The human forkhead protein FREAC-2 contains two functionally redundant activation domains and interacts with TBP and TFIIB.

Forkhead-related activator 2 (FREAC-2) is a human transcription factor expressed in lung and placenta that binds to cis-elements in several lung-specific genes. We have identified the parts of FREAC-2 responsible for trans-activation and found two functionally redundant activation domains on the C-terminal side of the DNA binding forkhead domain. Activation domain 1 consists of the most C-terminal 23 amino acids of FREAC-2 and contains a sequence motif conserved in an activation domain of another forkhead protein, FREAC-1. Activation domain 2 is built up by three synergistic subdomains in the central part of the FREAC-2 protein. FREAC-2 was shown to interact in vitro with TBP and TFIIB. The target site for FREAC-2 on TBP was localized to the N-terminal repeat in the core domain of TBP. TFIIB binds FREAC-2 close to the cleft between its two globular domains. The part of FREAC-2 that binds TBP was mapped to 21 amino acids in the C-terminal end of the forkhead domain. This sequence is well conserved among forkhead proteins, raising the possibility that interaction with TBP may be a general characteristic of this family of transcription factors. Overexpression of TFIIB potentiates activation by FREAC-2 in a manner dependent on the FREAC-2 activation domains. Nuclear localization of FREAC-2 was found to depend on sequences from both ends of the forkhead domain.

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.

Differential activation of lung-specific genes by two forkhead proteins, FREAC-1 and FREAC-2.

We describe the cDNA sequences for two human transcription factors, Forkhead RElated ACtivator (FREAC)-1 and -2, that belong to the forkhead family of eukaryotic DNA binding proteins. FREAC-1 and -2 are encoded by distinct genes, are almost identical within their DNA binding domains and in the COOH termini, but are otherwise divergent. Cotransfections with a reporter carrying FREAC binding sites showed that both proteins are transcriptional activators, and deletions located the activation domains to the COOH-terminal side of the forkhead domains. Expression of FREAC-1 and FREAC-2 is restricted to lung and placenta. We show that the promoters of genes for lung-specific proteins such as pulmonary surfactant proteins A, B, and C (SPA, SPB, and SPC) and the Clara cell 10-kDa protein (CC10) contain potential binding sites for FREAC-1 and FREAC-2. DNaseI footprinting verified that FREAC proteins bind to the predicted sites in the CC10 and SPB promoters. While an SPB promoter construct could be transactivated by both FREAC-1 and FREAC-2, CC10 was only activated by FREAC-1. Efficient activation of CC10 by FREAC-1 is shown to be specific for a lung cell line with Clara cell characteristics (H441) and to involve a region of the FREAC-1 protein unable to activate in other cell types.

Chromosomal localization of six human forkhead genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12).

The forkhead family of transcription factors has been shown to be involved in the regulation of embryonic development in organisms from Drosophila to humans. Genes encoding forkhead proteins are also emerging as a new class of oncogenes with relevance for human cancer. We have used a combination of fluorescence in situ hybridization and somatic cell hybrids to map the chromosomal localizations of five previously cloned human forkhead genes, freac-1, -3, -4, -5, and -6. (HGMW-approved symbols FKHL5, FKHL7, FKHL8, FKHL9 and FKHL10). We also report the sequence and the chromosomal position of a novel human forkhead gene, freac-8 (FKHL12). The freac genes have been localized to the following positions: freac-1, 16q24; freac-3, 6p25; freac-4, 5q12-q13; freac-5, 9 pericen; freac-6, 5q34, and freac-8, 1p32.

Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.

The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.