Identification of isoform-specific WT1 interaction partners

PROJECT SUMMARY Wilms’ tumor 1, or WT1, is a transcription factor that plays an essential role in both nephrogenesis and maintenance of the adult podocyte. Variants in WT1 can cause Wilms tumors, progressive glomerulopathy, and disorders of sex differentiation in humans. WT1 also plays a role as either a tumor suppressor or oncogene in a context-dependent manner in both hematological malignancies and solid tumors. There are at least 36 different WT1 isoforms generated by alternate start sites and splicing. Two common isoforms, designated WT1-KTS and +KTS, differ in the presence or absence of three amino acids, lysine-threonine-serine, between the protein’s third and fourth zinc fingers and result from alternate splice sites in intron 9. Interestingly these two isoforms exhibit different nuclear localization patterns and DNA-binding affinity, and have been shown to have distinct yet overlapping cellular roles. Although the role of WT1 in the developing and mature kidney has been the subject of many research endeavors, there remain several critical gaps in our understanding of WT1 biology: 1) the factors that regulate WT1 transcriptional activity are unknown; 2) the function of the WT1+KTS isoform has not been fully characterized; and 3) the mechanisms by which human WT1 mutations disrupt WT1+KTS function have not been studied. We aim to apply novel proteomic approaches including proximity labeling and quantitative mass spectrometry to address these knowledge gaps. In Specific Aim 1, we propose to apply proteomic techniques to identify overlapping and unique interaction partners of the WT1-KTS and WT1+KTS isoforms. In Specific Aim 2, we will apply similar techniques in addition to other relevant cell biological assays to determine how WT1 patient variants disrupt protein-protein interactions and cause disease. Completion of these aims would provide the first successful proteomic approach to identify WT1 interaction partners, thereby providing a better understanding of the factors that modulate WT1 transcriptional activity, insight into the differential functions of the WT1-KTS and WT1+KTS isoforms, and, ultimately, to the development of novel therapeutic strategies for patients with WT1-related kidney disease.