title: Expression of the Op18 gene is maintained by the CCAAT-binding transcription factor NF-Y.
abstract: Op18 (Oncoprotein 18, Stathmin) is a mitotic regulator that is highly expressed in many cancers. We have characterized four functional CCAAT boxes in the Op18 gene located at positions: -980, -745, -599 and -65, relative to the transcriptional start site. NF-Y is a ubiquitously expressed CCAAT-binding transcription factor that regulates a number of cell cycle controlled genes. We have used promoter-reporter assays and mobility shift assays to functionally examine these CCAAT boxes. All sites contribute to the basal expression of Op18, with the sites at -980 and -599 being repressive and the sites at -745 and -65 being stimulatory. Mobility shift assays indicate that all CCAAT box sites bind factors in nuclear extracts from Hek293. However, only the repressive site at -599 and the stimulatory site at -65 are competent to bind NF-Y, suggesting that NF-Y may play a role in promoting both activation and repression of Op18 expression. The NF-Y site at -65 accounts for greater than 60% of the Op18 gene expression. EMSA competition studies indicate that NF-Y binds with a much higher affinity to the -65 site than to the -599 site, suggesting that in asynchronously growing cells NF-Y functions only to stimulate expression through the -65 binding site. These data suggest that NF-Y is a major transcription factor promoting expression of Op18.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, 55455, USA.
authors.forename: Houda
authors.initials: H
authors.lastname: Benlhabib
authors.forename: Julio E
authors.initials: JE
authors.lastname: Herrera
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D050777:Stathmin / Q000235:genetics*; D014162:Transfection
chemical_list: D023081:CCAAT-Binding Factor; D050777:Stathmin; D004247:DNA; D008156:Luciferases