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(Received for publication, September 13, 1996, and in revised form, October 25, 1996)
From the Renal Division, Beth Israel Hospital and Harvard Medical
School, Boston, Massachusetts 02215
We performed deletion analysis of
WT1-reporter constructs containing up to 24 kilobases of
5
Volume 272, Number 5,
Issue of January 31, 1997
pp. 2901-2913
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
-flanking and first intron WT1 sequence in stably
transfected cultured cells as an unbiased approach to identify cis
elements critical for WT1 transcription. Although not a
tissue-specific element, a proximate 9-base pair CTC repeat accounted
for ~80% of WT1 transcription in this assay. Enhancer activity of the element and mutated versions correlated completely with
their ability to form a DNA-protein complex in gel shifts. Antibody
supershift, oligonucleotide competition, and Southwestern studies
indicated that the CTC-binding factor is the transcriptional activator
Sp1. Sp1 binds the CTC repeat with an affinity, KD = 0.37 nM, at least as high as the consensus GC box.
Similar CTC repeats are found in promoters of other growth-related
genes. Because Sp1 is important for WT1 expression, we
examined Sp1 immunohistochemistry in fetal and adult kidney. In a
pattern that precedes that of WT1 message, Sp1
immunostaining was highest in uninduced mesenchyme, early tubules,
developing podocytes, and mature glomeruli, but was minimal in mature
proximal tubules. This work suggests abundant Sp1 may be a prerequisite
for WT1 expression, and that Sp1 may have a wider role in
nephrogenesis.
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