The genetic pathways that underlie the development of the pediatric kidney cancer Wilms tumor provide unique insight into the link between normal development of the kidney and its deregulation during malignant transformation. Inactivating mutations in the WT1 tumor suppressor, a zinc finger transcription factor encoded by multiple alternative splicing variants, underlie a subset of Wilms tumors, pointing to critical transcriptional targets that contribute to kidney development and tumorigenesis. We have used cells with inducible expression of the transcription allyactive isoform, WT1 (-KTS), combined with expression profile analysis to identify physiologically regulated target genes, and we have established a model for WT1-directed cellular differentiation using hematopoietic precursors. Here, we propose to focus on the functional properties of the most abundant isoform, WT1 (+KTS), whose function is unknown.
In Aim I, we will search for endogenous genes whose expression is regulated by WT1 (+KTS), using both hybridization to microarrays and subtractivePCR approaches. The mechanisms by which this WT1 isoform regulates expression of its target genes (transcriptional as well as postulated post-transcriptional mechanisms) will be studied. The functional properties of these downstream effectors and their potential contribution to renal differentiation and Wilms tumorigenesis will be addressed.
In Aim II, we will pursue protein-protein interactions that have been implicated in WT1 (+KTS) function, as well as in its characteristic subnuclear localization within """"""""speckles"""""""". A combination of yeast-two hybrid assays and mass spectrometry sequencing of coprecipitated proteins will be employed, and the functional significance of confirmed interactors will be addressed.
In Aim III, we will study another recently isolated transcription factor, BF2, which is also essential for kidney development, but through a distinct mechanism. Remarkably, BF2 is expressed only in stromal cells of the fetal kidney, yet its inactivation in the mouse suppresses epithelial differentiation without affecting stromal cells themselves. Using inducible expression of BF2 and expression profile analysis, we have identified downstream targets, including secreted growth factors, whose contribution to renal differentiation and to stromal-epithelial interactions will be explored. Taken together, this proposal aims at identifying downstream targets of two transcription factors that are essential to kidney development, defining the mechanism by which these targets are regulated, and studying their potential contribution to Wilms tumorigenesis.
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