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• W2023766038 abstract "The neuropoietic cytokine ciliary neurotrophic factor (CNTF) potently induces transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair (bp) cytokine response element (CyRE) in the VIP promoter. We have previously shown that CNTF induction of STAT and AP-1 protein binding within the CyRE is necessary to mediate CNTF induction of VIP gene transcription. We now show that a third, previously uncharacterized site at the 3′-end of the CyRE is also critical to CNTF induction of CyRE transcription. A 4-bp mutation in this 3′-region reduced CNTF-mediated induction of transcription ∼80%. Whereas mutations in both the STAT and AP-1 sites substantially reduced CNTF induction of transcription, mutations in these sites together with the novel 3′-site completely abolished the ability of CNTF to induce CyRE-mediated transcription. Gel shift analysis indicated that a complex in neuroblastoma cells bound specifically to this 3′-site. This complex was not altered by CNTF treatment. Mutations in an 8-bp sequence (TTACTGGA) eliminated binding of this protein complex and markedly reduced transcriptional activation of the CyRE by CNTF. Thus, we have identified a protein complex binding to a novel DNA sequence that is necessary for full CNTF induction of VIP gene transcription. The neuropoietic cytokine ciliary neurotrophic factor (CNTF) potently induces transcription of the vasoactive intestinal peptide (VIP) gene through a 180-base pair (bp) cytokine response element (CyRE) in the VIP promoter. We have previously shown that CNTF induction of STAT and AP-1 protein binding within the CyRE is necessary to mediate CNTF induction of VIP gene transcription. We now show that a third, previously uncharacterized site at the 3′-end of the CyRE is also critical to CNTF induction of CyRE transcription. A 4-bp mutation in this 3′-region reduced CNTF-mediated induction of transcription ∼80%. Whereas mutations in both the STAT and AP-1 sites substantially reduced CNTF induction of transcription, mutations in these sites together with the novel 3′-site completely abolished the ability of CNTF to induce CyRE-mediated transcription. Gel shift analysis indicated that a complex in neuroblastoma cells bound specifically to this 3′-site. This complex was not altered by CNTF treatment. Mutations in an 8-bp sequence (TTACTGGA) eliminated binding of this protein complex and markedly reduced transcriptional activation of the CyRE by CNTF. Thus, we have identified a protein complex binding to a novel DNA sequence that is necessary for full CNTF induction of VIP gene transcription. ciliary neurotrophic factor vasoactive intestinal peptide leukemia inhibitory factor Janus kinase signal transducer and activator of transcription mitogen-activated protein kinase base pair(s) cytokine response element CAAT/enhancer-binding protein polymerase chain reaction electrophoretic mobility shift assay Rous sarcoma virus cAMP response element-binding protein CREB-binding protein hepatocyte nuclear factor-1 high mobility group Ciliary neurotrophic factor (CNTF),1 a gp130 cytokine with neurotrophic activity, performs many functions in the central and peripheral nervous systems. CNTF mediates cell survival in several different neuronal populations including motor and sensory neurons (1Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar, 2Martinou J.C. Martinou I. Kato A.C. Neuron. 1992; 8: 737-744Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 3Ernsberger U. Sendtner M. Rohrer H. Neuron. 1989; 2: 1275-1284Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 4Nakashima K. Wiese S. Yanagisawa M. Arakawa H. Kimura N. Hisatsune T. Yoshida K. Kishimoto T. Sendtner M. Taga T. J. Neurosci. 1999; 19: 5429-5434Crossref PubMed Google Scholar), induces reactive gliosis (5Winter C.G. Saotome Y. Levison S.W. Hirsh D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5865-5869Crossref PubMed Scopus (148) Google Scholar), and may stimulate differentiation of precursors toward the astrocytic lineage (4Nakashima K. Wiese S. Yanagisawa M. Arakawa H. Kimura N. Hisatsune T. Yoshida K. Kishimoto T. Sendtner M. Taga T. J. Neurosci. 