Functional roles of E2F in cell cycle regulation

The E2F family of transcription factors play a key role in G1-S progression. A dominant negative mutant (E2F97) of E2F1 containing the DNA binding domain of E2F1 under the control of a tetracycline-responsive promoter was constructed. Stable transfectants were produced in the pRb-lacking SaOS-2 cell line and SV40-transformed VA-13 cell line, respectively. Induction of E2F97 by tetracycline withdrawal resulted in strong inhibition of the E2F transcriptional activity and a decreased percentage of cells in S-phase, To understand the mechanism(s) by which E2F97 exerts its effect on the cell cycle, the effect of E2F97 on expression of various cell cycle proteins was examined. Upon induction of E2F97, a significant decrease in the levels of both dihydrofolate reductase and thymidylate synthase was observed in transfectants derived from both cell lines. Induction of E2F97 also led to a decrease in cyclin A and D1 protein levels. Regulation of cyclin A by E2F97 occurred at the transcriptional level. In addition, in VA13 cells, induction of E2F97 resulted in downregulation of the tumor suppressor protein p53. These data suggest that E2F regulates both G1 and S-phase cyclins and that there may be a potential positive feedback regulatory loop between E2F and cyclin D1.