been identified by expression cloning techniques w3x. Functional analysis studies have shown that the characterization of type II transporter correspond to the well-known properties of proximal tubular brush border membrane of Pi transport w3x. Indeed, expression of type II NaqrPi cotransporter is observed only in kidney, while type I is primarily expressed in kidney, liver, and brain w3x. However, an increased Naq-dependent Pi uptake was also observed after stable transfection of the type I transporter into MDCK cells w4x and in Xenopus oocyte expression analysis w5x. The immunoreactive protein of the type I has a exclusively proximal tubular brush border location w6,7x. In addition, type I NaqrPi
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 2 3 1 - 5
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cotransporters have been identified subsequently by homology-based screening procedures in mouse ŽNpt-1. w8x, rat ŽRNaPi-1. w9x, rabbit ŽNaPi-1. w5x and human ŽNPT-1. w10,11x kidney. Homologous protein was also identified in rat brain-specific NaqrPi cotransporter ŽBNP-1. w12,13x. Antisense hybrid depletion strategy and oocytes injection experiment provided that types I and II cotransporters might account for full NaqrPi cotransport activity w11x. Recent studies have shown that the content of type II NaqrPi cotransporter protein expressed in brush border membrane and the related mRNA were reduced in X-linked hypophosphatemic Ž Hyp . mice showing also reduced brush border membrane NaqrPi cotransport activity w3x. In contrast, type I NaqrPi transporter seems to be unaffected in Hyp mice w8x. Interestingly, the expression of type I is altered by fasting and streptozotocin-induced diabetic states w14x. Insulin has a significant effect on renal Pi transport activity and type I NaqrPi cotransporter expression w1,2x. Thus two types of NaqrPi cotransporters are regulated by different factors and each subtype may be involved in a specific cellular function. Recently, we have characterized the human type II NaqrPi cotransporter Ž NaPi-3 . chromosomal gene and analyzed its promoter function w15x. To further clarify the differences in the functional role between type I and type II NaqrPi cotransporters, the 5X flanking region of the human type I NPT-1 gene have been characterized in this study. Cloning of 5X-flanking region of the human NPT-1 gene. We established the 5X end of NPT-1 mRNA sequence reported initially by Chong et al. w10x using the 5X RACE PCR technique with the 5X RACE ready cDNA library as previously described w15x. Two sequential polymerase chain reaction Ž PCR. amplification reactions were employed to identify the 5X end of the NPT-1 mRNA and to generate DNA fragment useful for initial sequencing. Sequence determination of the PCR products of these sequential reactions established the 5X end of the NPT-1. The cloning and DNA sequence analysis of the resulting product suggested that the original cDNA sequence reported by Chong et al. w10x was incomplete at the 5X end by 147 bases. We utilized the 5X RACE-derived DNA fragment to map the exon 1. However, we could not find any clones that contains the 5X end of NPT-1 cDNA. To further isolate clones containing exon 1, we pre-
Fig. 1. Cloning of the 5X flanking region of the NPT-1 gene. Ža. Sequences of Sau 3AI cassette w23x and used primers. Žb. Amplification of a probe containing a new position of the 5X flanking region of the NPT-1 gene from BamHI-digested genomic DNA in Sau 3AI cassettes Žopen box. by two round of PCR with the indicated primers ŽC1, C2, NPT-1-R, and NPT-1-R3.. Žc. A restriction map of the 5X flanking region of the NPT-1 gene containing exon 1 and intron 1. Nucleotides are numbered with the major transcription initiation site as q1. Restriction enzymes: H, HindIII; E, EcoRI.
