The goal of this proposal is to understand how cell cycle transit and cellular phenotype is controlled by the transcription factor E2F1. E2F1 is normally expressed at the end of GO/G1 phase and regulates genes whose products are needed for movement through S phase. Yet the mechanisms by which E2F1 exerts this regulation are not well understood. Constitutive overexpression of E2F1 in NIH3T3 fibroblasts primarily alters GO transit. In contrast, constitutive overexpression of a mutant E2F1, lacking an amino terminal domain (NTD), has profound affects on growth rate, contact inhibition of growth, S phase duration, morphology and sensitivity of the cells to chemotherapeutic agents. Initial studies indicate that the NTD of E2F1 exerts a potent and novel transcriptionally repressing effect on the protein and that loss of this domain results in a significant upregulation of a number of specific E2F1 target genes. The NTD therefore modulates the ability of E2F1 to regulate expression of genes whose products affect cell structure, cell growth and aspects oncogenic transformation. The data indicate that this repressing effect is likely due to an association of cellular factors with the NTD. The initial goal of this grant will be to identify the cellular factors that associate with the NTD of E2F1 and identify the subregions within the NTD to which they bind. This information will be used to construct mutants in E2F1 that lack specific protein binding sites within the NTD. Expression of these mutants in NIH3T3 cells will be used to determine if loss of specific protein binding sites are essential for the upregulation of gene expression and for the generation of the profound phenotypic effects on the cells. In this way it will be possible to directly correlate protein binding to the NTD with E2F1 function in vivo. As a corollary to these studies, cDNA clones have been isolated that code for proteins that bind to the NTD.These cDNA clones will be analyzed in detail and their role in modulating the activity of E2F1 assessed. It is probable that these proteins have growth suppressing effects in cells since they are likely to inhibit the ability of E2F1 to transcriptionally transactivate. The results of these studies will further our understanding of how a domain on the transcription factor E2F1 regulates its function and in the process coordinately controls aspects of both cellular phenotype and cell proliferation.
Showing the most recent 10 out of 22 publications
