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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4138-4149
Selective Sp1 Binding Is Critical for Maximal Activity of the
Human c-kit Promoter
Gyeong H. Park,
Howard K. Plummer III, and
Geoffrey W. Krystal
From the Division of Hematology/Oncology, Departments of Medicine and
Microbiology/Immunology, Medical College of Virginia/Virginia
Commonwealth University and McGuire VA Medical Center, Richmond, VA.
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.
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