pared a DNA probe by PCR with the Sau 3AI cassette cloning system ŽFig. 1a,b.. An ; 2-kb DNA fragment containing the 5X flanking region and intron 1 of the NPT-1 gene was thus amplified by PCR, labeled with 32 P, and used to screen a lEMBL-3 human genomic DNA library. A single clone, lNPT11, was obtained and was digested with HindIII or EcoRI, and the resulting fragments were subcloned into plasmid pBluescript II SKŽq. for sequencing. The restriction enzyme map of the 5X flanking region of the NPT-1 gene is shown in Fig. 1c. Identification of the transcription start site. The transcription initiation site of NPT-1 mRNA was determined by primer extension analysis and S1 nuclease mapping. A 32 P-labeled antisense oligonucleotide derived from NPT-1 cDNA was hybridized to total RNA from human kidney cortex and then extended by reverse transcriptase. By comparing with a
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major transcription site was identified as a guanosine residue 109 or a adenosine residue 104 bases upstream of the 3X end of the exon 1 Ž Fig. 3. . In addition, S1 nuclease protection assay was performed to identify the transcription initiation sites. In a experiment with 148 bp probe, a major protected fragment of 104 bp was observed in RNA from human kidney cortex ŽFig. 2b.. These results confirmed that the
Fig. 2. Primer extension analysis and SI nuclease mapping. Ža. Primer extension analysis was carried out essentially as described w15,24x. An oligonucleotide NPT-1-R3 was labeled at its 5X terminus with w g- 32 PxATP and T4 polynucleotide kinase. The labeled primer Ž100,000 counts per minute. was then hybridized at 428C for 1 h to total RNA Ž10 m g. isolated from human kidney cortex Žhuman kidney. or yeast t-RNA Žyeast t-RNA. in a reaction mixture Ž20 m l. containing 10 mM Tris–HCl ŽpH 8.3., 250 mM KCl, and 1 mM EDTA. Reverse transcription and electrophoresis were then performed as described w15,24x. The major products of primer extension are indicated by arrows. The products length was estimated with reference to molecular weight marker ŽM.. Žb. S1 nuclease mapping was carried out essentially as described w25x. Probe was prepared by synthesis with Klenow fragment of DNA polymerase I, w a- 32 PxdCTP, NPT-1-R2 primer Ž5X-CTGATTCGGGACAAAAAACAAGTG, corresponding to nucleotides q84 to q104. and HindIII digested NPT-1 DNA fragment containing exon 1 as template. The probes were hybridized with 50 m g of total RNA from human kidney cortex Žhuman kidney RNA. or yeast t-RNA Žyeast t-RNA. in a hybridization buffer Ž20 m l. containing 80% formamide, 40 mM PIPES ŽpH 6.4., 400 mM NaCl, and 1 mM EDTA. S1 nuclease reaction and electrophoresis were then performed as described w25x. The digest is indicated by arrow and analyzed the size with reference to sequence ladders obtained with the same primer using for probe preparation.
molecular weight marker, the length of the two products was 200 and 195 nucleotides including the primer with additional restriction site Ž Fig. 2a. . Thus, the
Fig. 3. Nucleotide sequence of the 5X flanking region to exon 1 of the NPT-1 gene. Nucleotides are numbered with the major transcription initiation site, which is shown by arrow, as q1. Lower case letters show intron sequences, and bold lower case letters show consensus sequence of exon–intron junction known as GT–AG rule. Upper case letters show the sequences of exon or 5X flanking region. Various cis-regulatory elements are indicated as follows: CrEBP, CCAATrenhancer-binding protein binding site; AP-1, AP-1 binding site; AP-2, AP-2 binding site; HNF-1, hepatic nuclear factor-1 binding site; HNF-5, hepatic nuclear factor-5 binding site; GRE, glucocorticoid responsive element; TATA-like, a sequence weakly homologous to TATA box. The arrow head indicates the 5X end nucleotide derived from sequencing a major 5X RACE product. The underlined sequence indicates the translation initiation site.
