The receptor tyrosine kinase c-kit is necessary for normal hematopoies
is, the development of germ cells and melanocytes, and the pathogenesi
s of certain hematologic and nonhematologic malignancies. To better un
derstand the regulation of the c-kit gene, a detailed analysis of the
core promoter was performed. Rapid amplification of cDNA ends (RACE) a
nd RNase protection methods showed two major transcriptional initiatio
n sites. Luciferase reporter assays using 5' promoter deletion-reporte
r constructs containing up to 3 kb of 5' sequence were performed in he
matopoietic and small-cell lung cancer cell lines which either did or
did not express the endogenous c-kit gene, This analysis showed the re
gion 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-Sp
l antibody confirmed specific Spl binding. Site-directed mutagenesis o
f 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 expressi
on 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 Soc
iety of Hematology.