Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene

Abstract

von Willebrand factor (vWF) gene expression is restricted to endothelial cells and megakaryocytes. Previous results demonstrated that basal transcription of the human vWF gene is mediated through a promoter located between base pairs −89 and +19 (cap site: +1) which is functional in endothelial and non endothelial cells. Two DNA repeats TTTCCTTT correlating with inverted consensus binding sites for the Ets family of transcription factors are present in the −56/−36 sequence. In order to analyse whether these DNA elements are involved in transcription, human umbilical vein endothelial cells (HUVEC), bovine calf pulmonary endothelial cell line (CPAE), HeLa and COS cells were transfected with constructs containing deletions of the −89/+19 fragment, linked to the chloramphenicol acetyl transferase (CAT) reporter gene. The −60/+19 region exhibits significant promoter activity in HUVEC and CPAE cells only. The −42/+19 fragment is not active. Mutations of the −60/+19 promoter fragment in the 5′ (−56/−49) Ets binding site abolish transcription in endothelial cells whereas mutations in the 3′ (−43/−36) site does not. The −60/−33 fragment forms three complexes with proteins from HUVEC nuclear extracts in electrophoretic mobility shift assay which are dependent on the presence of the 5′ Ets binding site. Binding of recombinant Ets-1 protein to the −60/−33 fragment gives a complex which also depends on the 5′ site. The −60/+19 vWF gene core promoter is transactivated in HeLa cells by cotransfecting with Ets-1 or Erg (Ets-related gene) expression plasmids. In contrast to the wild type construct, transcription of the 5′ site mutants is not increased by these expressed proteins. The results indicate that the promoter activity of the −60/+19 region of the vWF gene depends on transcription factors of the Ets family of which several members like Ets-1, Ets-2 and Erg are expressed in endothelium. Cotransfection of Ets-1 and Erg expression plasmids is sufficient to induce the −60/+19 vWF promoter activity in HeLa cells.