Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein

Abstract

In a study of the role of transcription factor Sp1 in the formation of tumors by human fibrosarcoma cell lines that overexpress it [Cancer Res., 65 (2005) 1007], we found that expression of an Sp1-specific ribozyme, not only reduced the level of Sp1 protein, but also that of Sp3 protein, and that when the protein levels of these two transcription factors in the fibrosarcoma cell lines were reduced to near that found in normal human fibroblasts, the cell lines could no longer form tumors. An Sp1-specific ribozyme could reduce the level of expression of both Sp1 protein and Sp3 protein if the promoter of the Sp1 gene and that of the Sp3 gene both have Sp1/Sp3 transcription factor binding sites and if such sites are critically responsible for the level of expression of both Sp1 and Sp3 protein in the cells. The Sp1 minimal promoter has been identified and it has two Sp1/Sp3 sites [J. Biol. Chem. 276 (2001) 22126]. To characterize the Sp3 promoter, we isolated 2.1 kb of the 5'-flanking region of the Sp3 gene, which contains Sp1/Sp3 binding sites, and using an expression reporter assay, showed that it has promoter activity. We then systematically reduced the size of the 5' flanking region, and determined that the nt-339 to nt-39 fragment, which contains an Sp1/Sp3 binding site at nt-181 and another at nt-168, retained the same promoter activity as the 2.1 kb region. Electrophoretic mobility shift assays indicated that both Sp3 protein and Sp1protein bind to these two sites. By mutating either or both of these binding sites, we showed using the reporter assay that each site is required for full promoter activity. We then designed an Sp3-specific ribozyme, expressed it in a human fibrosarcoma cell line in which Sp1 protein and Sp3 protein are expressed at high levels, and found that, indeed, the level of expression of both proteins was significantly reduced.