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major initiation site was the adenosine residue located at 104 bases upstream of the 3X end of exon 1. Characterization of the NPT-1 gene promoter region. The nucleotide sequences of exon 1 and its 5X flanking region are shown in Fig. 3. Although the proximal ; 1.5 kb of 5X flanking of the NPT-1 gene does not contain typical TATA or GC boxes, a TTTTAA sequence, which shows weak homology to a TFIID binding site, is apparent at y41 to y36 bp relative to the transcription initiation site ŽFig. 3.. This sequence may play the role of a TATA box. Several well-characterized cis-acting elements including two AP-1 at positions y1151 and y418, two AP-2 sites at positions y886 and y846, a CCAATrenhancer binding protein binding site ŽCrEBP sites. at positions y637. In addition, two HNF-1 and two HNF-5 sequences at position y1287, y1091, y1405, y1219 were detected in the promoter. Furthermore, we determined the sequences of exon–intron junctions of 5X and 3X end of intron 1 and 5X and of intron 2. These sequences conserved the GT–AG rule w16x Ž Fig. 3. .
Promoter actiÕity of the 5X flanking region of the NPT-1 gene. Reporter constructs were made in which the 5X upstream sequences of the human NPT-1 gene was fused to a luciferase reporter gene. When transfected into OK-cells derived from opossum kidney proximal tubule, a construct containing sequences y1419 to q104 from the pNPT1.4 was able to detect expression of luciferase activity which on average was 1.5-fold compared with those in the vector alone. Progressive deletion to positions y670 and y45 resulted in an increase in activity, reaching a maximum 300% of the pNPT1.4 construct. In contrast, the pNPT1.4rev was decreased ; 60% of the maximum activity. These results suggest the presence of multiple negative regulatory elements localized between y1419 and y45 in the NPT-1 gene promoter. In addition, deletion of the proximal sequence y45 to q104 ŽpNPTD 0.04. completely abolished the induction of luciferase activity. The reverse orientation of the promoter Žy45 to q104. relative to the reporter did not produce luciferase activity Ždata not shown.. Thus, a 45-bp region of proximal to exon 1,
Fig. 4. Functional analysis of the human NPT-1 gene promoter. Various truncated promoter fragments were prepared by PCR and digestion with appropriate restriction enzymes indicated in top of figure, and was ligated into a pGL-3 luciferase reporter plasmid. These reporter plasmids were named as indicated. Numbers indicated distance in base pairs from the transcription initiation site. Restriction enzyme cleavage sites ŽS, SacI; H, HindIII. and putative regulatory elements were also shown at the top of figure. The b-galactosidase expression vector pCMV-b was used as an internal control. OK-cells were provided from American Type Cell Collection ŽCRL1840. and cultured at 378C under 5% CO 2 in F-12rDMEM Ž1:1. containing 10% fetal bovine serum. Transfection of OK-cells was performed by Lipofectamine with 0.5 m g of the NPT-1 promoter-luciferase reporter vector and 0.5 m g of pCMV-b per 5 = 10 5 cells. After transfection, the cells were incubated under standard conditions for 48 h. Cells were then harvested in cell lysis buffer w24x and the lysate assayed for luciferase activity. b-galactosidase activity, and protein concentration w24x. Luciferase activity was corrected for differences in transfection efficiency between experiments by normalizing to b-galactosidase activity values. Data are means" S.E.M. of triplicate determinations. These results were representative of three separate experiments.
