Abstract
Endothelin-1 is a 21-amino acid peptide first characterized as a potent vasoactive compound synthesized by endothelial cells. Because of its high level cell-restricted pattern of expression, we have employed this gene as a model for investigating the DNA and protein elements that mediate endothelial cell-specific gene expression. In this study we have identified a complex positive regulatory region located at base pairs -364 to -320 in the murine endothelin-1 gene. This region consists of three functionally dependent elements, ETE-C, ETE-D, and ETE-E, which are all required for full activity. When a 43-base pair fragment containing these three elements was employed in heterologous promoter experiments, this sequence was capable of increasing transcriptional activity in an endothelial cell-specific fashion. None of the elements contains a recognized consensus sequence known to bind transcriptional regulatory proteins in higher eukaryotes; however, each element does appear to mediate protein binding. The combination of all three elements promotes binding of a protein complex that is endothelial cell-specific. This is the first evidence for an endothelial cell-specific DNA regulatory element and cognate binding proteins.
Highlights
Endothelin-1 (ET-1)1 is a potent vasoconstrictor and smooth muscle cell mitogen expressed in endothelial cells of the vascular wall
Transcriptional activation by GATA cannot account for cell-restricted expression of human ET-1 (hET-1), since GATA-2 appears to be expressed in many cell types in the adult [6, 9]
Identification of an Upstream Positive Regulatory Region— Transgenic experiments had shown that a 7-kbp region of the murine ET-1 (mET-1) gene, including 5.9 kbp of 5Ј-flanking sequence and the first exon and intron, was sufficient to confer high level expression in endothelial cells in vivo [17]
Summary
Reporter Gene Construction—Lambda phage clones containing the mET-1 gene were cloned from a 129SV/J genomic library, employing a mET-1 cDNA cloned in this laboratory. The plasmids p286ϩETE-C, p286ϩETE-C/D, p286ϩETEC/D/E were generated by cloning annealed complementary oligonucleotides encoding one copy of the ETE-C, ETE-C/D, or ETE-C/D/E elements into KpnI and SacI sites of p286. The complementary oligonucleotides ETE-C/mD/E and ETE-C/D/mE encoding the mutant D and E elements were cloned into p286 at KpnI and SacI sites, respectively, to generate the plasmids p286ϩETE-C/mD/E and p286ϩETE-C/D/mE. The reporter plasmids pSV40ϩ(1X)ETE-C/D/E and pSV40ϩ(3X)ETE-C/D/E were prepared by cloning one copy or three copies of ETE-C/D/E oligonucleotides into KpnI and SacI sites of pGL2 promoter construct (Promega) containing the minimal SV-40 promoter. The relative luciferase activity was calculated as the ratio of light units to -galactosidase units These corrected light units have been divided by the value obtained with the promoterless pGL2-basic plasmid, which was included in all transfections. The specificity of binding was ascertained by competition with a 100-fold molar excess of cold oligonucleotides added to the reaction mixture
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