HIGH-AFFINITY BINDING-SITES FOR THE WILMS-TUMOR SUPPRESSOR PROTEIN WT1

The Wilms' tumour suppressor protein (WT1) is a putative transcription al regulatory protein with four zinc fingers, the last three of which have extensive sequence homology to the early growth response-1 (EGR-1 ) protein. Although a peptide encoding the zinc finger domain of WT1[- KTS] can bind to a consensus 9 bp EGR-1 binding site, current knowledg e about the mechanisms of zinc finger-DNA interactions would predict a more extended recognition site for WT1. Using a WT1[-KTS] zinc finger peptide (WT1-ZFP) and the template oligonucleotide GCG-TGG-GCG-NNNNN in a binding site selection assay, we have determined that the highest affinity binding sites for WT1[-KTS] consist of a 12 bp sequence GCG- TGG-GCG-(T/G)(G/ A/T)(T/G). The binding of WT1-ZFP to a number of the selected sequences was measured by a quantitative nitrocellulose filte r binding assay, and the results demonstrated that these sequences hav e a 4-fold higher affinity for the protein than the nonselected sequen ce GCG-TGG-GCG-CCC. The full length WT1 protein regulates transcriptio n of reporter genes linked to these high affinity sequences. A peptide lacking the first zinc finger of WT1[-KTS], but containing the three zinc fingers homologous to EGR-1 failed to select any specific sequenc es downstream of the GCG-TGG-GCG consensus sequence in the binding sit e selection assay. DNA sequences in the fetal promoter of the insulin- like growth factor II gene that confer WT1 responsiveness in a transie nt transfection assay bind to the WT1-ZFP with affinities that vary ac cording to the number of consensus bases each sequence possesses in th e finger 1 subsite.