E2F-3 accumulation is regulated by polypeptide stability

E2F is a complex family of transcription factors which appears to regulate the transcription of genes required for the S phase of the mammalian cell cycle. In the present work, we have examined the mechanisms regulating E2F-3 accumulation in mouse fibroblasts. We have determined that E2F-3 DNA binding activity is restricted to the G1/S transition and S phase in both normal BALB/c-3T3 fibroblasts and in an SV40 virus-transformed BALB/c-3T3 derivative. Immunoblot analysis indicates that G0 and G1 cells have little or no E2F-3 polypeptide and that the increase in the DNA binding activity of E2F-3 at the G1/S boundary is reflected by an increase in total E2F-3 protein. In contrast to the E2F-3 polypeptide, RNAse protection assays demonstrate that the E2F-3 mRNA is clearly present in G0 and G1 cells. Finally, pulse/chase experiments indicate that the half-life of E2F-3 is approximately 40-fold greater in cells blocked in S phase relative to asynchronously growing cells. Together, these results indicate that the accumulation E2F-3 at S phase may be regulated, at least in part, at the level of protein stability.