Functional Characterization of the 5'-Regulatory Region of the Human Thrombomodulin Gene1

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.