Regulation of E2f1-target gene expression during Drosophila embryogenesis

Cell proliferation is tightly controlled during animal development, and a key aspect of this control is cell cycle exit prior to terminal differentiation. Since most cells exit the cell cycle during G1, it is important to understand how the activities of the transcription factors and Cyclin/Cyclin-dependent kinase (Cyc/Cdk) complexes that promote S phase entry are restrained. In this thesis, I used both genetic and cell biological tools to examine the mechanisms of G1 cell cycle exit during embryogenesis in Drosophila melanogaster. In the Drosophila embryo, G1 arrest first appears in epidermal cells during midembryogenesis and multiple mechanisms contribute to a stable G1 quiescence in these cells. Cyclin E/Cdk2 (CycE/Cdk2) kinase activity is essential for S phase in flies and must be down-regulated by the cyclin-dependent kinase inhibitor (CKI) p27Dacapo to initiate G1 arrest. The E2f1 transcription factor induces the expression of replication factors and must be inhibited to maintain G1 arrest. This is accomplished by a Drosophila retinoblastoma family member called Rbf1, whose activity is controlled by phosphorylation. Hypo-phosphorylated Rbf1 can bind the E2f1 transcription factor and repress target gene expression. Phosphorylation of Rbf1 by CycE/Cdk2 results in the dissociation of Rbf1-E2f1 repressor complexes and the expression of replication factors Here, I show that embryonic Rbf1 activity is regulated by phosphorylation, and that the conversion of Rbf1 to an active repressor just prior to G1 arrest occurs by the developmental induction of p27Dacapo, which inhibits the Rbf1 kinase CycE/Cdk2. Surprisingly, however, the initial termination of E2f1-target gene expression occurs independently of Rbf1. Rather, my data suggest that the S phase-coupled destruction of E2f1 protein likely contributes to this initial termination of target gene expression. Since biochemical data in mammals suggest that type 1 protein phosphatases (PP1) regulate pRb activity, I tested genetically whether PP1 regulates Rbf1 activity in Drosophila. Interestingly, PP1 is not required for Rbf1-mediated repression during G1 arrest in the embryonic epidermis or for the regulation of Rbf1 activity during the endocycle in the embryonic midgut or larval salivary gland. This suggests that another phosphatase family member may counteract CycE/Cdk2 inhibition of Rbf1 in flies