Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

Abstract

E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G₀ phase of the cell cycle and release of the pocket protein in late G₁, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G₀ and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grant CA45240 (to P.J.F.) and training grants CA09681 (J.W.) and CA09135 (K.E.B. and C.J.F.) from the National Institutes of Health.

We thank Kathleen Schell, Kristin Elmer, and Janet Lewis for excellent technical assistance with flow cytometry analysis; members of the Farnham lab for helpful discussions; and Rick Maser for critical reading of the manuscript. We also express our gratitude to the laboratories of Mark Biggen, David Allis, and Richard Treisman for sharing cross-linking protocols.

ADDENDUM

While the manuscript was under review, a similar study reporting different results was published by Takahashi et al. (35a). It is possible that the discrepancies between the two studies are due to promoter-specific variations. However, the b-myb promoter was analyzed in both studies, and we detected robust binding of only E2F4 to the b-myb promoter in serum-starved and G₁-phase cells which disappeared as cells entered S phase. In contrast, Takahashi et al. observed very low levels of E2F binding to the b-myb promoter in G₀ and early G₁ cells. We would like to emphasize that our cross-linking results are in agreement with previous in vivo footprinting analyses showing occupancy of the E2F site within the b-myb promoter from G₀ through G₁ phase in serum-synchronized NIH 3T3 cells (44). Strikingly, Takahashi et al. reported no binding of E2F4 to promoters of target genes during S phase, while we observed robust S-phase binding of E2F4 to several target gene promoters. Since different antibodies had been used in the two studies, we performed an additional cross-linking experiment to directly compare the immunoprecipitation efficiencies of the E2F4 antibody which we used for the results presented here (sc-866X; Santa Cruz) and of that used by Takahashi et al. (sc-1082X; Santa Cruz). Our results indicated that both antibodies detected binding of E2F4 to several different promoters in synchronized NIH 3T3 cells but that the sc-1082X antibody generated a noticeably weaker signal on some promoters, and this difference was most pronounced in S-phase cells (data not shown). Finally, the differences in our results and those of Takahashi et al. could be species specific, as we used immortalized murine cells (NIH 3T3) while they used a human glioblastoma cell line (T98G). Recent cross-linking experiments (data not shown) have confirmed binding of E2F proteins, particularly E2F4, to multiple promoters during S phase in aphidicolin-synchronized HeLa and Raji cells, both of which are human tumor cell lines. It remains possible that expression of E2F target genes in T98G cells may be mediated by different E2F proteins than those in other human or murine cell lines.