Characterization of the human forkhead gene FREAC-4. Evidence for regulation by Wilms' tumor suppressor gene (WT-1) and p53.
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.
Ernstsson
S
S
Department of Molecular Biology, The Lundberg Laboratory, Göteborg University, Medicinaregatan 9C, S-413 90 Göteborg, Sweden.
Pierrou
S
S
Hulander
M
M
Cederberg
A
A
Hellqvist
M
M
Carlsson
P
P
Enerbäck
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D051858:Forkhead Transcription Factors; D005786:Gene Expression Regulation / Q000235:genetics*; D016162:Genes, Wilms Tumor; D016158:Genes, p53; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012260:Ribonucleases / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D012260:Ribonucleases