Functional analysis of the cell cycle regulator Rca1 in Drosophila melanogaster

Tight regulation of APC/C activity is essential for cell cycle progression. An important class of negative APC/C regulators are the Rca1/Emi1 family proteins. All members of the Rca1/Emi1 family share a conserved zinc binding region (ZBR) which is essential for their inhibitory activity. The Rca1/Emi1 proteins belong to the class of F-box proteins that are known to act as substrate recognition subunits in SCF-E3-ligase complexes. Emi1 and Rca1 interact in vitro with members of the Skp family via the F-box. However, no F-box dependent function has been ascribed to these proteins. In Drosophila, Rca1 is required in G2 to prevent premature activation of the APC/C by Fzr. Loss of Rca1 results in an arrest during G2 of the 16th embryonic cell cycle due to premature cyclin degradation. In order to map the essential domains for Rca1 function, a series of deletion constructs was tested for their ability to inhibit APC/C-Fzr activity in vivo. A C-terminal Rca1 fragment including the ZBR was sufficient to restore mitosis 16 in rca1 mutant embryos. This observation confirms that the ZBR is the only protein motif essential for APC/C-Fzr inhibition by Emi1/Rca1. Moreover, this result indicates that the F-box is dispensable for APC/C-Fzr inhibition during embryogenesis. However, analysis of Rca1 function during larval development revealed that Rca1 has a secondary role as an F-box protein. Using the MARCM technique, wing disc cells were generated in which endogenous Rca1 was replaced by an Rca1 construct lacking the F-box. These cells displayed a reduced proliferation rate and prolonged G1-phase. Conversely, overexpression of Rca1 accelerates the G1-S transition in imaginal discs in an F-box dependent manner. Hence, it is likely that Rca1 regulates S-phase entry as part of a yet uncharacterized SCF-complex. In addition, the effect of Rca1 on endoreplication was analyzed. Overexpression of Rca1 during salivary gland development leads to a reduction of polyploidization. This phenotype also depends on a functional F-box. Endoreplication cycles are driven by oscillating waves of Cyclin E/Cdk2 activity, whereas Cdk1 and the mitotic cyclins are transcriptionally downregulated. Furthermore, APC/C-Fzr activity seems not to be required once the endoreplication program has been initiated. Cells overexpressing Rca1 displayed elevated levels of Cyclin E, although Cyclin E is not a target of the APC/C-Fzr complex. It has been shown that continuous expression of Cyclin E interferes with DNA-licensing. Thus, the reduced DNA content in Rca1 overexpressing cells might be due to elevated Cyclin E levels. Additionally, Rca1 overexpressing cells displayed markers for mitotic cells such as Cdk1 and nuclear Cyclin A. The accumulation of Cdk1, Cyclin A and Cyclin E cannot simply be explained by APC/C inhibition. It rather appears that Rca1 activates the transcription of these genes by an unknown mechanism. Nevertheless it cannot be excluded that the APC/C-Fzr complex indirectly contributes to this process. Altogether, Rca1 might act as an F-box protein in an SCF complex that is involved in maintaining diploidy

Upregulation of ribosome biogenesis via canonical E-boxes is required for Myc-driven proliferation

The transcription factor Myc drives cell growth across animal phyla and is activated in most forms of human cancer. However, it is unclear which Myc target genes need to be regulated to induce growth and whether multiple targets act additively or if induction of each target is individually necessary. Here, we identified Myc target genes whose regulation is conserved between humans and flies and deleted Myc-binding sites (E-boxes) in the promoters of fourteen of these genes in Drosophila. E-box mutants of essential genes were homozygous viable, indicating that the E-boxes are not required for basal expression. Eight E-box mutations led to Myc-like phenotypes; the strongest mutant, ppan(Ebox-/-), also made the flies resistant to Myc-induced cell growth without affecting Myc-induced apoptosis. The ppan(Ebox-/-) flies are healthy and display only a minor developmental delay, suggesting that it may be possible to treat or prevent tumorigenesis by targeting individual downstream targets of Myc.Peer reviewe

Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development

The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.Peer reviewe

Translational control of E2f1 regulates the Drosophila cell cycle

E2F transcription factors are master regulators of the eukaryotic cell cycle. In Drosophila, the sole activating E2F, E2F1, is both required for and sufficient to promote G1 -> S progression. E2F1 activity is regulated both by binding to RB Family repressors and by posttranscriptional control of E2F1 protein levels by the EGFR and TOR signaling pathways. Here, we investigate cis-regulatory elements in the E2f1 messenger RNA (mRNA) that enable E2f1 translation to respond to these signals and promote mitotic proliferation of wing imaginal disc and intestinal stem cells. We show that small upstream open reading frames (uORFs) in the 5' untranslated region (UTR) of the E2f1 mRNA limit its translation, impacting rates of cell proliferation. E2f1 transgenes lacking these 5'UTR uORFs caused TOR-independent expression and excess cell proliferation, suggesting that TOR activity can bypass uORF-mediated translational repression. EGFR signaling also enhanced translation but through a mechanism less dependent on 50'TR uORFs. Further, we mapped a region in the E2f1 mRNA that contains a translational enhancer, which may also be targeted by TOR signaling. This study reveals translational control mechanisms through which growth signaling regulates cell cycle progression.Peer reviewe

Novel genetic loci underlying human intracranial volume identified through genome-wide association

Intracranial volume reflects the maximally attained brain size during development, and remains stable with loss of tissue in late life. It is highly heritable, but the underlying genes remain largely undetermined. In a genome-wide association study of 32,438 adults, we discovered five novel loci for intracranial volume and confirmed two known signals. Four of the loci are also associated with adult human stature, but these remained associated with intracranial volume after adjusting for height. We found a high genetic correlation with child head circumference (ρgenetic=0.748), which indicated a similar genetic background and allowed for the identification of four additional loci through meta-analysis (Ncombined = 37,345). Variants for intracranial volume were also related to childhood and adult cognitive function, Parkinson’s disease, and enriched near genes involved in growth pathways including PI3K–AKT signaling. These findings identify biological underpinnings of intracranial volume and provide genetic support for theories on brain reserve and brain overgrowth

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

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp–Cullin–F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C–Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1–S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1–S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry

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 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 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 CRL4CDT2 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 plants1,2,8, indicating that elements of this mechanism act in most growth-dependent cell cycles