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Research Papers
Massachusetts General Hospital, Cancer Center, Charlestown 02129, USA.
Abstract
Genetic studies have shown that cyclin E and dE2F are critical regulators of S-phase entry during Drosophila embryogenesis. Whereas the ectopic expression of cyclin E activates dE2F-dependent transcription, it has been proposed that cyclin E does not act directly on dE2F but targets a negative regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins (pRB, p107, and p130) that associate with E2F in humans; however, extensive efforts have failed to find such homologs in Drosophila. We have developed a two-hybrid approach that allows transcription activators to be used as bait for interacting proteins. From a screen using Drosophila E2F (dE2F and dDP) as bait, we identified a novel gene, RBF. RBF combines several of the structural features of pRB, p107, and p130, suggesting that it may have evolved from a common ancestor to the three human genes. RBF associates with dE2F and dDP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF was phosphorylated by a cyclin E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF plays a critical role in the regulation of cell proliferation in Drosophila and show that analogous pathways regulate S-phase entry in a diverse range of species.
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