1999; 19: 5429-5434Crossref PubMed Google Scholar, 6Yoshida T. Satoh M. Nakagaito Y. Kuno H. Takeuchi M. Brain Res. Dev. Brain Res. 1993; 76: 147-150Crossref PubMed Scopus (46) Google Scholar, 7Bonni A. Sun Y. Nadal-Vicens M. Bhatt A. Frank D.A. Rozovsky I. Stahl N. Yancopoulos G.D. Greenberg M.E. Science. 1997; 278: 477-483Crossref PubMed Scopus (861) Google Scholar). CNTF also initiates an adrenergic-to-cholinergic switch in the neurotransmitter phenotype of primary sympathetic neurons (3Ernsberger U. Sendtner M. Rohrer H. Neuron. 1989; 2: 1275-1284Abstract Full Text PDF PubMed Scopus (259) Google Scholar, 8Saadat S. Sendtner M. Rohrer H. J. Cell Biol. 1989; 108: 1807-1816Crossref PubMed Scopus (306) Google Scholar, 9Rao M.S. Tyrrell S. Landis S.C. Patterson P.H. Dev. Biol. 1992; 150: 281-293Crossref PubMed Scopus (141) Google Scholar, 10Fan G. Katz D.M. Development. 1993; 118: 83-93PubMed Google Scholar, 11Lewis S.E. Rao M.S. Symes A.J. Dauer W.T. Fink J.S. Landis S.C. Hyman S.E. J. Neurochem. 1994; 63: 428-429Google Scholar). As part of this phenotypic switch, CNTF induces the expression of various neuropeptide genes including vasoactive intestinal peptide (VIP) (9Rao M.S. Tyrrell S. Landis S.C. Patterson P.H. Dev. Biol. 1992; 150: 281-293Crossref PubMed Scopus (141) Google Scholar, 10Fan G. Katz D.M. Development. 1993; 118: 83-93PubMed Google Scholar, 12Patterson P.H. Nawa H. Cell/Neuron. 1993; 72/10 (suppl.): 123-138Google Scholar). To identify the molecular mechanisms by which the gp130 cytokines regulate gene transcription in the nervous system, we have examined CNTF induction of VIP gene expression. All the gp130 cytokines (interleukin-6, leukemia inhibitory factor (LIF), oncostatin M, cardiotrophin-1, and interleukin-11) activate similar intracellular signaling pathways (13Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Abstract Full Text PDF PubMed Google Scholar, 14Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (611) Google Scholar, 15Johnson J. Nathanson N. J. Biol. Chem. 1994; 269: 18856-18863Abstract Full Text PDF PubMed Google Scholar, 16Frank D.A. Greenberg M.E. Perspect. Dev. Neurobiol. 1996; 4: 3-18PubMed Google Scholar) by virtue of shared components of their receptor complex. CNTF binding to CNTF receptor-α, a glycosyl-phosphatidylinositol-linked subunit, leads to association of CNTF receptor-α with LIF receptor-β and then with gp130 to form a trimeric or hexameric receptor complex (14Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Abstract Full Text PDF PubMed Scopus (611) Google Scholar,17Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (593) Google Scholar, 18De Serio A. Graziani R. Laufer R. Ciliberto G. Paonessa G. J. Mol. Biol. 1995; 254: 795-800Crossref PubMed Scopus (49) Google Scholar, 19Carpenter L.R. Yancopoulos G.D. Stahl N. Adv. Protein Chem. 1998; 52: 109-140Crossref PubMed Google Scholar). LIF also signals through LIF receptor-β and gp130, but does not require CNTF receptor-α. LIF and CNTF share the transmembrane subunits gp130 and LIF receptor-β and thus have similar intracellular signal transduction mechanisms. Various signaling moieties are activated by the gp130 cytokines, including members of the JAK/STAT pathway (20Akira S. Nishio Y. Inoue M. Wang X.-J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Abstract Full Text PDF PubMed Scopus (877) Google Scholar, 21Narazaki M. Witthuhn B.A. Yoshida K. Silvennoinen O. Yasukawa K. Ihle J.N. Kishimoto T. Taga T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2285-2289Crossref PubMed Scopus (255) Google Scholar, 22Stahl N. Yancopoulos G.D. J. Neurobiol. 1994; 25: 1454-1466Crossref PubMed Scopus (205) Google Scholar); the Ras/MAPK pathway (13Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Abstract Full Text PDF PubMed Google Scholar, 23Schwarzschild M.A. Dauer W.T. Lewis S.E. Hamill L.K. Fink J.S. Hyman S.E. J. Neurochem. 