PAX 8 Regulates Human WT1 Transcription through a Novel DNA Binding Site
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.30678
ISSN1083-351X
AutoresGail Fraizer, Ryuji Shimamura, Xiaohong Zhang, Grady F. Saunders,
Tópico(s)Renal cell carcinoma treatment
ResumoThe Wilms' tumor gene (WT1) is an essential gene for kidney and gonadal development, although how WT1 expression is induced in these tissues is not known. One kidney transcription factor likely to play a role in this regulation is PAX 8. The co-expression of WT1 and PAX 8 during kidney development and in Wilms' tumors with an epithelium predominant histology suggested a possible interaction, and indeed, we identified potential core PAX-binding sites in the WT1 promoter. Endogenous PAX 8 plays an important role in the activation of the WT1promoter, since promoter activity is much stronger in cells with PAX 8 than without. Using binding assays, we searched for evidence of PAX 8-DNA interactions throughout the 652-base pair human WT1promoter and found only one functional PAX 8 site with DNA binding activity, located 250 base pairs 5′ of the minimal promoter. The responsiveness of the PAX 8 site was confirmed by assessing its ability to function as an enhancer significantly activating the minimal promoter in a position- and orientation-independent manner. Using transfection assays, we demonstrated that either endogenous or exogenously added PAX 8 activated the WT1 promoter and that this promoter up-regulation depended upon the presence of an intact PAX 8-binding site. In contrast, the previously reported core PAX 8-binding sites identified by computer analysis of the WT1 promoter failed to specifically bind in vitro translated PAX 8 protein or activate the minimal promoter. Thus, we identified a novel functional binding site for the transcription factor PAX 8, suggesting that part of its role in kidney development may be as a modulator of WT1 expression in the kidney. The Wilms' tumor gene (WT1) is an essential gene for kidney and gonadal development, although how WT1 expression is induced in these tissues is not known. One kidney transcription factor likely to play a role in this regulation is PAX 8. The co-expression of WT1 and PAX 8 during kidney development and in Wilms' tumors with an epithelium predominant histology suggested a possible interaction, and indeed, we identified potential core PAX-binding sites in the WT1 promoter. Endogenous PAX 8 plays an important role in the activation of the WT1promoter, since promoter activity is much stronger in cells with PAX 8 than without. Using binding assays, we searched for evidence of PAX 8-DNA interactions throughout the 652-base pair human WT1promoter and found only one functional PAX 8 site with DNA binding activity, located 250 base pairs 5′ of the minimal promoter. The responsiveness of the PAX 8 site was confirmed by assessing its ability to function as an enhancer significantly activating the minimal promoter in a position- and orientation-independent manner. Using transfection assays, we demonstrated that either endogenous or exogenously added PAX 8 activated the WT1 promoter and that this promoter up-regulation depended upon the presence of an intact PAX 8-binding site. In contrast, the previously reported core PAX 8-binding sites identified by computer analysis of the WT1 promoter failed to specifically bind in vitro translated PAX 8 protein or activate the minimal promoter. Thus, we identified a novel functional binding site for the transcription factor PAX 8, suggesting that part of its role in kidney development may be as a modulator of WT1 expression in the kidney. The Wilms' tumor gene (WT1), 1The abbreviations used are: WT1, Wilms' tumor 1; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyl transferase; EMSA, electrophoretic mobility shift assay. a tumor suppressor gene, was isolated by positional cloning of the 11p13 region (1Call K.M. Glaser T. 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EMBO J. 1995; 14: 4662-4675Crossref PubMed Scopus (311) Google Scholar, 17Adachi Y. Matsubara S. Pedraza C. Ozawa M. Tsutsui J. Takamatsu H. Noguchi H. Akiyama T. Muramatsu T. Oncogene. 1996; 15: 2197-2203Google Scholar, 18Shimamura R. Fraizer G. Trapman J. Lau Y.F. Saunders G.F. Clinical Cancer Res. 1997; (in press)PubMed Google Scholar) and functions as a tumor suppressor gene (19Knudson A.G. Strong L.C. J. Natl. Cancer Inst. 1972; 48: 313-324PubMed Google Scholar). WT1 is essential for the differentiation of mesenchymal blastema to epithelial and stromal structures during kidney development. Kidney development is arrested in transgenic mice containing a homozygous deletion in the WT1 gene (20Pritchard-Jones K. Fleming S. Davidson D. Bickmore W. Porteous D. Gosden C. Bard J. Buckler A. Pelletier J. Housman D.E. van Heyningen V. Hastie N. Nature. 1991; 346: 194-197Crossref Scopus (769) Google Scholar). Homozygous WT1mutations result in embryonic lethality and developmental defects in the urogenital system, heart, lungs, and mesothelia. During embryogenesis, WT1 is expressed at high levels in the genitourinary system (21Park S. Schalling M. Bernard A. Maheswaran S. Shipley G. Roberts D. Fletcher J. Shipman R. Rheinwald J. Demetri G. Griffin J. Minden M. Housman D. Haber D. Nat. Genet. 1993; 4: 415-420Crossref PubMed Scopus (189) Google Scholar) and mesothelia (22Armstrong J. Pritchard-Jones K. Bickmore W. Hastie N. Bard B. Mech. Dev. 1992; 40: 85-97Crossref Scopus (501) Google Scholar) and at lower levels in fetal hematopoietic organs (1Call K.M. Glaser T. Ito C.Y. Buckler A.J. Pelletier J. Haber D.A. Rose E.A. Kral A. Yeger H. Lewis W.H. Jones C. Housman D.E. Cell. 1990; 60: 509-520Abstract Full Text PDF PubMed Scopus (1676) Google Scholar, 3Huang A. Campbell C.E. Bonetta L. McAndrews-Hill M.S. Chilton-MacNeill S. Coppes M.J. Law D.J. Feinberg A.P. Yeger H. Williams B.R.G. Science. 1990; 250: 991-994Crossref PubMed Scopus (136) Google Scholar, 23Fraizer G.C. Patmasiriwat P. Zhang X.-H. Saunders G.F. Blood. 1995; 86: 4704-4707Crossref PubMed Google Scholar, 24Pelletier J. Schalling M. Buckler A.J. Rogers A. Haber D.A. Housman D.E. Genes Dev. 1991; 5: 1345-1356Crossref PubMed Scopus (386) Google Scholar). In the adult, WT1 is expressed in the testes, mesothelia, and immature hematopoietic progenitor cells,e.g. CD34+ bone marrow cells (22Armstrong J. Pritchard-Jones K. Bickmore W. Hastie N. Bard B. Mech. Dev. 1992; 40: 85-97Crossref Scopus (501) Google Scholar, 24Pelletier J. Schalling M. Buckler A.J. Rogers A. Haber D.A. Housman D.E. Genes Dev. 1991; 5: 1345-1356Crossref PubMed Scopus (386) Google Scholar, 25Kreidberg J.A. Sariola H. Loring J.M. Maeda M. Pelletier J. Housman D. Jaenisch R. Cell. 1993; 74: 679-691Abstract Full Text PDF PubMed Scopus (1672) Google Scholar). Differential WT1 expression during development suggests that WT1 expression is tightly regulated by tissue-specific transcription factors. PAX 8 encodes a developmentally important paired box transcription factor that is expressed in the developing kidney, among other tissues (26Stuart E.T. Gruss P. Cell Growth Differ. 1996; 7: 405-412PubMed Google Scholar). The expression of PAX 8 in the developing kidney precedes WT1 expression. Its expression initiates in the induced condensing mesenchyme, peaks in the S-shaped bodies, and finally declines in the epithelial cells of the glomerulus (27Poleev A. Fickenscher H. Mundlos S. Winterpacht A. Zabel B. Fidler A. Gruss P. Plachov D. Development. 