Molecular Analysis of a Novel Winged Helix Protein, WIN
1997; Elsevier BV; Volume: 272; Issue: 32 Linguagem: Inglês
10.1074/jbc.272.32.19827
ISSN1083-351X
AutoresKwok‐Ming Yao, Sha Mi, Zhijian Lu, Gordon G. Wong,
Tópico(s)Genomics and Chromatin Dynamics
ResumoWe have cloned a novel winged helix factor, WIN, from the rat insulinoma cell line, INS-1. Northern blot analysis demonstrated that WIN is highly expressed in a variety of insulinoma cell lines and rat embryonic pancreas and liver. In adults, WIN expression was detected in thymus, testis, lung, and several intestinal regions. We determined the DNA sequences bound in vitro by baculovirus-expressed WIN protein in a polymerase chain reaction-based selection procedure. WIN was found to bind with high affinity to the selected sequence 5′-AGATTGAGTA-3′, which is similar to the recently identified HNF-6 binding sequence 5′-DHWATTGAYTWWD-3′ (where W = A or T, Y = T or C, H is not G, and D is not C). We have isolated human WIN cDNAs by library screening and 5′-rapid amplification of cDNA ends. Sequence analysis indicates that the carboxyl terminus of human WIN has been previously isolated as a putative phosphorylation substrate, MPM2-reactivephosphoprotein 2 (MPP2); WIN may be regulated by phosphorylation. Alignment of the rat and human WIN cDNAs and their comparison with mouse genomic sequence revealed that the WIN DNA binding domain is encoded by four exons, two of which (exons 4 and 6) are alternatively spliced to generate at least three classes of mRNA transcripts. These transcripts were shown by RNase protection assay to be differentially expressed in different tissues. Alternative splicing within the winged helix DNA binding domain might result in modulation of DNA binding specificity. We have cloned a novel winged helix factor, WIN, from the rat insulinoma cell line, INS-1. Northern blot analysis demonstrated that WIN is highly expressed in a variety of insulinoma cell lines and rat embryonic pancreas and liver. In adults, WIN expression was detected in thymus, testis, lung, and several intestinal regions. We determined the DNA sequences bound in vitro by baculovirus-expressed WIN protein in a polymerase chain reaction-based selection procedure. WIN was found to bind with high affinity to the selected sequence 5′-AGATTGAGTA-3′, which is similar to the recently identified HNF-6 binding sequence 5′-DHWATTGAYTWWD-3′ (where W = A or T, Y = T or C, H is not G, and D is not C). We have isolated human WIN cDNAs by library screening and 5′-rapid amplification of cDNA ends. Sequence analysis indicates that the carboxyl terminus of human WIN has been previously isolated as a putative phosphorylation substrate, MPM2-reactivephosphoprotein 2 (MPP2); WIN may be regulated by phosphorylation. Alignment of the rat and human WIN cDNAs and their comparison with mouse genomic sequence revealed that the WIN DNA binding domain is encoded by four exons, two of which (exons 4 and 6) are alternatively spliced to generate at least three classes of mRNA transcripts. These transcripts were shown by RNase protection assay to be differentially expressed in different tissues. Alternative splicing within the winged helix DNA binding domain might result in modulation of DNA binding specificity. We are interested in the molecular basis of endocrine and exocrine pancreas formation. Gene expression studies suggest both pancreas compartments are derived from a band of endodermal cells in the foregut that comprises the pancreatic primordium. These specific endodermal cells can be identified prior to overt pancreas morphogenesis by their characteristic expression of Type II glucose transporter (Glut2) (1Pang K. Mukonoweshuro C. Wong G.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9559-9563Crossref PubMed Scopus (125) Google Scholar) and the homeobox gene PDX-1 (2Gus Y. Montminy M.R. Stein R.W. Leonard J. Gamer L.W. Wright C.V.E. Teitelman G. Development. 1995; 121: 11-18PubMed Google Scholar). A genetic deletion of thePDX-1 gene results in an almost surgical deletion of the pancreas (3Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1545) Google Scholar, 4Offield M.F. Jetton T.L. Labosky P.A. Ray M. Stein R.W. Magnuson M.A. