Target gene specificity of E2F and pocket protein family members in living cells.
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.
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
J
J
Wells
K E
KE
Boyd
C J
CJ
Fry
S M
SM
Bartley
P J
PJ
Farnham
D016475:3T3 Cells; D000818:Animals; D002352:Carrier Proteins; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D050684:E2F Transcription Factors; D005786:Gene Expression Regulation; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D009687:Nuclear Proteins; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors