Target gene specificity of E2F and pocket protein family members in livingcells

E2F-mediated transcription is thought to involve binding of an E2F-pocket p rotein complex to promoters in the G(0) phase of the cell cycle and release of the pocket protein in late G(1), 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 comple xes which change as cells progress through the cell cycle. We also find tha t 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 m embers may regulate transcription in both G(0) and S phases. Finally, we fi nd that a given promoter can be bound by one of several different E2F-pocke t protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, proces s.