Control of Drosophila endocycles by E2F and CRL4(CDT2).

Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. 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 biomass. 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-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4(CDT2) ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). 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 overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling 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 plants, indicating that elements of this mechanism act in most growth-dependent cell cycles.

Analysis of novel alleles of brother of tout-velu, the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome.

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovoD dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w + mW.hs ), or rosy (ry+ ) and neomycin (neoR ) transgenes are in common use. Parallel ovoD lines were developed using w + mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as ry+ , neoR FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry+ , neoR FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w + mW.hs FRT. Here we report the creation of a new ovoD line for the ry+ , neoR FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.

The anaphase-promoting complex/cyclosome (APC/C) is required for rereplication control in endoreplication cycles.

Endoreplicating cells undergo multiple rounds of DNA replication leading to polyploidy or polyteny. Oscillation of Cyclin E (CycE)-dependent kinase activity is the main driving force in Drosophila endocycles. High levels of CycE-Cdk2 activity trigger S phase, while down-regulation of CycE-Cdk2 activity is crucial to allow licensing of replication origins. In mitotic cells relicensing in S phase is prevented by Geminin. Here we show that Geminin protein oscillates in endoreplicating salivary glands of Drosophila. Geminin levels are high in S phase, but drop once DNA replication has been completed. DNA licensing is coupled to mitosis through the action of the anaphase-promoting complex/cyclosome (APC/C). We demonstrate that, even though endoreplicating cells never enter mitosis, APC/C activity is required in endoreplicating cells to mediate Geminin oscillation. Down-regulation of APC/C activity results in stabilization of Geminin protein and blocks endocycle progression. Geminin is only abundant in cells with high CycE-Cdk2 activity, suggesting that APC/C-Fzr activity is periodically inhibited by CycE-Cdk2, to prevent relicensing in S-phase cells.

Overgrowth caused by misexpression of a microRNA with dispensable wild-type function.

MicroRNAs (miRNAs) represent an abundant class of non-coding RNAs that negatively regulate gene expression, primarily at the post-transcriptional level. miRNA genes are frequently located in proximity to fragile chromosomal sites associated with cancers and amplification of a miRNA cluster has been correlated with the etiology of lymphomas and solid tumors. The oncogenic potential of a miRNA polycistron has recently been demonstrated in vivo. Here, we show that misexpression of the Drosophila miRNA mirvana/mir-278 in the developing eye causes massive overgrowth, in part due to inhibition of apoptosis. A single base substitution affecting the mature miRNA blocks the gain-of-function phenotype but is not associated with a detectable reduction-of-function phenotype when homozygous. This result demonstrates that misexpressed miRNAs may acquire novel functions that cause unscheduled proliferation in vivo and thus exemplifies the potential of miRNAs to promote tumor formation.