No abstract

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.

The temporal activation of E2F ansptional activity appears to be an important component of the mechanisms that prepare m an cells for DNA replication. Regulation of E2F actvity appears to be a highly complex process, and the dissection of the E2F pathway will be greatly faclitated by the ability to use genetic approaches. [a-32P]dCTP by random primer extension (34) and used in low-stringency hybridization. Positive clones were plaque purified and cDNA inserts were subcloned into pBluescript SK (Stratagene) for sequencing. The inserts were digested with exonuclease III and S1 nuclease to generate nested sets of deletions, which were sequenced with Sequenase 2.0 (United States Biochemical). To isolate dDP, the same library was screened with a probe to the putative DNA-binding domain of DP-1 (17).Plasmids. pBS-dE2F was made by subcloning a 4.4-kb EcoRI fragment (the entire cDNA) from A phage 16 into pBluescript SK(+). pBS-dE2F.ATG is a modified form of pBS-dE2F constructed by use of PCR to delete the first 849 bp of pBS-dE2F. pBS-dDP was made by subcloning the entire cDNA insert on an EcoRI fragment from A phage 3 into EcoRI-cut pBluescript SK(+). The expression plasmid Act-PPA, the internal control plasmid copia-lacZ (35), and the chloramphenicol acetyltransferase (CAT) reporter (E2F)4-BCAT (17) used in transfection experiments have been described. Act-dE2F and Act-dDP contain the entire coding regions of the genes.Cell Culture and Transfections. Schneider line 2 (SL2) cells (36) (generously supplied by Jayne Kassel, Massachusetts General Hospital Cancer Center, Boston) were maintained at room temperature in Schneider's Drosophila medium and Abbreviations: GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase.

Both the heterodimeric transcription factor, E2F, and the G1 cyclin, cyclin E, are required for the GI-S transition at the start of the metazoan cell cycle. It has been established that cyclin E can act as an upstream activator of E2F. In addition to this action, we show here that cyclin E has an essential role in DNA replication distinct from activating E2F. We have created transgenic Drosophila capable of inducible, ectopic production of E2F activity. Simultaneous overexpression of both Drosophila E2F subunits, dE2F and dDP, in embryos stimulated the expression of multiple E2F-target genes including cyclin E, and also caused the initiation of S phase. Mutation of cyclin E prevented the initiation of S phase after overexpression of dE2F/dDP without affecting induction of target gene expression. Thus, E2F-directed transcription cannot bypass loss of cyclin E in Drosophila embryos.