Abstract
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 Sp1 sites and a single transcription start site, although analysis 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, and mutagenesis of the KDR/flk-1 Inr reduces promoter activity to 28% of the wild-type promoter in transient transfection assays. Gel shift assays confirm that nuclear proteins specifically bind the Inr, and competition experiments 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 extracts prior to in vitro transcription assays abolishes transcription from the KDR/flk-1 Inr, an effect that can be rescued by adding back purified TFII-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 that is bound by TFII-I and that both the functional Inr and TFII-I activity are essential for transcription.
Highlights
Two families of transmembrane tyrosine kinase receptors that are expressed uniquely in endothelial cells and are essential for the development of the embryonic vascular system have recently been identified [1]
As a first step to determine whether specific nuclear proteins interact with the KDR/flk-1 initiator element (Inr), we examined whether primary culture human umbilical vein endothelial cells (HUVEC) contain a binding activity that will associate with the fragment from Ϫ10 to ϩ10 by Electrophoresis Mobility Shift Assays (EMSA), since HUVECs are known to express KDR/ flk-1 mRNA and protein at high levels [12, 31]
To prove more definitively that TFII-I bound the KDR/ flk-1 Inr, we examined the interaction of purified native TFII-I to the KDR/flk-1 Inr, and we compared complex formation with that of unfractionated HUVEC nuclear extract by EMSA (Fig. 4C)
Summary
Two families of transmembrane tyrosine kinase receptors that are expressed uniquely in endothelial cells and are essential for the development of the embryonic vascular system have recently been identified [1]. We demonstrate in in vitro transcription assays that the sequence from Ϫ10 to ϩ10 in the human KDR/flk-1 promoter contains Inr activity and that Inr activity is orientation- and position-dependent.
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