1992; 116: 611-623Crossref PubMed Google Scholar). The WT1 expression pattern is similar to the PAX 8 pattern, with its peak occurring after PAX 8 expression begins to decline and WT1 expression persists in the mature podocytes of the glomerulii (27Poleev A. Fickenscher H. Mundlos S. Winterpacht A. Zabel B. Fidler A. Gruss P. Plachov D. Development. 1992; 116: 611-623Crossref PubMed Google Scholar, 28Plachov D. Chowdhury K. Whalther C. Simon D. Guenet J.L. Gruss P. Development. 1990; 110: 643-651Crossref PubMed Google Scholar). Like WT1 expression, PAX 8 expression has also been found in the Wilms' tumors with an epithelial predominant histology 2S. Hamada and G. F. Saunders, unpublished observations. (27Poleev A. Fickenscher H. Mundlos S. Winterpacht A. Zabel B. Fidler A. Gruss P. Plachov D. Development. 1992; 116: 611-623Crossref PubMed Google Scholar, 29Miwa H. Tomlinson G.E. Timmons C.F. Huff V. Cohn S.L. Strong L.C. Saunders G.F. J. Natl. Cancer Inst. 1992; 84: 181-187Crossref PubMed Scopus (38) Google Scholar, 30Tagge E.P. Hanson P. Re G.G. Othersen Jr., H.B. Smith C.D. Garvin A.J. J. Pediatr. Surg. 1994; 29: 134-141Abstract Full Text PDF PubMed Scopus (27) Google Scholar). The presence of PAX 8 mRNA in these tumors with high WT1 expression is consistent with its postulated role as an activator of WT1 expression. PAX 8 paralogues include PAX 2 and PAX 5. UnlikePAX 2 and PAX 8, PAX 5 is expressed in developing hematopoietic cells (pre-B cells). While in vivodownstream target genes have not been identified for thesePAX genes, several genes whose expression is regulated by PAX 5 and PAX 8 in vitro have been identified, and consensus binding sites have been defined by oligonucleotide selection methods. PAX 5 can regulate expression of the B cell-specific surface protein CD19 (31Kozmik Z. Kurzbauer R. Dorfler P. Busslinger M. Mol. Cell. Biol. 1993; 13: 6024-6035Crossref PubMed Scopus (143) Google Scholar), and PAX 8 can regulate expression of thyroperoxidase and thyroglobin (32Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar). Recently, engrailed-2 was shown to be a target gene for Pax 2, Pax 5, and Pax 8 (which are co-expressed with Engrailed-2) in mouse embryos during mid-hindbrain development (33Song D.-L. Chalepakis G. Gruss P. Joyner A. Development. 1996; 122: 627-635PubMed Google Scholar). PAX 2, PAX 5, and PAX 8 each have only a partial homeodomain, so DNA binding is mediated by the paired domain, which is composed of three α-helices. The NH2-terminal subdomain of the paired domain is highly conserved and binds a core recognition sequence of the PAX-binding site. However, the COOH-terminal subdomain also influences site-specific binding (34Jun S. Desplan C. Development. 1996; 122: 2639-2650Crossref PubMed Google Scholar). While the WT1 promoter coupled to the SV40 enhancer strongly activates transcription in all cell lines tested (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar), its basal activity varies among cell lines, suggesting differential transactivation by tissue-specific factors. In constructs lacking the SV40 enhancer, the transcriptional activity of the WT1promoter is much lower, as is typical of a TATA-less, CCAAT-less, GC-rich promoter. The ubiquitous transcription factor Sp1 has been shown by footprint analysis to bind at many positions throughout theWT1 promoter and to transactivate the promoter region in cotransfection assays (36Hofmann W. Royer H.-D. Drechsler M. Schneider S. Royer-Pokora B. Oncogene. 1993; 8: 3123-3132PubMed Google Scholar, 37Cohen H.T. Bossone S.A. Zhu G. McDonald G.A. Sukhatme V.P. J. Biol. Chem. 1997; 272: 2901-2913Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). WT1 protein also binds at many positions throughout the WT1 promoter, and the least abundant isoforms lacking the KTS insertion strongly repress theWT1 promoter in cotransfection assays (38Rupprecht H.D. Drummond I.A. Madden S.L. Rauscher III, F.J. Sukhatme V.P. J. Biol. Chem. 1994; 269: 6198-6206Abstract Full Text PDF PubMed Google Scholar, 39Hewitt S.M. Fraizer G.C. Wu Y.