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which contained a TATA-like sequence, was sufficient for promoting luciferase expression in OK-cells ŽFig. 4.. In a previous study, we isolated a type I NaqrPi cotransporter Ž NPT-1. gene from a human kidney cDNA library w11x. Functional analysis in Xenopus oocytes showed NPT-1 to be a high affinity NaqrPi cotransporter which suggest that at least two high affinity NaqrPi cotransporter Ž NPT-1 and NaPi-3. are present in the human renal cortex w11x. The structure of human type II Ž NaPi-3 . chromosomal gene has been determined recently w15x. The organization of the genes was approximately 16-kb long and comprising 13 exons and promoter regions. There is a typical TATA box and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2, and SP-1 sites in the promoter. This region also contains three direct repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone superfamily, including vitamin D. Deletion analysis indicated that the upstream region Žfrom nt-2409 to nt-1259. in the NaPi-3 gene promoter is important in kidney specific gene expression w15x. However, we could not find out homologous region for specific gene expression in kidney between the NaPi-3 gene and the NPT-1 gene. As shown in Fig. 3, the NPT-1 gene promoter may belong to the family of promoter sequences that bind already known liver-enriched transactivators Ž i.e., CrEBP, HNF-1, HNF-5. w17,18x. Indeed, NPT-1 is abundant in the liver w11x. Moreover, the levels of HNF-1 mRNA are known to be reduced in diabetic rat w19x. Since insulin and glucose stimulate the expression of rat type I NaqrPi cotransporter gene w14x, changes in the amount of HNF-1 protein may affect the expression of the NPT-1 gene in a model animals with diabetes mellitus. More recently, Pontoglio et al. w20x reported that HNF-1 knockout mice manifested renal Fanconi syndrome characterized by phosphaturia, polyuria, glucosuria, and generalized aminoaciduria. This syndrome is the results from multiple transport dysfunctions mainly in the renal proximal tubules. Most of these transports rely on the Naq-dependent cotransporter w20x. The reduced number of Naqrglucose cotransporter molecule was detected in the mutant mice w20x. These observations suggest that HNF-1 may regulate the expression of
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apical Naq-dependent cotransporter genes in the kidney. Interestingly, the existence of overlaps between HNF-5 and a glucocorticoid binding site at a position y1231 to y1213 is observed in the NPT-1 gene promoter. This element is important in glucocorticoid receptor action in the rat tyrosine aminotransferase ŽTAT. gene promoter w21x. Rat TAT gene is expressed specially in liver where its transcription rate is increased by glucocorticoids w22x. These observations suggest that glucocorticoids may be important regulators of NPT-1 in the liver. Finally, these features of the NPT-1 gene promoter will lead to a better understanding of the mechanism of tissue-specific expression and the physiological regulation by the metabolic and hormonal factors. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Setsuro Fujii Memorial Foundation, the Uehara Memorial Foundation, and the Salt Science Research Foundation. References w1x H. Murer, J. Biber, in: D.W. Seldin, G. Giebisch ŽEds.., The Kidney: Physiology and Pathophysiology, 2nd edn., Raven Press, New York, 1992, pp. 2481–2509. w2x T.J. Berndt, F.G. Knox, in: D.W. Seldin, G. Giebisch ŽEds.., The Kidney: Physiology and Pathophysiology, 2nd edn., Raven Press, New York, 1992, pp. 2511–253. w3x H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 433 Ž1997. 379–389. w4x E.S. Quabius, H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 430 Ž1995. 132–136. w5x A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, H. Murer, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 9608– 9612. w6x M. Levi, S.A. Kempson, M. Lotscher, J. Biber, H. Murer, J. ¨ Membr. Biol. 154 Ž1996. 1–9. w7x J. Biber, M. Arar, B. Kaissling, H. Murer, J. Biber, Pflug. Arch. Eur. J. Physiol. 426 Ž1994. 5–11. w8x S.S. Chong, C.A. Kozak, L. Liu, K. Kristjansson, S.T. Dunn, J.E. Brourdeau, M.R. Hughes, Am. J. Physiol. 268 Ž1995. F1038–F1045. w9x H. Li, Z. Xie, Cell. Mol. Biol. Res. 41 Ž1995. 451–460. w10x S.S. Chong, K. Kristjansson, H.Y. Zoghbi, M.R. Hughes, Genomics 18 Ž1993. 335–339. w11x K. Miyamoto, S. Tatsumi, T. Sonoda, H. Yamamoto, H. Minami, Y. Taketani, E. Takeda, Biochem. J. 305 Ž1995. 81–85. w12x B. Ni, X.-W. Yan, J. Wang, S.M. Paul, J. Neurosci. 15 Ž1995. 5789–5799.
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