Characterization of the Human Forkhead Gene FREAC-4

• Published in: The Journal of biological chemistry Vol. 271; no. 35; pp. 21094 - 21099
• Main Authors: Ernstsson, Sveinn, Pierrou, Stefan, Hulander, Malin, Cederberg, Anna, Hellqvist, Marika, Carlsson, Peter, Enerbäck, Sven
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 30.08.1996
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.35.21094

We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4 , a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5′-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis -element that is capable of interacting with recombinant WT-1.