RBF, A NOVEL RE-RELATED GENE THAT REGULATES E2F ACTIVITY AND INTERACTS WITH CYCLIN-E IN DROSOPHILA

Genetic studies have shown that cyclin E and dE2F are critical regulat ors of S-phase entry during Drosophila embryogenesis. Whereas the ecto pic expression of cyclin E activates dE2F-dependent transcription, it has been proposed that cyclin E does not act directly on dE2F but targ ets a negative regulator of E2F activity. Such a regulator might be an alogous to the family of RE-related proteins (pRB, p107, and p130) tha t associate with E2F in humans; however, extensive efforts have failed to find such homologs in Drosophila. We have developed a two-hybrid a pproach that allows transcription activators to be used as bait for in teracting proteins. From a screen using Drosophila E2F (dE2F and dDP) as bait, we identified a novel gene, REP. RBF combines several of the structural features of pRB, p107, and p130, suggesting that it may hav e evolved from a common ancestor to the three human genes. RBF associa tes with dE2F and dDP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent trans cription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF was phosphorylat ed by a cyclin E-associated kinase in vitro, and loss-of-function cycl in E mutations enhanced an RBF overexpression phenotype, consistent wi th the idea that the biological activity of RBF is negatively regulate d 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 i n the regulation of cell proliferation in Drosophila and show that ana logous pathways regulate S-phase entry in a diverse range of species.