Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription

Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2+-dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a -2486/+24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions -49/-39) interacting with factors related to the Spl family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Spl family revealed that Spl and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GC;AA motifs, located at positions -93/-90 and -109/-106, were shown to interact with nuclear factors. Site-directed mutagenesis of these sequences demonstrated that these Ets binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the -109/-106 site.