SEQUENCE AND STRUCTURAL REQUIREMENTS FOR HIGH-AFFINITY DNA-BINDING BYTHE WT1 GENE-PRODUCT

The Wilms' tumor suppressor gene, WTI, encodes a zinc finger polypepti de which plays a key role regulating cell growth and differentiation i n the urogenital system. Using the whole-genome PCR approach, we searc hed murine genomic DNA for high-affinity WT1 binding sites and identif ied a 10-bp motif 5'GCGTGGGAGT3' (which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognize d by EGR-1 and is also suggested to function as a binding site for WT1 , setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affin ities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-h igher affinity for the WTE sequence compared with that of the EGR-1 bi nding motif. Mutational analysis of the WTE motif revealed a significa nt contribution to binding affinity by the adenine nucleotide at the e ighth position (5'GCGTGGGAGT3') as well as by the S'-most thymine (S'G CGTGGGAGT3'), whereas mutations in either flanking nucleotides or othe r nucleotides in the core sequence did not significantly affect the sp ecific binding affinity. Mutations within WT1 zinc fingers II to IV ab olished the sequence-specific binding of WT1 to WTE, whereas alteratio ns within the first WT1 zinc finger reduced the binding affinity simil ar to 10-fold but did not abolish sequence recognition. We have thus i dentified a WT1 target, which, although similar in sequence to the EGR -1 motif, shows a 20- to 30-fold-higher affinity for WT1. These result s suggest that physiological action of WT1 is mediated by binding site s of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is di scussed in the context of recent structural analysis.