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-995Crossref PubMed Google Scholar). However, many additional transcription factors including HB9, Isl1, Neuro D/Beta 2, Nkx6.1, Pax6, and PTF1 are expressed in some cells of the pancreatic primordium and developing pancreas and may be important for complete pancreas development (5Harrison K.A. Druey K.M. Deguchi Y. Tuscano J.M. Kehrl J.H. J. Biol. Chem. 1994; 269: 19968-19975Abstract Full Text PDF PubMed Google Scholar, 6Thor S. Ericson J. Brannstrom T. Edlund T. Neuron. 1991; 7: 881-889Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 7Naya F.J. Strellrecht C.M.M. Tsai M.-J. Genes & Dev. 1995; 9: 1009-1019Crossref PubMed Scopus (515) Google Scholar, 8Jensen J. Serup P. Karlsen C. Nielsen T.F. Madsen O.D. J. Biol. Chem. 1996; 271: 18749-18758Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 9Turque N. Plaza S. Radvanyi F. Carriere C. Saule S. Mol. Endocrinol. 1994; 8: 929-938Crossref PubMed Scopus (111) Google Scholar, 10Krapp A. Knofler M. Frutiger S. Hughes G.J. Hagenbuchle O. Wellauer P.K. EMBO J. 1996; 15: 4317-4329Crossref PubMed Scopus (190) Google Scholar). Recently, analysis of Isl1- and Pax4-deficient embryos indicates that both transcription factors are required for endocrine islet cell formation (11Ahlgren U. Pfaff S.L. Jessell T.M. Edlund T. Edlund H. Nature. 1997; 385: 257-260Crossref PubMed Scopus (579) Google Scholar, 12Sosa-Pineda B. Chowdhury K. Torres M. Oliver G. Gruss P. Nature. 1997; 386: 399-402Crossref PubMed Scopus (646) Google Scholar). Additional transcription factors may be involved. The prototypical winged helix (WH) 1The abbreviations used are: WH, winged helix; PCR, polymerase chain reaction; RT, reverse transcription; SAAB, selection and amplification of binding sites; bp, base pair(s); kb, kilobase(s); RACE, rapid amplification of cDNA ends; ORF, open reading frame; RPA, RNase protection assay; MOI, multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay. 1The abbreviations used are: WH, winged helix; PCR, polymerase chain reaction; RT, reverse transcription; SAAB, selection and amplification of binding sites; bp, base pair(s); kb, kilobase(s); RACE, rapid amplification of cDNA ends; ORF, open reading frame; RPA, RNase protection assay; MOI, multiplicity of infection; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay. factors (name based on the x-ray structure of HNF-3γ DNA-binding domain complexed to the transthyretin promoter) (13Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1076) Google Scholar), Drosophila melanogasterForkhead (Fkh) and rat HNF3 factors, are associated with the development of endodermal-derived tissues. Fkh mutants have an intestinal phenotype and HNF3 factors were initially isolated from the liver biochemically (14Weigel D. Jurgens G. Kuttner F. Seifert E. Jackle H. Cell. 1989; 57: 645-658Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 15Lai E. Prezioso V.R. Smith E. Litvin O. Costa R.H. Darnell Jr., J.E. Genes & Dev. 1990; 4: 1427-1436Crossref PubMed Scopus (404) Google Scholar, 16Lai E. Prezioso V.R. Tao W. Chen W.S. Darnell Jr., J.E. Genes & Dev. 1991; 5: 416-427Crossref PubMed Scopus (430) Google Scholar). The WH factors are likely to have a role in many endodermally derived organ including the pancreas. Recent methods of degenerate PCR and low stringency hybridization have expanded the WH gene family (17Hacker U. Grossniklaus U. Gehring W.J. Jackle H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8754-8758Crossref PubMed Scopus (103) Google Scholar, 18Clevidence D.E. Overdier D.G. Tao W. Qian X. Pani L. Lai E. Costa R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3948-3952Crossref PubMed Scopus (218) Google Scholar, 19Kaestner K.H. Lee K.-H. Schlondorff J. Hiemisch H. Monaghan A.P. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7628-7631Crossref PubMed Scopus (126) Google Scholar, 20Pierrou S. Hellqvist M. Samuelsson L. Enerback S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (367) Google Scholar). More than 80 members have been identified in different species. Their origins and functions have been reviewed extensively (21Hromas R. Costa R. Crit. Rev. Oncol. Hematol. 