The human KDR/flk-1 gene contains a functional initiator element that is bound and transactivated by TFII-I

KDR/flk-1, the receptor for vascular endothelial growth factor, is required for normal vascular development. KDR/flk-1 is a TATA-less gene, containing four upstream Spl sites and a single transcription start site, although an alysis of the start site sequence discloses only weak similarities with the consensus initiator element (Inr) sequence. In vitro transcription assays, however, demonstrate that the region from -10 to +10 relative to the start site contains Inr activity that is orientation- and position-dependent, an d mutagenesis of the KDR/flk-1 Inr reduces promoter activity to 28% of the wild-type promoter in transient transfection assays. Gel shift assays confi rm that nuclear proteins specifically bind the Inr, and competition experim ents demonstrate that TFII-I, a multifunctional Inr-binding nuclear protein , is a component of these DNA-protein complexes. TFII-I transactivates the wild-type KDR/flk-1 promoter, but not a promoter containing a mutated Inr, in transient transfection assays. Immunodepletion of TFII-I from nuclear ex tracts prior to in vitro transcription assays abolishes transcription from the KDR/flk-1 Inr, an effect that can be rescued by adding back purified TF II-I, reflecting the importance of TFII-I in KDR/flk-1 Inr activity. These experiments demonstrate that the KDR/flk-1 gene contains a functional Inr t hat is bound by TFII-I and that both the functional Inr and TFII-I activity are essential for transcription.