1994; 63: 1246-1254Crossref PubMed Scopus (17) Google Scholar, 24Schiemann W.P. Nathanson N.M. J. Biol. Chem. 1994; 269: 6376-6382Abstract Full Text PDF PubMed Google Scholar, 25Giordano V. De Falco G. Chiari R. Quinto I. Pelicci P.G. Bartholomew L. Delmastro P. Gadina M. Scala G. J. Immunol. 1997; 158: 4097-4103PubMed Google Scholar); SHP-2 tyrosine phosphatase (26Schiemann W.P. Bartoe J.L. Nathanson N.M. J. Biol. Chem. 1997; 272: 16631-16636Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar); protein phosphatase 2A (27Choi I. Lee M.J. Kim E.J. Kang H.S. Pyun K.H. Immunol. Lett. 1998; 61: 103-107Crossref PubMed Scopus (8) Google Scholar); Src, protein kinase C, and p70 S6 kinase (24Schiemann W.P. Nathanson N.M. J. Biol. Chem. 1994; 269: 6376-6382Abstract Full Text PDF PubMed Google Scholar); phosphatidylinositol 3-kinase (13Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Abstract Full Text PDF PubMed Google Scholar, 28Chen R.H. Chang M.C. Su Y.H. Tsai Y.T. Kuo M.L. J. Biol. Chem. 1999; 274: 23013-23019Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar); and inhibitors of cell signaling such as suppressors of cytokine signalling and protein inhibitor of activated STAT (29Bjorbaek C. El-Haschimi K. Frantz J.D. Flier J.S. J. Biol. Chem. 1999; 274: 30059-30065Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 30Bousquet C. Susini C. Melmed S. J. Clin. Invest. 1999; 104: 1277-1285Crossref PubMed Scopus (90) Google Scholar, 31Kim H. Baumann H. Mol. Cell. Biol. 1999; 19: 5326-5338Crossref PubMed Scopus (148) Google Scholar, 32Starr R. Hilton D.J. Bioessays. 1999; 21: 47-52Crossref PubMed Scopus (231) Google Scholar). Although many of the gp130-activated cytoplasmic signaling pathways have now been described, the mechanisms through which these pathways influence downstream factors that bind DNA and thus regulate transcription in response to the gp130 cytokines are not as well characterized. VIP is expressed in specific regions of the brain and the peripheral nervous system (33Gozes I. Shani Y. Rostene W.H. Brain Res. 1987; 388: 137-148Crossref PubMed Scopus (1) Google Scholar, 34Gozes I. Schachter P. Shani Y. Giladi E. Neuroendocrinology. 1988; 47: 27-31Crossref PubMed Scopus (56) Google Scholar, 35Symes A.J. Fink J.S. Said S.I. Pro-inflammatory and Anti-inflammatory Peptides. Marcel Dekker, Inc., New York1998: 293-306Google Scholar). In vitro, its expression in cultured sympathetic neurons can be enhanced by CNTF or LIF (10Fan G. Katz D.M. Development. 1993; 118: 83-93PubMed Google Scholar, 11Lewis S.E. Rao M.S. Symes A.J. Dauer W.T. Fink J.S. Landis S.C. Hyman S.E. J. Neurochem. 1994; 63: 428-429Google Scholar). Likewise, these cytokines induce an 8–10-fold increase in VIP mRNA levels in the neuroblastoma cell line NBFL (36Symes A.J. Rajan P. Corpus L. Fink J.S. J. Biol. Chem. 1995; 270: 8068-8075Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). By analyzing successive deletions of the VIP promoter upstream of a luciferase reporter, we were able to map a 180-bp cytokine response element (CyRE) 1.15 kilobases upstream of the VIP transcription start site, which mediates the transcriptional response to CNTF in NBFL cells (37Symes A.J. Rao M.S. Lewis S.E. Landis S.C. Hyman S.E. Fink J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 572-576Crossref PubMed Scopus (58) Google Scholar,38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar). The VIP CyRE is a complex regulatory element composed of binding sites for a variety of different transcription factors. We have previously reported that there are functional STAT and AP-1 sites within the VIP CyRE (36Symes A.J. Rajan P. Corpus L. Fink J.S. J. Biol. Chem. 1995; 270: 8068-8075Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar, 39Symes A.J. Corpus L. Fink J.S. J. Neurochem. 