1995; 20: 129-140Crossref PubMed Scopus (85) Google Scholar, 22Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (571) Google Scholar). WH genes may have diverse roles evident by their expression beyond endodermal derivatives. Functional diversity is evident in the wide spectrum of phenotypes associated with mutations of WH genes. HCM1 and FHL1 were isolated as suppressors of calmodulin and RNA polymerase III mutations, respectively, in yeast (23Zhu G. Muller E.G.D. Amacher S.L. Northrop J.L. Davis T.N. Mol. Cell. Biol. 1993; 13: 1779-1787Crossref PubMed Scopus (51) Google Scholar, 24Hermann-Le Denmat S. Werner M. Sentenac A. Thuriaux P. Mol. Cell. Biol. 1994; 14: 2905-2913Crossref PubMed Scopus (86) Google Scholar). Genetic analysis revealed thatD. melanogaster croc and slp1,2 are required for proper segmentation in early embryogenesis (25Hacker U. Kaufmann E. Hartmann C. Jurgens G. Knochel W. Jackle H. EMBO J. 1995; 14: 5306-5317Crossref PubMed Scopus (75) Google Scholar, 26Grossniklaus U. Pearson R.K. Gehring W.J. Genes & Dev. 1992; 6: 1030-1051Crossref PubMed Scopus (189) Google Scholar) andCaenorhabditis elegans lin-31 is essential for normal vulva development (27Miller L.M. Gallegos M.E. Morisseau B.A. Kim S.K. Genes & Dev. 1993; 7: 933-947Crossref PubMed Scopus (147) Google Scholar). In rodents, natural mutations at the nude locus, which resulted in abnormal hair growth and thymus development, were shown to be due to the disruptions of the whn WH gene (28Nehls M. Pfeifer D. Schorpp M. Hedrich H. Boehm T. Nature. 1994; 372: 103-107Crossref PubMed Scopus (561) Google Scholar). The knockout phenotypes of at least three WH genes have been reported. The knockout of HNF3β led to defective node formation and the absence of notochord (29Ang S.-L. Rossant J. Cell. 1994; 78: 561-574Abstract Full Text PDF PubMed Scopus (862) Google Scholar, 30Weinstein D.C. Ruiz i Altaba A. Chen W.S. Hoodless P. Prezioso V.R. Jessell T.M. Darnell Jr., J.E. Cell. 1994; 78: 575-588Abstract Full Text PDF PubMed Scopus (691) Google Scholar). Brain abnormalities were detectable in knockout mice lacking expression of the neurally expressed BF-1 and BF-2 genes (31Xuan S. Baptista C.A. Balas G. Tao W. Soares V.C. Lai E. Neuron. 1995; 14: 1141-1152Abstract Full Text PDF PubMed Scopus (453) Google Scholar, 32Hatini V. Huh S.O. Herzlinger D. Soares V.C. Lai E. Genes & Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (409) Google Scholar). Moreover, loss of BF-2, which is also expressed in the stromal mesenchyme of the kidney, led to abnormal kidney morphogenesis (32Hatini V. Huh S.O. Herzlinger D. Soares V.C. Lai E. Genes & Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (409) Google Scholar). In this paper, we describe the analysis of WH gene expression in a rat pancreatic endocrine cell line, INS-1, by RT-PCR and the subsequent isolation and characterization of a novel WH gene, named WIN. WIN has about 40% amino acid identity within the WH domain and was found to be highly expressed in different insulinoma cell lines and embryonic pancreas and liver. In adult tissues, WIN expression was high in testis and thymus and lower in lung and intestine. A histidine-tagged WIN fusion protein was used to select the WIN binding sites in vitro by following a modified PCR-based selectionand amplification of binding sites (SAAB) procedure. WIN has a unique binding specificity. We isolated human WIN cDNAs and found that a region outside of the WH domain was previously isolated as a partial 3′ cDNA encodingMPM2-reactive phosphoprotein 2 (MPP2) (33Westendorf J.M. Rao P.N. Gerace L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 714-718Crossref PubMed Scopus (237) Google Scholar). MPP2 was isolated by expression cloning with the MPM2 monoclonal antibody, which bound its phosphorylated epitopes. WIN may be regulated by phosphorylation at the carboxyl terminus. WIN function may also be regulated by differential splicing. Analysis of multiple human and rat WIN cDNAs indicated that differential splicing occurs within the WH DNA binding domain at regions important for directing DNA binding specificity (34Overdier D.G. Porcella A. Costa R.H. Mol. Cell. Biol. 1994; 14 (2nd Ed.): 2755-2766Crossref PubMed Scopus (327) Google