DNA-binding transcription factors play essential roles in many biological processes, including cell growth regulation, development and differentiation. Defects in some of these proteins have been correlated with developmental abnormalities and tumorigenesis, as exemplified by the Wilms' tumor suppressor protein WT1. Inactivation of WT1 is correlated with the incidence of Wilms' tumor, a pediatric kidney cancer, and the Denys-Drash syndrome which is characterized by nephropathy, intersex disorders and Wilms' tumor. WT1 is a zinc finger-containing transcription factor that can repress or activate transcription of its target genes. The loss of WT1 function and/or an imbalance of its repression and activation activities may lead to deregulated cell growth that contributes to the formation of Wilms' tumor. The intent of this application is to study mechanisms that control the dual transcriptional activities and tumor suppressor functions of WT1. The proposed studies will provide novel insight into mechanisms of tumorigenesis as well as transcriptional regulation in mammalian cells. The transcriptional activities of WT1 are affected by cellular proteins that it interacts with. We have recently cloned a WT1-interacting protein (Ciao 1) that is related to the yeast co-repressor Tup1, raising the possibility that Ciao 1 may be a co-factor for WT1. We will explore the possibility that Ciao 1 modulates the transcriptional activities of WT1 via protein/protein interactions by transfection experiments and mutagenesis studies. We will examine the interaction of Ciao 1 with naturally occurring, repression-defective WT1 mutants as lack of interactions will suggest a functional role for Ciao 1 in WT1-mediated transcriptional repression. We will also determine the effect of Ciao 1 on the tumor suppressor function of WT1, i.e., its ability to suppress colony formation in a Wilms' tumor cell line. Lastly, WT1 mutations were identified in only 10% of the Wilms' tumor. We will examine the possibility that defects in WT1-interacting proteins may also contribute to the formation of Wilms' tumor, by analyzing Ciao 1 mRNA and proteins in Wilms' tumor samples. Ultimately, we wish to identify the molecular components involved in WT1-mediated transcription, as the regulation of the transcriptional activities of WT1 is critical for its tumor suppressor functions. As an important step towards this goal, we will establish an in vitro transcription system to study transcriptional repression/activation by WT1. With the in vitro system, we will determine the steps in the transcriptional process at which WT1 acts to repress/activate transcription. We will study the functions of WT1 mutants and assess the functional importance of the WT1/Ciao 1 interactions in vitro.
