The E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S-phase transition of the mammalian cell cycle (see reference 16 for an extensive review of the E2F regulatory pathway). E2F binding sites are found in the promoters of genes whose products are required for nucleotide synthesis (e.g., dihydrofolate reductase [DHFR] and thymidine kinase [TK]), for DNA replication (e.g., DNA polymerase α and cdc6), and for cell cycle progression (e.g., cyclin E, cyclin D1, c-myc, b-myb, and cdc2). Transcription from each of these promoters increases during late G1 or early S phase, and this regulation is mediated by protein binding to one or more E2F binding sites (3, 11, 22, 30, 34, 43, 44). To date, eight members of the E2F family have been identified: six E2F proteins (E2F1 to -6) and two DP proteins (DP1 and DP2). The E2F and DP proteins bind to DNA as a heterodimer and can function as activators of transcription. Alternatively, E2F-DP heterodimers can also repress transcription when complexed with members of the Rb family of pocket proteins (pRb, p107, and p130) due to the ability of the pocket proteins to bind to and mask the E2F transactivation domain and to recruit histone deacetylases (6, 17, 25). Individual E2Fs preferentially bind to different pocket proteins. E2F1, E2F2, and E2F3, for example, bind to pRb, while E2F4 predominantly binds to p130 and p107 and E2F5 binds to p130. A popular model for how E2F family members regulate G1/S-phase-specific gene expression invokes a complex pattern of protein-protein and protein-DNA interactions that change as cells progress through the cell cycle (Fig. (Fig.1A).1A). As depicted, transcription from E2F site-containing promoters is thought to be repressed in G0 phase due to the binding of a trimolecular E2F-DP-pocket protein complex and recruitment of histone deacetylase activity by the pocket protein component (6, 17, 25). As cells progress through the cell cycle, various cyclin–cyclin-dependent kinase (cdk) complexes phosphorylate the pocket proteins, causing release of the hyperphosphorylated pocket protein and associated proteins from the DNA-bound E2F-DP heterodimer (1, 2, 7). Finally, traversal through S phase is thought to be accompanied by cyclin-cdk-mediated phosphorylation of the DP subunit of E2F1-3–DP complexes, resulting in release of the heterodimers from the promoter DNA (15, 21, 42). In some cells, E2F4-containing complexes are thought to be inactivated by relocation to the cytoplasm (28, 38). FIG. 1 E2F-mediated transcriptional regulation. (A) The current model of E2F-mediated transcriptional regulation is thought to involve binding of an E2F-pocket protein complex to promoters in G0 phase of the cell cycle and release of the pocket protein in late ... Although this model is attractive, it is largely based upon circumstantial data, and several important questions remain unanswered. For example, the transcriptional activity of complexes containing E2F4 and E2F5 may be shut off during mid- to late S phase by association with unphosphorylated p107 or p130, rather than by relocation to the cytoplasm (10). Additionally, it is not known if E2F target gene specificity exists or if all six E2Fs bind to and regulate every target gene or if Rb family members display target gene specificity. Determination of target gene specificity has been difficult due to the fact that most cells studied to date contain all of the E2Fs and pocket proteins. Thus, most analyses of E2F and pocket protein binding specificity have been performed using in vitro systems or by overexpression of an individual E2F or pocket protein in cells. In vitro systems, however, cannot recapitulate the complex environment of living cells, and altering the relative amounts of individual proteins through overexpression may abolish important protein-protein interactions. Therefore, we wished to determine which E2Fs and pocket proteins bind to and regulate expression of specific target genes in intact cells under physiological conditions. We felt that the use of an unperturbed in vivo system was of particular importance in the analysis of E2F target gene specificity since regulation occurs in the context of cell cycle progression which cannot be mimicked in vitro and which is often altered when individual E2F proteins are overexpressed. Toward this goal, we have used a formaldehyde cross-linking and immunoprecipitation system to monitor protein-DNA and protein-protein interactions on E2F target genes in living cells. Importantly, this procedure has allowed us to determine the in vivo patterns of E2F and pocket protein binding to specific E2F target genes as cells progress through a cell cycle. Our results indicate that, while some promoters fit the proposed model for E2F-mediated transcriptional regulation, others do not, necessitating individualization of the current model. Furthermore, our data suggest that cell cycle-regulated activity is a stochastic event, as cells have the ability to form several different E2F-pocket protein complexes on a given promoter at each stage of the cell cycle.