Transcriptional Repressor Functions of Drosophila E2F1 and E2F2 Cooperate To Inhibit Genomic DNA Synthesis in Ovarian Follicle Cells

Abstract

Individual members of the E2F/DP protein family control cell cycle progression by acting predominantly as an activator or repressor of transcription. In Drosophila melanogaster the E2f1, E2f2, Dp, and Rbf1 genes all contribute to replication control in ovarian follicle cells, which become 16C polyploid and subsequently undergo chorion gene amplification late in oogenesis. Mutation of E2f2, Dp, or Rbf1 causes ectopic DNA replication throughout the follicle cell genome during gene amplification cycles. Here we show by both reverse transcription-PCR and DNA microarray analysis that the transcripts of prereplication complex (pre-RC) genes are elevated compared to the wild type in E2f2, Dp, and Rbf1 mutant follicle cells. For some genes the magnitude of this transcriptional derepression is greater in Rbf1 than in E2f2 mutants. These differences correlate with differences in the magnitude of the replication defects in follicle cells, which attain an inappropriate 32C DNA content in both Rbf1 and Dp mutants but not in E2f2 mutants. The ectopic genomic replication of E2f2 mutant follicle cells can be suppressed by reducing the Orc2, Orc5, or Mcm2 gene dose by half, indicating that small changes in pre-RC gene expression can affect DNA synthesis in these cells. We conclude that RBF1 forms complexes with both E2F1/DP and E2F2/DP that cooperate to repress the expression of pre-RC genes, which helps confine DNA synthesis to sites of gene amplification. In contrast, E2F1 and E2F2 repressors function redundantly for some genes in the embryo. Thus, the relative functional contributions of E2F1 and E2F2 to gene expression and cell cycle control depends on the developmental context.

Identification of aDrosophilaMyb-E2F2/RBF transcriptional repressor complex

“…In Drosophila tissue culture cells, E2F2 appears to function primarily for repression of developmentally regulated genes , while E2F1/RBF1 complexes are involved in regulating genes involved in cell cycle progression. However, microarray studies performed in e2f2 and rbf1 mutant follicle cells indicate that both E2F2 and RBF1 are involved in the repression of several S-phase genes, including CDT1 and the ORC and MCM complex subunits (Cayirlioglu et al 2003). Therefore, it is likely that the set of genes regulated by the Myb-MuvB complex may change depending on the developmental context.…”

Pocket proteins and cell cycle control

“…These studies showed that the E2F1 and pRB homologs (dE2F1 and Rbf) bind the Drosophila origin recognition complex (DmORC) protein and the chorion gene cluster origin of replication, and thereby limit the physiological amplification of this cluster in ovarian follicle cells (Royzman et al, 1999;Bosco et al, 2001). dE2F1 and Rbf were also found to suppress genomic rereplication in Drosophila, yet later work indicated that this was mediated through transcriptional effects (Cayirlioglu et al, 2003). Recent studies suggest that pocket proteins also have replicative functions in vertebrates.…”

Cul4 and DDB1 regulate Orc2 localization, BrdU incorporation and Dup stability during gene amplification inDrosophilafollicle cells

“…Previous evidence suggests that high transcriptional activity of specific loci controls replication origin firing during the gene amplification stage (Claycomb and Orr-Weaver, 2005;Tower, 2004). Mutants for transcription factors such as E2f2, Dp, Rbf, Myb and Mip130 show increased mRNA and protein levels of replication factors, such as components of the Orc and MCM complexes, and ectopic genomic replication during the gene amplification stage (Bosco et al, 2001;Cayirlioglu et al, 2001;Cayirlioglu et al, 2003;Royzman et al, 1999). Mutant follicle cells for Cul4 displayed both Orc2 localization defects and abnormal genomic replication, implying that Cul4 is probably involved in both processes by modulating the transcriptional activity in DNA replication.…”

“…Moreover, the trans regulators Myb and E2F transcriptional complexes bind ACE3, and the Myb complex also binds Ori-β. Defects in those transcription factors result in impaired gene amplification (Beall et al, 2004;Beall et al, 2002;Bosco et al, 2001;Cayirlioglu et al, 2001;Cayirlioglu et al, 2003;Royzman et al, 1999).…”