Scholar). We demonstrated by RNase protection analysis that these unprecedented differential splicing events are regulated. Standard molecular biology techniques used are described by Sambrook et al. (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total RNAs were extracted by the guanidium isothiocyanate method (36Davis L.G. Dibner M.D. Battey J.F. Basic Methods in Molecular Biology. Elsevier, New York1986: 129-135Google Scholar) and poly(A)+ RNA prepared using the Promega Poly(A)Tract mRNA isolation system. PCR was done using Vent DNA polymerase (New England Biolabs, Inc.) unless specified otherwise. Sequencing was performed using the Sanger dideoxy chain termination method. The two sets of degenerate oligonucleotides, WH-1 (5′-AARCCHCCHTAWTCNTAYAT-3′) and WH-2 (5′-RTGYCKRATNGARTTCTGCCA-3′) were designed based on previous reports (18Clevidence D.E. Overdier D.G. Tao W. Qian X. Pani L. Lai E. Costa R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3948-3952Crossref PubMed Scopus (218) Google Scholar, 19Kaestner K.H. Lee K.-H. Schlondorff J. Hiemisch H. Monaghan A.P. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7628-7631Crossref PubMed Scopus (126) Google Scholar). RT-PCR used the Perkin-Elmer RT-PCR kit with poly(A)+ RNA from INS-1 cells as templates at an annealing temperature of 40 °C with random hexamers. The amplified DNA (∼153 bp) was isolated and subcloned into pBluescriptII (Stratagene) using the TA cloning vector from Invitrogen. A directional INS-1 cDNA library was constructed in plasmid vector, pJG4–5, using the Stratagene cDNA synthesis kit. The 3.0-kb rat WIN cDNA was isolated by screening one million colonies of this library using a 30-mer oligonucleotide (5′-GCCAGCCTGGCTTGGCAATGTGCTTAAAAT-3′). The human WIN cDNAs were isolated by screening human adenocarcinoma (Stratagene) and testis (CLONTECH) directional cDNA libraries using the rat WIN cDNA under high stringency conditions. 5′-RACE was performed using the Life Technologies, Inc. RACE kit with rat 18 days post coital pancreas total RNAs and human thymus total RNAs (CLONTECH) as templates. The longest 5′-RACE products were assembled with the rat and human partial cDNAs at unique EcoRV and BssH1 site, respectively. The predicted ORF within the assembled 3.4-kb rat cDNA (WIN-1) was tested by coupled in vitrotranscription/translation using the Promega TNT Coupled Reticulocyte Lysate System. The rat and human cDNA sequences were submitted to GenBank™ under the accession numbers U83112 and U83113, respectively. RNAs were electrophoresed on 1% agarose-formaldehyde gel and blotted onto nylon membrane (GeneScreen) and probed with32P-labeled WIN-1 cDNA. Blots were stripped and reprobed with rat γ-actin according to the GeneScreen manual. TheCLONTECH mouse and human endocrine system Multiple Tissue Northern blots were probed with WIN-1 as described by the manufacturer using high stringency washing conditions. For RPA, WIN DNA spanning exons 4, 5, and 6 was amplified by PCR and subcloned into pBluescript II SK− as DNA template for RNA synthesis. After linearizing with EcoRI, 32P-labeled antisense RNA probes (243 bases) were synthesized by in vitrotranscription using T7 polymerase (Ambion Maxiscript kit) and gel-purified, and RPA was performed with total RNAs using the Ambion RPA kit. RPA using cyclophilin as probe was also carried out for RNA quantitation. COS cells were transfected by the DEAE-dextran method (37Danielsen M. Northrop J.P. Ringold G.M. EMBO J. 1986; 5: 2513-2522Crossref PubMed Scopus (293) Google Scholar). Two days after transfection, nuclear extracts were prepared from the cells according to Schreiber et al. (38Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3902) Google Scholar). The BAC-TO-BAC Baculovirus expression system (Life Technologies Inc.) was used to express the WIN protein. The WIN gene was generated by a two-step PCR procedure using three primers: Primer 1 (5′-CATCATCATGGAGACGATGACGATAAGATGAGAACCAGCCCCCGGCGG-3′), Primer 2 (5′-GTTGTTGGATCCACCATGGGACACCATCACCATCATCATGGAGACGATGAC-3′), and Primer 3 (5′-GTTGTTCTCGAGCTATCGCAGCTCAGGGATGAACTG-3′). PCR was performed first with Primers 1 and 3 for 10 cycles. The PCR product was purified using the Promega