We performed deletion analysis of WT1-reporter constructs containing u
p to 24 kilobases of 5'-flanking and first intron WT1 sequence in stab
ly transfected cultured cells as an unbiased approach to identify cis
elements critical for WT1 transcription. Although not a tissue-specifi
c element, a proximate 9-base pair CTC repeat accounted for similar to
80% of WT1 transcription in this assay. Enhancer activity of the elem
ent and mutated versions correlated completely with their ability to f
orm a DNA-protein complex in gel shifts. Antibody supershift, oligonuc
leotide competition, and Southwestern studies indicated that the CTC-b
indiug factor is the transcriptional activator Sp1. Sp1 binds the CTC
repeat with an affinity, K-D = 0.37 nM, at least as high as the consen
sus GC box. Similar CTC repeats are found in promoters of other growth
-related genes. Because Sp1 is important for WT1 expression, we examin
ed Sp1 immunohistochemistry in fetal and adult kidney. In a pattern th
at precedes that of WT1 message, Sp1 immunostaining was highest in uni
nduced mesenchyme, early tubules, developing podocytes, and mature glo
meruli, but was minimal in mature proximal tubules. This work suggests
abundant Sp1 may be a prerequisite for WT1 expression, and that Sp1 m
ay have a wider role in nephrogenesis.