SP1 IS A CRITICAL REGULATOR OF THE WILMS TUMOR-1 GENE

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.