ROLE OF E2F IN REGULATION OF CELL CYCLE AND TUMOR DEVELOPMENT

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The developmental and tumor-suppressive properties of the retinoblastoma protein (pRB) both appear to be at least partially dependent upon its ability to regulate the transcriptional activity of a discrete subset of the E2F transcription factors. Animal models will be used to study the developmental role of the pRB-specific E2Fs and determine how their regulation contributes to the properties of the retinoblastoma protein. Novel mouse strains with germline mutations in the E2F-2 and E2F-3 genes will be created using standard gene targeting technology. Analysis of these mice will establish how loss of either single or multiple pRB- specific E2Fs affects the development of embryonic and adult mice. Crossbreeding will then be used to determine whether mutation of the these genes is sufficient to block either the tumor formation that occurs in the Rb+/- mice or the widespread apoptosis that occurs in the Rb-/- embryos. This will help to establish how E2F-regulation contributes to the developmental and tumor-suppressive properties of pRB. The effect of these mutations will also be examined at the molecular level using the single and compound mutant mouse strains to generate primary cells lines. In each case, the spectrum of E2F complexes, the degree and timing of activation of E2F responsive genes and growth properties of these mutant cells will be analyzed. These studies will help to link the pRB-specific E2Fs to the activation of specific target genes and determine how these individual events contribute to growth stimulatory properties of the endogenous E2F. Finally, we will continue to develop in vitro transcription assays that will allow us to study the direct consequences of the interaction between pRB and its target E2Fs. In particular, this system will be optimized to study the mechanism by which pRB abolishes the basal transcription of promoters to which it is bound. The goal of these in vitro studies is to separate the E2F-specific and global repression properties of pRB, to establish the molecular mechanisms by which these effects are mediated, and to determine how each one contributes to the growth suppressive properties of this protein.