E2F transcription factors regulate the expression of numerous genes involved in cell cycle progression and have been implicated in the processes of S phase entry, oncogenic transformation, and apoptosis. It is now known that E2F is composed of a family of closely related proteins encoded by at least five distinct genes: E2F-1,E2F-2,E2F-3,E2F-4,and E2F- 5. Each E2F isoform has the ability to dimerize with a protein termed DP-1, bind to consensus E2F sites, and activate transcription but it is unclear whether the various E2Fs have different transcriptional activation properties or activate distinct sets of genes. One difference that is clear among the E2F species is their abilities to bind and be regulated by members of the Rb tumor suppressor protein family. Expression during the cell cycle also differs among E2F species. Although these findings, as well as structural differences, suggest that there may be unique functions for various E2Fs, a careful examination of the relative activities of E2F family members has not been performed. In preliminary studies we compared the abilities of E2F-1,-4, and -5, the species showing the greatest potential variation, to activate transcription from a variety of gene promoters. We also examined their DNA-binding activities and the strength of their transcriptional activation domains by creating fusions with Gal4. In addition, the ability of E2F-1,-4 and -5 to induce anchorage-independent growth of 3T3 cells was also assayed in order to correlate transcriptional activation capacity with oncogenic potential. Our findings suggest that there are clear differences in the activities of these E2F isoforms. The experiments outlined in this proposal will test the hypothesis that different E2F family members have different transcriptional and growth regulatory properties which reflect distinct cellular functions. Assays will be developed to examine the abilities of individual E2F isoforms to activate transcription, stimulate S phase entry, contribute to oncogenic transformation, and induce apoptosis. E2F mutants and chimeras will then be used to map differences observed in these assays to specific protein domains to gain an understanding of how E2F participates in each process. Using E2F mutants with dominant-negative phenotypes, we will also examine the role E2F plays in cell cycle progression, transformation, and apoptosis induced by other factors. This comprehensive study of the functional activities of E2F family members should provide insight into the unique roles of individual E2F isoforms and help define the relationship between transcriptional activation, S phase induction, transformation, and apoptosis mediated by E2F, as well as by other factors.
