CLONING AND CHARACTERIZATION OF 7 HUMAN FORKHEAD PROTEINS - BINDING-SITE SPECIFICITY AND DNA BINDING

The forkhead domain is a monomeric DNA binding motif that defines a ra pidly 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 stringe ncy hybridization to isolate clones from human cDNA and genomic librar ies that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from s pecific for a single tissue to nearly ubiquitous. The DNA binding spec ificities of four of the FREAC proteins were determined by selection o f binding sites from random sequence oligonucleotides. The binding sit es for all four FREAC proteins share a core sequence, RTAAAYA, but dif fer in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as resp onsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins t o their cognate sites results in bending of the DNA at an angle of 80- 90 degrees.