A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase.
Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753, USA.
Tetsuo
T
Maruyama
Andrea
A
Farina
Anup
A
Dey
JaeHun
J
Cheong
Vladimir P
VP
Bermudez
Tomohiko
T
Tamura
Selvaggia
S
Sciortino
Jon
J
Shuman
Jerard
J
Hurwitz
Keiko
K
Ozato
D016475:3T3 Cells; D000818:Animals; D001973:Bromodeoxyuridine / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002455:Cell Division; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D051818:Replication Protein C; D016196:S Phase; D013997:Time Factors; D014157:Transcription Factors; D014162:Transfection; D000092468:Bromodomain Containing Proteins
D001973:Bromodeoxyuridine; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D011994:Recombinant Proteins; D051818:Replication Protein C; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C486694:Brd4 protein, mouse