Control of Drosophila endocycles by E2F and CRL4CDT2

Abstract

Endocycles are variant cell cycles comprised of DNA Synthesis (S)- and Gap (G)- phases but lacking mitosis1,2. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world’s biomass3,4. DNA replication in endocycling Drosophila cells is triggered by Cyclin E/Cyclin Dependent Kinase 2 (CycE/Cdk2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase5,6. How CycE/Cdk2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modeling, we show that Drosophila’s endocycles are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4Cdt2 ubiquitin ligase, and this in turn mediates the destruction of E2F17. We propose that it is the transient loss of E2F1 during S-phases that creates the window of low Cdk activity required for preRC formation. In support of this model over-expressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by de-regulating target genes including CycE, as well as Cdk1 and mitotic Cyclins. Moreover, we find that altering cell growth by changing nutrition or TOR signaling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants1,2,8, suggesting that elements of this mechanism act in most growth-dependent cell cycles.

Endoreplication and polyploidy: insights into development and disease

“…A good example is provided by polyploid Drosophila follicle cells, which require many copies of the chorion genes to produce sufficient protein for eggshell biosynthesis (Calvi and Spradling, 1999). However, increased gene copy number does not always correlate with increased gene expression (Kim et al, 2011). Thus, the idea that polyploidy primarily increases biosynthetic capacity is probably too simplistic.…”

“…However, Rbmediated regulation of E2f1 is not essential for endoreplication in Drosophila salivary glands, perhaps because of the action of the E2f2-containing Myb-MuvB complex in repressing Cyclin E expression during G phase (Maqbool et al, 2010;Weng et al, 2003). Zielke et al (Zielke et al, 2011) provide data in support of a model whereby E2f1 accumulation during G phase results in activation of the Cyclin E/Cdk2 complex, which triggers S phase, which in turn causes the subsequent inactivation of E2f1 via the action of CRL4…”

“…Recently, mathematical modeling of endoreplication oscillations helped guide experiments demonstrating that cyclic accumulation of the transcription factor E2f1 (E2f -FlyBase) is essential for endoreplication in the highly polyploid Drosophila salivary gland (Zielke et al, 2011). E2F transcription factors are potent stimulators of S-phase entry and control the expression of genes required for DNA synthesis, including Cyclin E. Their activity is regulated by the Rb family of tumor suppressors, which bind to and inhibit E2F proteins during G1 phase (van den Heuvel and Dyson, 2008).…”

Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication

“…S phase) Cyclin E-Cdk2 activity are required for repeated rounds of endocycle S phase (Follette et al, 1998;Weiss et al, 1998). The mechanisms that control oscillations of Cyclin E-Cdk2 activity in the Drosophila endocycle operate at many levels, including those that activate Cyclin E-Cdk2, such as the transcriptional induction of the Cyclin E gene by E2f1 (Duronio and O'Farrell, 1995), and those that inhibit Cyclin E-Cdk2, such as destruction of Cyclin E protein by the SCF Ago E3 ubiquitin ligase (Moberg et al, 2001;Shcherbata et al, 2004;Zielke et al, 2011). Therefore, in order to fully understand the endocycle, all of the mechanisms that contribute to oscillations of Cyclin E-Cdk2 activity must be determined.…”

Direct Role for Proliferating Cell Nuclear Antigen in Substrate Recognition by the E3 Ubiquitin Ligase CRL4Cdt2

“…Cdt2 targets the transcription factor E2F in flies (to shut off G 1 transcription in S phase and to regulate endocycles) (19,20), the translesion DNA polymerase in worms (perhaps to restrict access of this mutagenic polymerase to undamaged DNA) (21), and the ribonucleotide reductase inhibitor Spd1 in fission yeast (to activate nucleotide synthesis in S phase and after DNA damage) (22). Other substrates of CRL4…”