-J. Rauscher III, F.J. Saunders G.F. J. Biol. Chem. 1996; 271: 8588-8592Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The autoregulatory sites in the WT1 promoter may be essential for the down-regulation of WT1 expression observed during the differentiation of leukemic cells (40Sekiya M. Adachi M. Hinoda Y. Imai K. Yachi A. Blood. 1994; 83: 1876-1882Crossref PubMed Google Scholar, 41Phelan S.A. Lindberg C. Call K.M. Cell Growth Differ. 1994; 5: 677-686PubMed Google Scholar). Using deletion analysis, we previously identified the minimal promoter region necessary and sufficient for promoter activity in K562 and HeLa cells (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar). This 104-bp minimal promoter is GC-rich (79% G + C); contains seven overlapping potential transcription factor binding sites within a core of 30 bp, including Sp1, AP4, WT1, and CACCC binding sites; and has half the transcriptional activity of the full-length WT1promoter. To better understand the mechanism for differential promoter activity in various cell lines, derived from different tissues, we examined the 5′-flanking region of the full-length human WT1 promoter. Sequence analysis identified potential binding sites for the zinc finger GATA factors (42Evans T. Reitman M. Felsenfeld G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5976-5980Crossref PubMed Scopus (361) Google Scholar) and two paired box transcription factors, PAX 8 (32Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar) and PAX 2 (43Epstein J. Cai J. Glaser T. Jepeal L. Mass R. J. Biol. Chem. 1994; 269: 8355-8361Abstract Full Text PDF PubMed Google Scholar). The latter two are co-expressed with WT1 in the kidney and may contribute to kidney expression of WT1 in vivo. Recently, the murine WT1 promoter was shown to be transactivated by both Pax 2 and Pax 8 (44Dehbi M. Pelletier J. EMBO J. 1996; 15: 4297-4306Crossref PubMed Scopus (62) Google Scholar, 45Dehbi M. Ghahremani M. Lechner M. Dressler G. Pelletier J. Oncogene. 1996; 13: 447-453PubMed Google Scholar). We have identified a novel consensus site 250 bp 5′ of the minimal promoter that strongly bound PAX 8 and mediated potent PAX 8 transactivation of the human WT1 promoter. This is in contrast to the potential PAX 8-binding sites previously identified in the human WT1 promoter (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar), which were unable to bind PAX 8 or mediate transactivation ofWT1. Cell lines 293 (derived from adenovirus 5-transformed human embryonic kidney cells) (CRL1573; American Type Culture Collection (ATCC), Rockville, MD), ACHN (human renal carcinoma cells) (CRL1611, ATCC), Madin-Darby canine kidney cells (CCL34, ATCC), and HeLa (a human cervical carcinoma cell line) (CCL2, ATCC) were grown in Eagle's minimal essential medium. Renal carcinoma cells Caki-1 and Caki-2 (HTB46 and -47, respectively; ATCC) were grown in McCoy's 5a medium. BHK, a cell line derived from baby hamster kidney cells (CRL10, ATCC) was grown in Dulbecco's modified Eagle's medium. TM4, a mouse Sertoli cell line (CCL1715, ATCC) was grown in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium. K562, a cell line derived from a human chronic myelogenous leukemia in blast crisis (ATCC, CCL243), was maintained in RPMI 1640 medium. All cells were maintained in media supplemented with 10% fetal calf serum. Monolayers were seeded, and the suspension culture (K562) was split 48–72 h before transfection. The cells were transfected while in