Wizard PCR Preps DNA Purification System, followed by PCR using Primers 2 and 3 for 20 additional cycles. The 5′-primers (Primers 1 and 2) led to the introduction of a BamHI site, and then a sequence based on the Kozak rule for optimal protein expression in-frame with an initiating methionine with a glycine spacer followed by nucleotides coding for the (HIS)6 tag and the enterokinase consensus sequence, followed by the first 21 nucleotides of the WIN gene. The 3′-primer (Primer 3) contains 24 nucleotides of 3′ WIN sequence and allowed the introduction of a XhoI site. The PCR product was digested by BamHI and XhoI and ligated into identical sites of the donor plasmid pFASTBAC-1. The ligation product was transformed into DH10BAC Escherichia coli cell. The transformants were plated out in Luria agar plates containing kanamycin, gentamycin, tetracycline, bluo-gal, and isopropyl-1-thio-β-d-galactopyranoside. Four white colonies were selected after 48 h of transformation. Mini DNA preparations were prepared, and the isolation of recombinant baculovirus DNA was confirmed by PCR. Transfection of Sf9 insect cells was by Cell-fectamine (Life Technologies, Inc.). The recombined virus was harvested after 7 days of transfection, and the virus stock was amplified by infecting Sf9 cells using low viral MOIs (1 MOI/cell). For WIN protein production, Sf9 cells were seeded to 90% confluence in two T175 flasks (Falcon), and the cells were infected with a high MOI (about 10 MOI/cell) from the viral stock. Infected cells were harvested after 96 h of infection and lysed in Tris buffer, pH 8.0, containing 0.5 m NaCl, 0.1% Nonidet P-40, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin A, 0.2 μg/ml aprotinin, and 2 mm phenylmethylsulfonyl fluoride. Lysed cells were then sonicated briefly and centrifuged at 10,000 × g for 30 min. The supernatant was used for binding to an Ni column (Qiagen). The WIN protein was eluted out using 200 mm imidazole. WIN protein purification was confirmed by SDS-PAGE gels stained by Coomassie Blue. EMSA was conducted using the Bandshift kit from Pharmacia Biotech Inc. A typical DNA binding reaction contained ∼2 ng of 32P-labeled DNA and 2 μl of nuclear extract or purified WIN in 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 3 mm dithiothreitol, 5 mm MgCl2, 0.05% Nonidet P-40, 10% glycerol, 1 μg poly (dI-dC), 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin A, 0.2 μg/ml aprotinin, and 2 mm phenylmethylsulfonyl fluoride at a total reaction volume of 20 μl. Both DNA binding and gel electrophoresis were carried out at 4 °C. The DNA sequences recognized by WIN was determined using a modified SAAB procedure. The random oligonucleotide, 5′-CAGTGCTCTAGAGGATCCGTGAC(N13)CGAAGCTTATCGATCCGAGCG-3′, and PCR primers (Primer 4, 5′-CGCTCGGATCGATAAGCTTCG-3′ Primer 5, 5′-CAGTGCTCTAGAGGATCCGTGAC-3′) were designed according to Kunschet al. (39Kunsch C. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1992; 12: 4412-4421Crossref PubMed Google Scholar). The random DNA pool for selection was generated by annealing of 32P-labeled Primer 4 with the random oligonucleotide followed by Klenow extension. 500,000 cpm (∼150 ng) of the labeled DNA was subjected to WIN binding and EMSA. In the first two rounds of selection, there was no discernible band shift, gel pieces above the unbound DNA were excised, and the DNA was eluted in TE (10 mm Tris, 1 mm EDTA, pH 8) with 50 mm NaCl. ∼120 of the eluted DNA was amplified by PCR using Primers 4 and 5 for 30 cycles. After phenol/chloroform extraction, the amplified DNA was concentrated and washed in Microcon 100 concentrator (Amicom), followed by purification in a 12% native PAGE gel. The purified DNA was then radiolabeled by kinasing and subjected to subsequent round of WIN selection. After five rounds of WIN selection, the PCR-amplified DNA was digested with BamHI and HindIII and subcloned into pBluescript II SK− for sequencing. The insulinoma cell line INS-1 expresses many of the properties of isolated primary rat islet beta cells and is a ready source of material for gene expression analysis. We sought to characterize the WH genes expressed in INS-1 by PCR with two sets of degenerate oligonucleotides, WH-1 and WH-2, that span two conserved blocks of sequence homology within the WH DNA binding domain (Fig.1 A). PCR products of about 150 bp were generated, subcloned, and sequenced (Fig. 1 B). 