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 Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the -93/-84 Sp1 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 Sp1 expression vector showed an Sp1 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 Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.
Park
G H
GH
Division of Hematology/Oncology, Departments of Medicine and Microbiology/Immunology, Medical College of Virginia/Virginia Commonwealth University and McGuire VA Medical Center, Richmond, VA, USA.
Plummer
H K
HK
Krystal
G W
GW
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D016329:Sp1 Transcription Factor; D019009:Proto-Oncogene Proteins c-kit