Requirement of a GT Box (Sp1 Site) and Two Ets Binding Sites for Vascular Endothelial Cadherin Gene Transcription*
- Sylvie Gory,
- Jacques Dalmon,
- Marie-Hélène Prandini,
- Thierry Kortulewski,
- Yvan de Launoit§ and
- Philippe Huber¶
- From the CEA, Laboratoire de Transgénèse et Différenciation Cellulaire, Département de Biologie Moléculaire et Structurale, 17 rue des Martyrs, 38054 Grenoble and§UMR 319 CNRS, Institut Pasteur de Lille, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille, France
Abstract
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 Sp1 family. Point mutations abolished the binding of nuclear proteinsin vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA 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.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) Y10887.
-
↵¶ To whom correspondence should be addressed: CEA, Grenoble, DBMS-TDC, 17 rue des Martyrs, 38054 Grenoble, France. Tel.: (33)476884118; Fax: (33)476884964; E-mail:huber{at}carre.ceng.cea.fr.
-
↵1 The abbreviations used are: VE cadherin, vascular endothelial cadherin; EMSA, electrophoretic mobility shift assay; BAEC, bovine aortic endothelial cells; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; WT, wild-type.
-
↵2 S. Gory, M. Vernet, M. Laurent, E. Dejana, J. Dalmon, and P. Huber, submitted for publication.
-
- Received September 9, 1997.
- Revision received November 24, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