35 clones were picked randomly and found to encode WH proteins; 51% of the clones showed identity to the HNF3γ DNA binding domain, 6% to the rat homolog of human FREAC, and 43% or 15 of 35 clones contained an identical novel WH sequence. Because the novel WH sequence was cloned from INS-1 RNAs, we named the novel gene WIN (Winged helix from INS-1 cells). Northern blot analysis of INS-1 RNAs indicated that the full-length cDNA gene for rat WIN should be about 3.5 kb (see Fig.3 A). We designed a 30-mer oligonucleotide from the novel WIN sequence and used it to screen a INS-1 cDNA library. A single clone with an insert of about 3 kb was isolated. DNA sequence analysis revealed an ORF of 651 amino acids containing the identified novel WH DNA binding domain, however without an initiating methionine. 5′-RACE with rat 18 dpc pancreas RNA generated a 900-bp fragment (RACE2.1) 5′ of the EcoRV site present in the 3-kb cDNA (Fig.2 A).Figure 2Sequence analysis of rat WIN. A, sequence of the rat WIN cDNA and encoded protein. Positions of in-frame stop codons are denoted by asterisks. The WIN WH DNA binding domain identified by sequence comparison isunderlined. Three restriction enzyme sites are indicatedabove the sequences. Also above the sequences arearrowheads and numbers that show the positions of the introns, predicted based on the comparison of the cDNA sequence against mouse genomic WIN sequence2 and the corresponding assigned exons. B and C, comparison between WIN and 10 conserved WH proteins within the DNA binding domain using the Pileup comparison program (GCG). B, the sequences were aligned against rat HNF3α as a reference. The prefix letter in each sequence name denotes the sequence source: r, rat;m, mouse; x, frog; d, fruit fly;h, human; y, yeast. Within parenthesesare the references of the sequences. Dots denote identical amino acids, and dashes represent gaps inserted in the sequences to optimize homology. The percentage of identity between any sequence against rHNF3α is indicated on the right. Predicted structures and divergent regions previously described within the WH domain (13Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1076) Google Scholar) are shown above and below the sequence alignment. C, dendrogram analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Sequence analysis of rat WIN. A, sequence of the rat WIN cDNA and encoded protein. Positions of in-frame stop codons are denoted by asterisks. The WIN WH DNA binding domain identified by sequence comparison isunderlined. Three restriction enzyme sites are indicatedabove the sequences. Also above the sequences arearrowheads and numbers that show the positions of the introns, predicted based on the comparison of the cDNA sequence against mouse genomic WIN sequence2 and the corresponding assigned exons. B and C, comparison between WIN and 10 conserved WH proteins within the DNA binding domain using the Pileup comparison program (GCG). B, the sequences were aligned against rat HNF3α as a reference. The prefix letter in each sequence name denotes the sequence source: r, rat;m, mouse; x, frog; d, fruit fly;h, human; y, yeast. Within parenthesesare the references of the sequences. Dots denote identical amino acids, and dashes represent gaps inserted in the sequences to optimize homology. The percentage of identity between any sequence against rHNF3α is indicated on the right. Predicted structures and divergent regions previously described within the WH domain (13Clark K.L. Halay E.D. Lai E. Burley S.K. Nature. 