the log phase of growth, i.e. the monolayers were <75% confluent, and the suspension cultures were at <5 × 105 cells/ml. The 652-bpHindIII-PstI fragment comprising theWT1 promoter contains potential binding sites for the PAX, GATA, E2A, Sp1, and WT1 transcription factors (Fig. 1). To determine the role of the PAX core binding sites, we subcloned the 5′-flanking region containing two potential PAX 8 core sites (CTGCCC) and the distal potential PAX 2 core site (GTTCCC) but lacking the most 5′ novel PAX site, WT1 PAX CON. The 652-bp WT1 promoter was used as a template for PCR amplification of the 130-bp fragment with primers altered to insert BamHI sites (altered bases are in boldface type) as described elsewhere (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar). The forward PCR primer (5′-CTGCGGGATCCTGAAGTTCC-3′) (bp 20–39) and the reverse PCR primer (5′-TTCAGGTAAGCAGTGGATCCG-3′) (bp 128–149) were derived from the 5′-flanking region of the promoter sequence (beginning 20 bp 3′ of the HindIII site). The PCR product was digested with BamHI, and the resulting 104-bp BamHI fragment was purified on a Qiagen column (Qiagen, Chatsworth, CA) and cloned in the reverse orientation into the BamHI site of theWT1 minimal promoter construct pcb.1 to create the 5′-flanking construct, pcb.1e.1. The orientation and sequence of the 5′-flanking region were confirmed by sequence analysis. To determine if the novel WT1 PAX CON site was sufficient for PAX 8 transactivation of WT1, we cloned the 30-bp double-stranded oligonucleotide used in EMSA (see below) 3′ of the chloramphenicol acetyltransferase (CAT) gene in pcb.l, the minimal WT1promoter construct. Initially, BamHI linkers were ligated to the 30-mer, and then both the double-stranded oligonucleotide insert and promoter construct were digested with BamHI. The free linkers were removed from the insert by column chromatography, and the insert was ligated to phosphatase-treated pcb.1, the minimalWT1 promoter construct, to create the PAX 8-enhancer construct, pcb.le.05. While the WT1 promoter construct pcb.7PH contains several potential PAX 8-binding sites, the only site able to bind PAX 8 in EMSA is at the most 5′-end of the 652-bp promoter. The mutant transactivation reporter target construct was created by PCR amplification of the 652-bp WT1 promoter in pcb.7PH by using a forward PCR primer (5′-TATGACCAAGCTTACGCCAAGATTGTCTGAGTTCTTTCTG-3′) containing the WT1 promoter 5′-flanking sequence (underlined) with a mutant PAX 8 site (as discussed below) and pCAT®-Basic 5′-flanking sequence containing a 2-bp mismatch to create a HindIII site (altered bases are in boldface type). The reverse PCR primer S3 (5′-CTCCTGAAAATCTCGCCAAGC-3′) was derived from pCAT®-Basic and is adjacent to the PstI cloning site. The PCR-amplified fragment was digested with HindIII andPstI, and the resulting 661-bp promoter fragment was cloned into the HindIII and PstI-digested pCAT®-Basic vector to create mpcb.7PH (replacing the wild-type promoter in pcb.7PH). The mutagenized binding site and its orientation in the clone were verified by DNA sequence analysis. K562, 293, and HeLa cells were transfected with pcb.1, pcb.1e.1, pcb.le.05, pcb.7PH, and mutant pcb.7PH as described previously (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar, 46Wu Y. Fraizer G.C. Saunders G.F. J. Biol. Chem. 1995; 270: 5944-5949Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). TM4 cells were electroporated at 250 V in 200 μl of serum-free medium containing 10 μg of plasmid DNA. As an internal control for transfection efficiency, the cells were cotransfected with 5 μg of the β-galactosidase control DNA, pSV40β-Gal (Promega Corp., Madison, WI). The cells were harvested, cytoplasmic extracts were prepared, and β-galactosidase activity was determined as described previously (35Fraizer G.C. Wu Y.