The tumor suppressor, pRB, and its related proteins, p107 and p130, play a pivotal role in the regulation of terminal differentiation, proliferation and tumorigenesis through their ability to control the E2F transcription factors. This proposal focuses on understanding how E2F4, the most abundant of the E2F family members, contributes to the growth suppressive properties of the pRB family of proteins. In the last few years, the Lees lab has generated a panel of compound mutant mouse strains, E2f4;Rb, E2f4;p107;p130 and E2f4;E2f5. The preliminary analysis of these animals shows that E2F4 has a profound influence on the regulation of cell cycle exit, the terminal differentiation of numerous cell lineages and the formation of pRB-deficient tumors. They also suggest that the E2F4 complexes are regulating differentiation through two distinct mechanisms: (i) they repress E2F-responsive genes to mediate cell cycle exit, and (ii) they directly influence the transcriptional activity of genes that promote the differentiation process. The role of E2F4 in cell cycle control, differentiation and tumorigenesis will be investigated. The analysis of mouse embryonic fibroblasts will allow dissection of how the E2F4 complexes contribute to the regulation of E2F-responsive gene transcription, the induction of cell cycle exit and the inhibition of apoptosis. Whole animal and in vitro differentiation studies will be used to determine how E2F4 complexes participate in the differentiation process. Finally, through a combination of tumor analysis and biochemical studies, the mechanism by which E2F4-loss suppresses the development of pRB-deficient tumors will be determined. Preliminary studies suggest that this occurs through a novel mechanism that suggests alternative strategies for the design of chemotherapeutic agents.