Cloning and characterization of freac-9 (FKHL17), a novel kidney-expressed human forkhead gene that maps to chromosome 1p32-p34.

We describe the cloning of a near full-length cDNA of 4258 nucleotides encoding freac-9 (HGMW-approved symbol FKHL17), a novel human forkhead gene. The 5' untranslated region is unusual since it is very long, 2127 nucleotides, and contains 15 upstream AUG codons. Hybridization to a panel consisting of RNA derived from 50 different tissues showed that freac-9 is transcribed exclusively in the kidney. The kidney-derived cell lines COS-7 and 293 are shown to express freac-9. A combination of fluorescence in situ hybridization and somatic cell hybrids localizes freac-9 to the chromosomal region of 1p32-p34. The conceptual translation product predicts a protein of 372 amino acids with an N-terminal domain rich in acidic amino acids and with a high likelihood of forming an amphipatic helix, a DNA binding forkhead domain, and a C-terminal region that has a high probability of forming an amphipatic beta-sheet. The amino acid sequence of the DNA binding forkhead motif of FREAC-9 is identical to that of another forkhead protein, FREAC-4, whereas 12 substitutions are present at the nucleotide level. There are no similarities in regions outside of the DNA binding domains of FREAC-9 and FREAC-4 and since freac-4 maps to a different chromosome (5q12-q13) it is likely that an evolutionary selection has acted to maintain identical DNA binding domains between these two kidney expressed transcription factors.

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.