The primary response transcription factor, early growth response-1 (Egr-1), is rapidly activated by a variety of extracellular stimuli. Egr-1 binds to a sequence found in the promoters of genes involved in vascular injury, such as PDGF-A and tissue factor, and trans -activates their expression in en
The results indicate that the promoter activity of the 760/+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 su cient to induce the 760/+19 vWF promoter activity in HeLa cells.
The vascular endothelium, lining the blood vessel wall, is constantly exposed to wall shear stresses generated by flowing blood. Gene regulation, critical for endothelial cell function, depends on complex interactions at the promoter level and utilizes overlapping signal transduction cascades to activate the expression of genes involved in many biological processes.
The early growth response factor-1 (Egr-1) gene encodes a zinc finger transcription factor which is critical for cell proliferation and differentiation. The human Egr-1 promoter comprises regulatory elements including two Sp1 sites, an AP1 site, two cAMP response elements and an Egr-1 binding site. In addition to these transcription factor binding sites, the promoter harbours five serum response elements (SREs) and associated binding sites for the Ets transcription factor family, previously identified from partial sequence data (Sakamoto et al, Oncogene 6; 867-871, 1991). We now report the full sequence of the human Egr-1 promoter and confirm the presence of a fifth serum response element. This element is functionally active in a minimal promoter vector in response to the MAP kinase kinase MEK1.
Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5' of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5' end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5'-flanking region interact to regulate vWF gene expression.
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