-J. Hewitt S.M. Maity T. Ton C.C.T. Huff V. Saunders G.F. J. Biol. Chem. 1994; 269: 8892-8900Abstract Full Text PDF PubMed Google Scholar). Extracts containing equivalent amounts of β-galactosidase activity were assayed for CAT activity (47Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). The protein content of the cell extracts was also determined by the Bradford assay (Bio-Rad), and the CAT assays were performed with extracts containing 25–50 μg of protein (for K562 cells) or 50–100 μg of protein (for the other cell lines). After thin layer chromatography, the acetylated [14C]chloramphenicol was quantitated by measuring the radioactivity with a Betascope 603 blot analyzer (Betagen Corp., Waltham, MA). The relative activities shown are the averages from at least three different experiments. For transactivation assays PAX 8 was co-transfected into HeLa cells as described above except that increasing amounts of the human PAX 8 cDNA expression construct (48Hewitt S.M. Hamada S. Monarres A. Kotticap L.V. Saunders G.F. McDonnell T.J. Anticancer Res. 1997; (in press)PubMed Google Scholar), pSVK3PAX8, were added to each transfection of 5 μg of reporter pcb.7PH and 2 μg of pSV40β-Gal. The PAX 8 expression construct contains a 1.4-kilobase pair PAX8 cDNA including the entire open reading frame of the predominant isoform PAX 8a (32Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar). This cDNA was initially obtained by screening a human fetal kidney cDNA library (CLONTECH, Palo Alto, CA) and was cloned into the expression vector pSVK3 (Pharmacia Biotech Inc.) (48Hewitt S.M. Hamada S. Monarres A. Kotticap L.V. Saunders G.F. McDonnell T.J. Anticancer Res. 1997; (in press)PubMed Google Scholar). The mutant clone containing a mutagenized PAX 8-binding site mut.pcb.7pH, the empty pCAT®-Basic vector (Promega), the minimal promoter pcb.1e.1, and the PAX 8 enhancer construct pcb.1e.05 were also cotransfected with 5 or 10 μg of the PAX 8 expression construct and 2 μg of pSV40β-Gal. Total cellular RNA was extracted from 2-day-old mouse kidneys and all cell lines described above by the method of Chomczynski and Sacchi (49Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) with STAT-60 (Tel Test, Friendswood, TX) according to the manufacturer's recommendations. First strand cDNA synthesis and PCR amplification were performed as described previously (50Patmasiriwat P. Fraizer G.C. Claxton D. Kantarjian H. Saunders G.F. Leukemia. 1996; 10: 1127-1133PubMed Google Scholar). PCR amplification of cDNA encoding the Pro-Ser-Thr transactivation domain and carboxyl terminus of the PAX 8 gene in human samples was performed by using the primer pairs: forward PAX 8 (5′-TCCACCCCTTCCTCTTTATCT-3′) and reverse PAX 8 (5′-AGTCCTCCTGTTGCTCAGTCG-3′). As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was amplified by using forward GAPDH (5′-TTGCCATCAATGACCCCTTCA-3′) and reverseGAPDH (5′-CCAGTGAGCTTCCCGTTCAGC-3′). The cDNA encoding the octapeptide, homeodomain, and 5′-end of the Pro-Ser-Thr transactivation domain of the PAX 8 gene in non-human samples was amplified by PCR with the murine Pax 8 primer pairs forward murine Pax 8 (5′-AAGTCTCTGAGCCCAGGACA-3′) and reverse murinePax 8 (5′-GGATCTGCAGCAAGTCGGCT-3′). PCR amplification of cDNA encoding the WT1 zinc finger region and β-actin gene in human and non-human samples was carried out using the human and murine primer pairs as described (23Fraizer G.C. Patmasiriwat P. Zhang X.-H. Saunders G.F. Blood. 