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Target gene specificity of E2F and pocket protein family members in living cells

By Julie Wells, Kathryn E. Boyd, Christopher J. Fry, Stephanie M. Bartley, Peggy and J. Farnham


E2F-mediated transcription is thought to involve binding of an E2F-pocket protein 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 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 G 0 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. The E2F family of transcription factors plays an important role in the regulation of gene expression at the G 1/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 � 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 G 1 or early S phase, and this regulation is mediated by protei

Year: 2000
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