1995; 65: 1926-1933Crossref PubMed Scopus (19) Google Scholar, 40Symes A.J. Gearan T. Eby J. Fink J.S. J. Biol. Chem. 1997; 272: 9648-9654Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). CNTF treatment of NBFL cells activates STAT and AP-1 proteins to bind two distinct sites within the CyRE (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar, 40Symes A.J. Gearan T. Eby J. Fink J.S. J. Biol. Chem. 1997; 272: 9648-9654Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Mutation of the STAT site within the wild-type CyRE reduces CNTF induction of CyRE transcription by ∼80% (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar). Mutation of the AP-1 site alone reduces CNTF-mediated induction by ∼50% (40Symes A.J. Gearan T. Eby J. Fink J.S. J. Biol. Chem. 1997; 272: 9648-9654Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), suggesting that both these sites are important to the CNTF induction of VIP transcription through the CyRE. However, our previous deletion studies of the VIP CyRE suggested that an additional region in the 3′-CyRE, distinct from the STAT and AP-1 sites, also contributes considerably to CNTF-mediated induction of CyRE-directed transcription. In this study, we examined the proteins binding to the 3′-region and characterized the sequences to which they bind to understand the combinatorial mechanisms through which CNTF induces VIP gene expression. Cell culture reagents were obtained from Mediatech (Herndon, VA); fetal bovine/horse serum was from Life Technologies, Inc.; and culture plates were from Costar (Corning, NY). Recombinant human CNTF was a gift from Regeneron Pharmaceuticals (Tarrytown, NY). Oligonucleotides encoding the consensus sites for the transcription factors STAT, C/EBP, ETS, NFAT, AP-1, AP-2, AP-3, OCT-1, and nuclear factor-1 were purchased from Promega (Madison, WI). The remaining oligonucleotides were synthesized on a PE Applied Biosystems 394 synthesizer by the Uniformed Services University of the Health Sciences in-house oligonucleotide facility. This included all PCR and mutagenic primers listed in Table I and the electrophoretic mobility shift assay (EMSA) probes G9 (GGG CAG GAT ATT CTT TTA CTG GAT CAG TCT GA), G10 (GGG CAT AGC AGG ATA TTC TTT TAC TGG), G11 (GGG TGG ATC AGT CTG ACT TTG AAC G), p28 (GGG TTT TAC TGG ATC AGT CTG ACT TTG AAC G), C/EBP (AGC TTG ATT AGG ACA TCG), acute phase response element (GGA CCA CAG TTG GGA TTT CCC AAC CTG ACC A), M6 (GGA CCA CAG TTG TGA TTT CAC AAC CTG ACC A), and CyB (GAA AAT ATG ATT AAG CAT AGA GCA GG).Table IPCR primers for site-directed mutagenesisPrimer5′ → 3′A1CCGGGTACCTAAAAAAGATTTCCTGGmA1CCGGGTACCTAAAAAAGAT G T A C T GGA4CACCTGCAGCGTTCAAAGTCAGACmG3CCG GGT ACC TAA AAA AGA T G T A C T GGT ATT AAG CCA CAG GAA CTC TGGa/s 1-aa/s, antisense. mG9CAG CTG CAG CGT TCA AAG TCA GAC TGA TC T TAG AAA AGA ATA TCC TGCa/s mS1CAG CTG CAG GTA TCA AAG TCA GAC TGA TCC AGT AAA Aa/s mS2CAG CTG CAG CGT AGT AAG TCA GAC TGA TCC AGT AAA Aa/s mS3CAG CTG CAG CGT TCA TTC TCA GAC TGA TCC AGT AAA Aa/s mS4CAG CTG CAG CGT TCA AAG ATT GAC TGA TCC AGT AAA Aa/s mS5CAG CTG CAG CGT TCA AAG TCA CTA TGA TCC AGT AAA Aa/s mS6CAG CTG CAG CGT TCA AAG TCA GAC ACT TCC AGT AAA Aa/s mS7CAG CTG CAG CGT TCA AAG TCA GAC TGA ATG AGT AAA Aa/s mS8CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC TCA AAA Aa/s mS9CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC AGT TTC Aa/s mS10CAG CTG CAG CGT TCA AAG TCA GAC TGA TCC AGT AAA TCG ATA T1-a a/s, antisense. Open table in a new tab NBFL cells were maintained and transfected as described previously (37Symes A.J. Rao M.S. Lewis S.E. Landis S.C. Hyman S.E. Fink J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 572-576Crossref PubMed Scopus (58) Google Scholar). Cells were plated at 1.5 or 4.5 × 105 cells/well in 6-well plates and transfected overnight by calcium phosphate precipitation. Each well received 1 μg of luciferase reporter construct, 0.5 μg of RSV-β-galactosidase, and 2.5 μg of carrier DNA. CNTF was added in serum-free medium 6 h after the DNA precipitate was removed and for 40 h before cell harvesting. Samples were assayed for luciferase activity (41Brasier A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar) and β-galactosidase activity (Galacto-Light Plus kit, Tropix Inc., Bedford, MA). Luciferase activity was normalized to β-galactosidase activity to control for transfection efficiency. The details of Cy1luc, Cy1mG3luc, Cy1mG2luc (previously termed m2G2Cyluc), and VIP1330luc have been described (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar,42Symes A.J. Stahl N. Reeves S. Farruggella T. Servedei T. Gearan T. Yancopoulos G.D. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The 3-bp substitution mutant VIPm1300luc was constructed using theCLONTECH Transformer site-directed mutagenesis kit with the mutagenic primer 5′-GCA GGA TAT TCT TTT TGA GGA TCA GTC TGA C-3′ and the selection primer 5′-GAG CTC CCA TCG CGA TGG ATG CAT AG-3′. To construct a multimeric G9 site, the G9 EMSA probe was synthesized with 5′-GATC overhangs, phosphorylated, and ligated. The ligated fragments were digested with BamHI and BglII to digest fragments containing oligonucleotides ligated in the wrong orientation and then subcloned into BamHI-digested pSP73 plasmid (Promega). Fragments containing four copies of the G9 site in both the correct (4G9) and reverse (R4G9) orientations were excised byKpnI/PstI digest and inserted intoKpnI/PstI-digested ΔeRSVluc. Formation of 4(G9)luc and R4(G9)luc was confirmed by sequencing. All other luciferase plasmids were constructed by PCR site-directed mutagenesis (43Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar) with primers containing either a 5′-KpnI or 3′-PstI digestion site (see Table I). Cy1mG9luc was prepared by PCR amplification of Cy1luc using primers A1 and mG9 (see Table I). A series of 3-bp substitution mutants were amplified from Cy1luc with primer A1 and one of primers mS1 to mS10 (see Table I). Cy1mG2luc was used as a template in PCR with mutant primers to construct the double mutants Cy1mG2mG3luc and Cy1mG2mG9luc (see Table I). Subsequently, Cy1mG2mG3luc was used as a template DNA to construct the triple mutant Cy1mG2mG3mG9luc (see Table I). In procedures where the template already contained an mG3 site, primer mA1 was used in place of primer A1 to maintain the mutation in G3. PCR products were gel-purified and ligated into KpnI/PstI-digested ΔeRSVluc. All plasmids were sequenced to confirm their identity. EMSAs were performed as described previously (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar). Briefly, nuclear extracts were prepared from untreated NBFL cells and incubated for 15 min at 4 °C with 0.5 ng of [α-32P]dCTP-labeled oligonucleotides. Binding reactions were electrophoresed on a 5% nondenaturing polyacrylamide gel in 0.5× Tris borate/EDTA buffer at 200 V. For the CNTF time course, NBFL cells were serum-starved overnight and then activated with CNTF for 0, 0.5, 1, 3, 6, and 24 h prior to the extraction of nuclear proteins. When used, competitor oligonucleotides (5–200 ng) were incubated with the nuclear extracts for 10 min at room temperature prior to adding probe. We have previously characterized the STAT and AP-1 sites within the VIP CyRE and shown both sites to contribute to the CNTF-mediated induction of CyRE-dependent transcription (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar, 40Symes A.J. Gearan T. Eby J. Fink J.S. J. Biol. Chem. 1997; 272: 9648-9654Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, our previous deletion studies on the VIP CyRE also revealed that a 28-bp region at the 3′-end of the CyRE, distinct from the STAT and AP-1 sites, contributes substantially to the transcriptional activity of the CyRE (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar). To determine the exact sequences within the 3′-end of the VIP CyRE that mediate the actions of this 28-bp 3′-region, we compared murine and human genomic sequences (38Symes A.J. Lewis S.E. Corpus L. Rajan P. Hyman S.E. Fink J.S. Mol. Endocrinol. 1994; 8: 1750-1763Crossref PubMed Google Scholar). Although the 180-bp CyRE is 84% homologous between mouse and human, the distal 28-bp 3′-sequence is only 53% conserved. However, one 4-bp motif (G9 site) is conserved between species, suggesting functional significance of this site. To investigate whether this 4-bp motif is important in mediating CNTF induction of CyRE transcription, we transfected NBFL neuroblastoma cells with a Cy1luc luciferase reporter plasmid mutated within these 4 bp (Cy1mG9luc) (TableI). CNTF induction of transcription driven by Cy1mG9luc was reduced to 28% of that mediated by Cy1luc (Fig. 1). These