High affinity binding sites for the Wilms' tumour suppressor protein WT1.
The Wilms' tumour suppressor protein (WT1) is a putative transcriptional 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 knowledge 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 filter binding assay, and the results demonstrated that these sequences have a 4-fold higher affinity for the protein than the nonselected sequence GCG-TGG-GCG-CCC. The full length WT1 protein regulates transcription 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 sequences downstream of the GCG-TGG-GCG consensus sequence in the binding site selection assay. DNA sequences in the fetal promoter of the insulin-like growth factor II gene that confer WT1 responsiveness in a transient transfection assay bind to the WT1-ZFP with affinities that vary according to the number of consensus bases each sequence possesses in the finger 1 subsite.
Hamilton
T B
TB
Department of Biochemistry and Microbiology, University of Victoria, BC, Canada.
Barilla
K C
KC
Romaniuk
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D025721:WT1 Proteins; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D007335:Insulin-Like Growth Factor II; D004247:DNA