application) The WT1 gene plays an essential role in normal kidney and gonadal development and has also been implicated in the pathogenesis of the embryonic kidney malignancy Wilms' tumor. This gene encodes a zinc finger protein that is produced as different isoforms generated primarily through alternative splicing. One major alternative splice leads to the insertion of three amino acids within the zinc finger domain (the +KTS insertion), and this splicing event appears to be well conserved through evolution from zebrafish to humans. In addition, mutations that affect the efficiency of the +KTS splicing event have been associated with Frasier syndrome characterized by male pseudo-hermaphroditism, progressive glomerulonephropathy with chronic renal failure, and an increased risk of gonadoblastoma. WT1 isoforms lacking the KTS insertion are believed to bind DNA and to function as transcriptional regulator proteins. The functional role of isoforms with the KTS insertion is less clear although some evidence suggests that these proteins may affect RNA splicing. The goal of this proposal is to identify protein-nucleic acid and protein-protein interactions in which the isoforms of WT1 participate and to thus better define the molecular mechanisms by which WT1 exerts its effects on the processes of development and oncogenesis. A combination of biochemical methods, sequence database searches, and a novel bacterial cell-based genetic method will be used to identify and characterize potential nucleic acid and protein interaction targets of WT1 isoforms. The physiologic relevance of these interactions will then be characterized using a variety of in vivo methods.
The Specific Aims of the proposal are as follows: (1) To identify and characterize the physiologic nucleic acid binding site of the WT1+KTS isoform; and (2) To identify proteins that interact with DNA-bound zinc finger domains of the various WT1 isoforms. The study of these specific aims will lead to a greater understanding of the molecular processes involved in kidney and gonadal development and oncogenesis. More detailed knowledge of the fundamental regulatory circuits in which WT1 plays a central role has important implications for the treatment and diagnosis of developmental disorders and cancer.