data suggest that this 4-bp motif may form part of a binding site for a protein complex that contributes to CNTF-induced transcription through the VIP CyRE. We wanted to investigate the relationship between the newly identified G9 site and the previously identified STAT and non-canonical AP-1 sites that contribute to CNTF-mediated induction of CyRE-driven transcription. To determine whether the 3′-G9 site acts independently of the STAT and AP-1 sites, we constructed a series of luciferase plasmids with individual, double, or triple substitution mutations at these sites within the wild-type Cy1luc plasmid. Cy1luc directed the highest level of transcription in unstimulated cells. CNTF induction of CyRE-mediated transcription was reduced in all of the mutant constructs. In the CyRE-luciferase construct with mutations in both STAT and AP-1 sites (Cy1mG2mG3luc), CNTF induced luciferase activity 9-fold (Fig. 2). This remaining CNTF inducibility mediated by a CyRE-luciferase plasmid without a functional AP-1 or STAT site supported the existence of additional functional sites such as the 3′-G9 site in the CyRE. CNTF induction of CyRE transcription was reduced to ∼17% of Cy1luc with single mutations in either the STAT or 3′-G9 site (Fig. 2). Mutations in both the STAT (G3) and 3′-G9 sites further reduced CNTF induction of transcription to 8% of that produced by the wild-type Cy1luc plasmid. Thus, the two sites each contribute to the CyRE-mediated CNTF response. Mutation of the AP-1 site (G2) reduced CNTF-induced transcription by 44%, and double mutants of the AP-1 site together with either the STAT or G9 site reduced CNTF induction of transcription by 90 and 84%, respectively. Introduction of mutations into all three sites (Cy1mG2mG3mG9luc) abrogated the response to CNTF. These data suggest that the STAT, AP-1, and 3′-G9 sites all independently contribute to CNTF induction of CyRE transcription. To investigate whether CNTF treatment of NBFL cells altered nuclear protein binding to the 3′-region of the CyRE, we performed EMSAs with four overlapping probes of the 3′-region of the CyRE (Fig.3 A). Three protein complexes of different mobility bound to different probes from the 3′-CyRE in nuclear extracts prepared from untreated NBFL cells (Fig.3 B). Complex A bound strongly to the G9 probe, complex B to the G10 probe, and complex C to the G11 probe (Fig. 3 B). CNTF treatment did not alter binding of any of these nuclear protein complexes, showing that these complexes are able to bind to the DNA sequences constitutively. We also investigated which complexes bound to a probe containing the entire distal 28 bp of the 3′-end (p28) and detected a single band with the same mobility as the G9-binding complex A, which did not change with CNTF treatment (Fig. 3 C and data not shown). The protein complex binding to p28 was competed by a 100-fold molar excess of G9, but not G10 or G11, suggesting that this complex was probably the G9-binding nuclear protein complex (Fig. 3 C). Therefore, nuclear protein binding to this region of DNA is complex; several protein complexes may be responsible for mediating the CNTF induction of CyRE-driven transcription at the 3′-end. To determine whether nuclear protein binding to the G9, G10, and G11 probes was specific and to identify whether similar proteins were bound to each of the probes, we performed EMSAs in the presence of varying concentrations of unlabeled competitor oligonucleotides. Binding of NBFL nuclear protein to each probe was specific, as its binding was competed by a 100-fold molar excess of unlabeled oligonucleotide (Fig.4). The mG9 oligonucleotide failed to compete for binding to the G9 probe (Fig. 4 A) or to the p28 probe (Fig. 3 C), showing that this mutation markedly reduces the ability of complex A to bind. As this oligonucleotide is mutated in the same 4 bp as the Cy1mG9luc plasmid (Fig. 1), these data suggest that complex A binding to the G9 site may contribute to the CNTF induction of CyRE transcription. The G9 oligonucleotide competed for the protein complexes binding to the G10 and G11 probes; in contrast, G10 