Overexpression of the E2F-1 cDNA in mammalian cells disrupts normal control of the cell cycle and drives cells into S phase. Whereas eliminating E2F activity would test its inferred involvement in the G~-S transition, elimination is complicated by the existence of gene families encoding mammalian E2F. Here we identify mutations in a single essential Drosophila gene, dE2F, that encodes a homolog of the mammalian E2F gene family. Embryos homozygous for null mutations of dE2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. We conclude that in most cells clE2F is essential for a G~-S transcriptional program and for G~-S progression.[Key Words: Drosophila; E2F; S phase; cell cycle; embryogenesis] Received April 14, 1995; accepted May 3, 1995.The E2F transcription factor was originally identified through its role in the regulation of the adenovirus E2 promoter during viral infection. E2F is a heterodimer composed of a polypeptide encoded by DP-1 (or DP-1-related genes) and a polypeptide encoded by E2F-1 (or E2F-l-related genes). Studies in mammalian cells have suggested that E2F provides a nexus between the cyclindependent kinases, the retinoblastoma tumor suppressor gene, and the cell cycle control of transcription. A favored model of E2F regulation (for review, see Nevins 1992;Helin and Harlow 1993;Farnham 1995) is summarized below.In mammalian cells, the transcriptional activity of E2F is constrained by its physical association with the retinoblastoma protein (pRB) and the pRB-related proteins p107 and p130. The pRB-E2F interaction is regulated through phosphorylation by cyclin-dependent kinases (cdks). pRB has been shown to be phosphorylated by cdks that become activated during G1 phase of the cell cycle, such as cyclin D/cdk4, cyclin D/cdk6, and cyclin E/cdk2 kinases. Phosphorylation of pRB is thought to lead to the release of E2F, the activation of E2F-dependent transcription, and the promotion of entry into S phase. In this model, E2F and pRB are proposed to act antagonistically to regulate entry into S phase. This scheme provides an appealing common rationale for the action of oncogenes and tumor suppressor genes. Functions that interfere with the inhibitory interaction of 3Corresponding author.

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.

The deregulation of E2F activity is thought to contribute to the uncontrolled proliferation of many tumor cells. While the effects of overexpressing E2F genes have been studied extensively in tissue culture, the consequences of elevating E2F activity in vivo are unknown. To address this issue, transgenic lines of Drosophila were studied in which ectopic expression of dE2F and dDP was targeted to the developing eye. The co‐expression of dDP or dE2F disrupted normal eye development, resulting in abnormal patterns of bristles, cone cells and photoreceptors. dE2F/dDP expression caused ectopic S phases in post‐mitotic cells of the eye imaginal disc but did not disrupt the onset of neuronal differentiation. Most S phases were seen in uncommitted cells, although some cells that had initiated photo‐receptor differentiation were also driven into the cell cycle. Elevated expression of dE2F and dDP caused apoptosis in the eye disc. The co‐expression of baculovirus p35 protein, an inhibitor of cell death, strongly enhanced the dE2F/dDP‐dependent phenotype. These results show that, in this in vivo system, the elevation of E2F activity caused post‐mitotic cells to enter the cell cycle. However, these cells failed to proliferate unless rescued from apoptosis.

The transcription factor E2F is a target of the retinoblastoma tumor suppressor protein (pRB) and may mediate pRB regulation of S phase entry in mammalian cells. The recent identification of mutant alleles of the Drosophila E2F gene (dE2F) has shown that dE2F is required for embryogenesis. dE2F‐mutant embryos lack a co‐ordinated program of gene expression which accompanies S phase entry and DNA synthesis declines to levels that are barely detectable. We have investigated the role of the dE2F gene at later stages of development. dE2F is expressed in several larval tissues and is required for cell proliferation in the eye imaginal disc. Surprisingly, dE2F expression persists in post‐mitotic cells of the eye disc of third‐instar larvae. The loss of dE2F function in these cells causes a novel phenotype, characterized by loss of photoreceptors and abnormal rhabdomere cell morphology. These results show that dE2F is required at multiple stages of development and suggest that E2F may have an important function in post‐mitotic cells in addition to its role during cell proliferation.