Terminal differentiation is a crucial developmental process in which cell cycle exit is coordinated with the expression of physiological functions. Understanding mechanisms that control the transformation of immature and proliferating epithelial cells into mature differentiating cell types is important for our understanding of renal development as well as mechanisms of repair and regeneration after injury. The tumor suppressor protein, p53, occupies a unique position among regulators of terminal differentiation due to its dual roles in cell cycle control and transcriptional regulation. The overall hypothesis to be tested in this proposal is that p53 plays a key role in the transcriptional control of terminal epithelial cell differentiation in the developing mammalian kidney.
The First Aim will test the hypothesis that p53 stimulates the biochemical differentiation of the renal epithelium; p53 will be examined for in vitro binding to and activation of promoters of renal function genes and other terminal differentiation genes using gel shift and transient transfection assays. Chromatin immunoprecipitation assays will test if p53 binding to target promoters is selectively activated during the process of terminal differentiation in vivo. Immunohistochemical studies will examine the cellular co-localization of endogenous p53 and target terminal differentiation genes.
The Second Aim will examine the role of p53 in the morphological and functional differentiation of the kidney. Developing p53 null mice bred on various genetic backgrounds will be assessed for signs of aberrant spatiotemporal expression of terminal differentiation markers, uncontrolled epithelial cell proliferation, tubular dysgenesis, and impaired renal function. The morphogenetic events leading to the renal dysgenesis will be identified using developmental stage-specific markers and immunohistochemistry/in situ hybridization techniques. To further confirm the role of p53 in renal cell differentiation, we will determine whether the p53 null renal phenotype can be recapitulated by transgenic over expression of dominant negative mutant p53 under the control of kidney-specific promoters. In addition, p53 null mice will be stressed by induction of renal ischemia/reperfusion injury and the capacity of the renal epithelium to undergo repair and regeneration will be examined.
The Third Aim will elucidate the role of p53 in specification of terminal differentiation fate. Reporter transgenic mice harboring wild-type or mutant p53-binding sites in the promoter of a selected terminal differentiation gene will be tested for appropriate spatio-temporal specification of transgene expression in vivo. The results should advance our understanding of the transcriptional program of epithelial cell differentiation and elucidate a novel function for p53 in regulating epithelial cell differentiation in the kidney.
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