Identification of functional elements in the promoter region of the human gene for thymidylate synthase and nuclear factors that regulate the expression of the gene

To identify the essential motifs of the promoter of the human gene for thymidylate synthase (TS), we constructed a set of deletion mutants from the 5'-terminal region of the human TS gene, From the results of assays of the expression of chloramphenicol acetyltransferase (CAT), we identified two functional elements with positive effects on the promoter activity: a CACCC box (CCACACCC) and an Spl-binding motif (GAGGCGGA) that was homologous to the Spl binding site in the mouse TS gene, In addition, negative regulatory sequences were identified between the two positive elements and in the region upstream of the CACCC box, The results of gel mobility shift analyses suggested that Spl binds to the Spl-binding motif of the human TS gene promoter and that multiple nuclear factors that are related to Spl bind to the CACCC box, Furthermore, the binding of Spl to mutated Spl-binding motifs in the promoter region of the human TS gene was correlated with the promoter activity, as measured by the CAT assay, Therefore, the Spl motif seems to be a major contributor to the basic promoter activity of the human TS gene, although multiple positive and negative regulatory elements are involved in the regulated expression of this gene.