The biological effects of the ski oncogene are pleiotropic because its complex molecular interactions allow it to impinge upon elaborate pathways that regulate cellular proliferation and differentiation. Retroviral-mediated overexpression of either v-ski or the proto-oncogene, c-ski, transforms avian embryo fibroblasts and converts them into skeletal myoblasts. v-Ski and c-Ski apparently produce these biological effects by binding specific DNA elements in combination with other proteins, and either activating or repressing transcription. The Ski-related protein SnoN dimerizes with Ski and shares its oncogenic and transcriptional regulatory activities. The overall goals of this work are to 1) identify genes regulated by Ski and Sno that are involved in their ability to induce oncogenic transformation and 2) characterize DNA and protein interactions that may underlie their transcriptional activities. To accomplish these goals we propose a combination of focused and genome-based approaches. The mRNA sequences that are induced and repressed in Ski transformed cells will be identified by PCR-based differential display using a high-density cloning and screening method. The roles of the identified genes in Ski-induced transformation will be studied in model in vitro systems. We will explore the role of Ski, and its phosphorylation, in the regulation of two genes whose expression is responsive to Ski (myogenin) or to sequence elements identified as Ski binding sites (PAI-2). A protein partner that may act as an effector of Ski's transcriptional regulatory function has been identified using the yeast two-hybrid protein interaction screen. This protein has functions in chromatin-mediated gene regulation; the effects of its interactions with Ski and Sno on gene regulation will be examined using model gene expression systems. A protein that co-binds a DNA element that mediates repression of gene expression by Ski/SnoN has been partially purified. Its identification and an analysis of its role in Ski transformation will also be pursued. Finally, a specially devised system for tight control of ski expression in a cell line from the ski knock-out mouse will be used to identify genes acutely regulated by Ski. This will involve the powerful oligonucleotide computer chip analysis system developed by Affymetrix to identify activated and repressed genes.
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