and G11 were unable to compete for the protein complexes binding to the G9 probe (Fig. 4, B and C). Thus, complex A binding to the G9 probe appears to require sequence additional to that in either G10 or G11, despite the considerable overlap between the oligonucleotide probes. Our results implicate complex A binding to the G9 site as critical for the CNTF induction of CyRE-mediated transcription. In our initial attempts to identify the components of the G9-binding complex A, we used a variety of known transcription factor-binding sites to compete for binding of complex A to the G9 probe. None of the oligonucleotides shown or oligonucleotides containing CREB, SMAD, or NF-κB consensus sites competed for complex A binding (Fig. 4 D and data not shown). We also competed nuclear protein binding to the CyRE probes with an AP-1 consensus oligonucleotide, as there is an AP-1-like site within this region (ATCAGTCT). The AP-1 oligonucleotide failed to compete for binding of the nuclear protein complexes specific to the G9 or G10 probe. However, it did compete partially for complex C binding to the G11 probe (Fig. 4 C). Supershift analysis with antibodies raised against several different members of the AP-1 protein family (c-Fos, c-Jun, JunB, JunD, and activating transcription factor-2) did not identify any known AP-1 proteins contributing to complex C (data not shown). Thus, several unrelated protein complexes are able to bind to sites within the 3′-CyRE. Complex A may represent a novel constitutive factor required for CNTF-mediated transcription. To identify which specific bases within the G9 oligonucleotide are required for complex A binding, we synthesized oligonucleotides containing a series of sequential 2- and 3-bp mutations in the G9 probe. EMSAs performed with NBFL nuclear extracts binding to these mutant oligonucleotides showed that 2- or 3-bp mutations within a large 17-bp region reduced or eliminated complex A binding to the probe (Fig.5 A). This region includes and extends from the 4-bp mutation in mG9. The m3, m4, m5, and m6 oligonucleotides, with mutations in the sequence TTACTGGA, were unable to bind complex A or to compete for its binding to the wild-type G9 probe (Fig. 5 B). The m2 and m7 oligonucleotides bound complex A weakly and were able to compete for complex A binding to G9, but with less affinity than either the wild-type G9 or m1 oligonucleotide. Interestingly the m2, m3, m5, and m6 oligonucleotides all bound a larger complex, whereas m4 bound none. Reducing the length of the G9 oligonucleotide by 8 bp while retaining the central core recognition sequence also reduced binding of complex A. Thus, the binding site for complex A extends over 17 bp of the G9 oligonucleotide and requires adjacent sequence for high affinity binding. However, the core 8-bp sequence TTACTGGA is critical for complex A binding. To determine the exact sequence within the 3′-end of the CyRE necessary to mediate CNTF-induced transcription, we made a series of CyRE-luciferase constructs containing sequential 3-bp mutations along the most distal 28 bp of the 3′-CyRE, within the context of wild-type Cy1luc. A notable reduction in CNTF-mediated luciferase activity was observed in cells transfected with Cy1mS7luc, Cy1mS8luc, or Cy1mS9luc (Fig. 6 A). The 3-bp mutations in Cy1mS7luc and Cy1mS8luc each overlap the 4 bp mutated in Cy1mG9luc, confirming our original observation as to the importance of this site (Fig. 1). Furthermore, the 3-bp mutations in Cy1mS7luc, Cy1mS8luc, and Cy1mS9luc are located within the sequence TTTACTGGA required for complex A binding (Fig.5 A). A direct comparison of the transfection data with an EMSA using a G9 probe containing the same mutations as in Cy1mS7luc, Cy1mS8luc, and Cy1mS9luc (Fig. 6, A andB) demonstrated a correlation between loss of CNTF-induced transcriptional activity of th" @default.
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• W2023766038 title "Identification of a Novel gp130-responsive Site in the Vasoactive Intestinal Peptide Cytokine Response Element" @default.
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