1995; 86: 4704-4707Crossref PubMed Google Scholar, 46Wu Y. Fraizer G.C. Saunders G.F. J. Biol. Chem. 1995; 270: 5944-5949Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The conditions for PAX 8and GAPDH co-amplification were 3 min of denaturation at 95 °C; 35 cycles of 94 °C denaturation for 1 min, 58 °C (human) or 64 °C (murine) annealing for 2 min, and 72 °C extension for 2 min; and a final 5-min extension at 72 °C. To compensate for the overabundance of GAPDH and β-actin mRNA, we used 110 the concentration of GAPDH and β-actin primers as PAX 8 primers (0.2 μm). The PCR products were analyzed on an ethidium bromide-stained 1.5% agarose gel. The size marker used was a 100-bp DNA ladder (Life Technologies, Inc.). The duplex PCR amplification resulted in PCR products of the expected sizes: 600 bp for GAPDH, 441 bp for human PAX 8, 558 bp for murine Pax 8, 330 bp for murineWT1, and 540 bp for β-actin. EMSAs were performed by using Caki-1 renal cell nuclear extracts and in vitro translated PAX 8 protein. The in vitro PAX 8 expression construct pBSPAX8 was derived from the PAX 8 expression construct pSVK3PAX8. The 1.4-kilobase pair PAX 8 coding sequence was released by EcoRI digestion, gel-purified, and subcloned into the EcoRI site in pBlueScript II (Stratagene). The adjacent T7 polymerase binding site was used forin vitro transcription according to the manufacturer's recommendations (Promega). PAX 8 protein was translated in vitro by coupled transcription and translation of 1 μg of the PAX 8 expression construct pBSPAX8 in a wheat germ extract system according to the manufacturer's recommendations (Promega). The molecular size of the PAX 8 protein product was confirmed by SDS-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts and compared with prestained molecular weight markers (Amersham Corp.). Nuclear miniextracts were prepared from exponentially growing K562 and Caki-1 cells by the method of Dignam et al. (51Dignam J.D. Martin P.L. Shastry B.S. Roeder R.G. Methods Enzymol. 1983; 101: 582-598Crossref PubMed Scopus (746) Google Scholar) as modified by Leeet al. (52Lee K.A.W. Bindereif A. Green M.R. Gene Anal. Technol. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar). The EMSAs were performed essentially as described by Singh et al. (53Singh H. Sen R. Baltimore D. Sharp P.A. Nature. 1988; 319: 154-158Crossref Scopus (624) Google Scholar). The 30-bp double-stranded oligonucleotides were labeled with [γ-32P]ATP by using T4 polynucleotide kinase. TheWT1 promoter PAX consensus probe (WT1 PAX CON), 5′-CGCTTCTTTGAAGCTTGACTGAGTTCTTTC-3′, was used to identify the PAX 8-binding site (predicted consensus PAX 8-binding sites are underlined). Radiolabeled control PAX 8 oligonucleotide CT (5′-TGATGCCCACTCAAGCTTAGACAGG-3′), which was derived from the thyroperoxidase gene (32Zannini M. Francis-Lang H. Plachov D. Di Lauro R. Mol. Cell. Biol. 1992; 12: 4230-4241Crossref PubMed Scopus (275) Google Scholar), was used as a positive control for comparison with the WT1 promoter probe WT1 PAX CON. One to two hundred fmol of labeled oligonucleotide (1–4 × 105 cpm) was incubated with either 6 μg of the nuclear protein extracts or 1 μl of a 50-μl reaction of in vitrotranslated PAX 8 protein lysate and 2 μg of poly(dI-dC) in 15 mm Tris-HCl (pH 7.5), 6.5% glycerol, 90 mmKCl, 0.7 mm EDTA, 0.2 mm dithiothreitol, and 0.1% bovine serum albumin. After a 30-min incubation at room temperature, the reaction mixture was analyzed by electrophoresis at 300 V on a 5% polyacrylamide/bisacrylamide gel (39:1) in 0.5 × TBE (45 mm Tris borate, and 1 mm EDTA, pH 8.0), dried, and visualized by autoradiography with Hyperfilm-AP film (Amersham). For competition assays, the unlabeled double-stranded PAX 8 oligonucleotide CT and consensus GATA oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) were used as positive and negative controls, respectively. To verify the importance of two in
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