Selective Sp1 binding is critical for maximal activity of the human c-kit promoter

The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5' promoter deletion-reporter constructs containing up to 3 kb of 5' sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene, This analysis showed the region 83 to 124 bp upstream of the 5' transcription initiation site was crucial for maximal core promoter activity, Sequence analysis showed several potential Spl binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Spl selectively bound to a single site within this region. Supershift studies using an anti-Spl antibody confirmed specific Spl binding. Site-directed mutagenesis of the -93/-84 Sp 1 binding site reduced promoter reporter activity to basal levels in c-kit-expressing cells. Cotransfection into Drosophila SL2 cells of a c-kit promoter reporter construct with an Spl expression vector showed an Spl dose-dependent enhancement of expression that was markedly attenuated by mutation of the -93/-84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Spl sites, Spl binding is a selective process that is essential for core promoter activity, (C) 1998 by The American Society of Hematology.