1993; 364: 412-420Crossref PubMed Scopus (1076) Google Scholar) are shown above and below the sequence alignment. C, dendrogram analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 3-kb cDNA and RACE2.1 were assembled at the EcoRV site to give a 3.4-kb cDNA (WIN-1), which was completely sequenced (Fig. 2 A). Conceptual translation revealed a 771-amino acid ORF that begins with two ATGs (at nucleotide 85). The absence of a purine in the −3 position of the first ATG would predict that the second ATG at nucleotide 88 is the initiating methionine. A similarly positioned methionine was found to be conserved in the human WIN cDNA sequence. Two in-frame stop codons are found 5′ to this ATG. WIN-1, when tested in a coupled in vitrotranscription/translation reaction, yielded a polypeptide with a SDS-PAGE mobility of 90-kDa (Fig. 1 C). A cDNA assembled using a shorter RACE fragment (RACE2.2) that starts at nucleotide 199 did not yield a translation product. The synthesis of WIN fusion protein of the predicted size using the baculovirus expression system also provides evidence that the predicted ORF was used in vivo. WIN-1 was searched against GenBank™ sequences. The only significant matches were gene sequences of the WH gene family and with MPP2 (see "Isolation of Human WIN"). From a comparison of the 10 most homologous WH genes, we found homology only in the WH DNA binding domain with no conservation of Regions II, III, and IV, previously identified as transcriptional activation domains in rodent HNF3s and other related WH proteins (21Hromas R. Costa R. Crit. Rev. Oncol. Hematol. 1995; 20: 129-140Crossref PubMed Scopus (85) Google Scholar, 40Pani L. Overdier D.G. Porcella A. Qian X. Lai E. Costa R.H. Mol. Cell. Biol. 1992; 12: 3723-3732Crossref PubMed Google Scholar). Both the alignment of the homologous WH domains against rat HNF3α (Fig. 2 B) and the dendrogram analysis (Fig. 2 C) indicate that WIN is distantly related to other WH proteins (less than 40% amino acid identity). The alignment also reveals the striking displacement of 12 amino acids in the center of Helix 3 of the WIN WH domain. The 36-bp DNA sequence corresponding to these 12 amino acids is absent from the original WIN PCR sequences. We questioned whether this 36-bp DNA sequence would be evident in the genomic DNA sequence of WIN. Phage genomic DNAs for murine WIN were isolated, subcloned, and sequenced. 2K.-M. Yao and G. G. Wong, unpublished data. A comparison of mouse and rat sequences revealed the intron and exon structure described in Fig. 2 A. The 36-bp sequence specific to the WH domain of the WIN gene is conserved in the mouse genomic WIN sequence and constitutes a single exon, exon 4. Moreover, RT-PCR analysis using primers flanking exon 4 and INS-1 poly(A)+ RNA as templates indicated that both transcripts with and without exon 4 are expressed by INS-1 cells. WIN-1 was used as a probe for Northern analysis of RNAs from rodent and human cells and tissues (Fig. 3). Species specific RNA band patterns were observed: a 3.5-kb doublet and a faint 4.3-kb band in rat (Fig. 3, A and B); two equally intense 3.5-kb and 4.3-kb bands in mouse (Fig. 3, A andC) and a 4-kb band in human (Fig. 3 D). WIN expression was detected in all the rat (INS-1, B2, 38, and RIN56A) and murine (alphaTC1, betaTC1, and beta TC6) endocrine cell lines analyzed (Fig. 3 A). PC12, a neuronal cell line, expressed a lower level of WIN. Rat RNAs prepared from e12, 14, 18, neonate and adult pancreas and livers were tested for WIN expression (Fig.3 B). Expression levels appeared to be high in the embryonic pancreas and liver but decreased to undetectable levels in the adult. The lack of detectable expression in HepG2 cells is consistent with the absence of expression in adult liver. However, expression of WIN could persist in islet endocrine cells and be diluted by its relatively low concentration in the adult pancreas. In adult tissues, high level WIN expression was apparent in testis and thymus (Fig. 3, C andD). A moderate level of WIN expression was also detected in lung and several intestinal regions (large intestine and duodenum; Fig.6 B and results not shown). The distant relationship of WIN to other WH proteins suggests that it may have a different DNA binding specificity. WIN-1, HNF3α, and HNF3γ cDNAs were heterologously expressed in COS-1 cells to generate nuclear extracts for DNA binding experiments. Nuclear extracts were prepared from transfected cells and tested for their ability to bind the known HNF3 binding sites in an EMSA. EMSA showed that nuclear extracts containing HNF3α and HNF3γ bound oligonucleotides corresponding to the HNF3 binding site TTR-S wi
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