Stabilized dE2F1 can induce apoptosis through transcriptional and non-transcriptional mechanisms.

<p>(1) During S-phase, a dE2F1 protein without a PIP-degron but with an intact DNA–binding domain will promote apoptosis through transcriptional activation of pro-apoptotic genes, such as <i>hid</i>. (2) A dE2F1 protein without a PIP-degron and lacking an intact DNA-binding domain can promote apoptosis through an unknown mechanism that requires a physical interaction with the RBF1 protein; this unknown mechanism is also dependent upon <i>hid</i>.</p

E2F and p53 network between mammals and flies.

<p>In mammalian cells, the activation of E2F1 is sufficient to activate p53. One of the key links between E2F and p53, is provided by p19/p14^ARF and mdm2. <i>Drosophila</i> lack any clear orthologs of p19/p14^ARF and mdm2 and lack of this connection may limit the crosstalk between E2F1 and p53. In the context of DNA damage, where dE2F1 and dp53 converge on a common set of pro-apoptotic target genes, dE2F1 and dp53 co-operate to promote cell death. While DNA damage is shown to activate E2F1 and p53 pathways in mammals, it remains uncertain whether dE2F1 is similarly activated by DNA damage.</p

DNA damage-induced cell death in eye discs is suppressed by the mutation of either <i>dDP</i> or <i>dp53</i>.

<p>Third instar larvae of indicated genotypes were treated with 40 Gy of ionizing irradiation. C3 immunostaining was used to detect apoptotic cells in eye imaginal discs either before (0 h) or 4 h after (4 h) irradiation.</p

Mutation of <i>dp53</i> has no effect on dE2F1/dDP-dependent cell death in <i>rbf1</i> mutant eye discs.

<p>Third instar eye discs of the indicated genotypes were stained with C3 to visualize apoptotic cells. A wave of cell death in <i>rbf1^120a</i> mutant eye discs was eliminated by the homozygous mutation of <i>dDP</i> but not by the homozygous mutation of <i>dp53</i>.</p

<i>hid</i> and <i>reaper</i> are deregulated, and functionally important, for DNA damage-induced apoptosis in <i>rbf1</i> mutant eye discs.

<p>(A) Eye discs from control and <i>rbf1</i> mutant third instar larvae were dissected before and three hours after ionizing irradiation. RNA samples were isolated from each genotype and the expression levels of indicated genes were measured by quantitative real-time PCR. Relative expression levels are shown from three independent experiments, each with triplicate samples. A similar analysis was performed using <i>dDP</i> and <i>dDP;dp53</i> mutant larvae. (B) Effects of <i>reaper</i> and <i>hid</i> mutation in <i>rbf1</i> mutant eye discs are shown. 4 h after irradiation treatment, apoptotic cells in the indicated eye discs were visualized by C3. <i>hid</i> Mutation completely suppresses DNA damage-induced cell death throughout both control and <i>rbf1</i> mutant eye discs. (C) The intensity of C3 staining signals in <i>rbf1</i> mutant and <i>rbf1;rpr</i> mutant eye discs was quantified using the Image J software. The fold change indicates that the <i>reaper</i> mutation results in a more significant reduction of the level of DNA damage-induced cell death posterior to the MF (region 1 indicated in B) compared to the MF region (region 2 indicated in B) in <i>rbf1</i> mutant discs.</p

dCAP-D3 is necessary for a proper immune response to bacterial infection.

<p>Adult female flies expressing dCAP-D3 (red) dsRNAs under the control of <i>yolk-GAL4</i> were infected with the Gram positive bacterium, <i>Staphylococcus aureus</i> (A) or the Gram negative bacterium, <i>Pseudomonas aeruginosa</i> (B). Flies expressing GFP dsRNAs under the control of <i>yolk-GAL4</i> (green) were used as “wild-type” controls. Eater mutants which are defective in phagocytosis (blue) or flies expressing IMD dsRNAs which are compromised in a major innate immune signaling pathway (yellow) were used as positive controls. Results demonstrate that flies expressing reduced levels of dCAP-D3 in the fat body cells are more susceptible to either type of infection than wild type controls. Three independent experiments are depicted with results of each experiment shown as the average of three sets of 10 infected adults per genotype. These experiments were also performed using a sterile needle dipped in PBS to rule out death as a result of wounding and survival curves matched those of yolk-GAL4 expressing flies (data not shown). Results are presented as cox regression models with statistical significance (p≤0.05) represented as shaded areas above and below the curves.</p

RBF1 and dCAP-D3 are necessary for the ability to clear bacteria <i>in vivo</i>.

<p>Adult female flies expressing RBF1 (purple) or dCAP-D3 (red) dsRNAs under the control of <i>yolk-GAL4</i> were infected with the Gram positive bacterium, <i>Staphylococcus aureus</i>. Flies expressing GFP dsRNAs under the control of <i>yolk-GAL4</i> (green) were used as “wild-type” controls. Eater mutants which are defective in phagocytosis (blue) or flies expressing IMD dsRNAs which are compromised in a major innate immune signaling pathway (yellow) were used as positive controls. Results demonstrate that at 15 hours following infection, flies expressing reduced levels of dCAP-D3 or RBF1 in the fat body cells exhibit increased numbers of bacteria in comparison to wild type controls. Three independent experiments are shown and results for each experiment are the average of three sets of three infected adults, per genotype, per timepoint. Asterisks emphasize statistical significance (p≤0.05) as determined by a students paired t-test.</p

RBF1 is necessary for a proper immune response to Gram positive bacterial infection.

<p>Adult female flies expressing RBF1 (purple) dsRNAs under the control of <i>yolk-GAL4</i> were infected with the Gram positive bacterium, <i>Staphylococcus aureus</i> (A) or the Gram negative bacterium, <i>Pseudomonas aeruginosa</i> (B). Flies expressing GFP dsRNAs under the control of <i>yolk-GAL4</i> (green) were used as “wild-type” controls. Eater mutants which are defective in phagocytosis (blue) or flies expressing IMD dsRNAs which are compromised in a major innate immune signaling pathway (yellow) were used as positive controls. Results demonstrate that flies expressing reduced levels of RBF1 in the fat body cells are more susceptible to infection with Gram positive bacteria (A) than wild type controls. Three independent experiments are depicted with results of each experiment shown as the average of three sets of 10 infected adults per genotype. Results are presented as cox regression models with statistical significance (p≤0.05) represented as shaded areas above and below the curves. In the third experiment in (A), which is highlighted by a star, the survival endpoint becomes significant when the confidence level is changed to 90% (p≤0.10) instead of 95% (p≤0.05). These experiments were also performed using a sterile needle dipped in PBS to rule out death as a result of wounding and survival curves matched those of yolk-GAL4 expressing flies (data not shown).</p

Complete AMP induction following bacterial infection depends on dCAP-D3 and RBF1.

<p>Adult female flies expressing RBF1 (purple) or dCAP-D3 (red) dsRNAs under the control of <i>yolk-GAL4</i> were infected with the Gram positive bacteria, <i>Staphylococcus aureus</i>. A) qRT-PCR analyses for transcript levels of the <i>Drosomycin</i> AMP gene in these flies show that while flies expressing GFP dsRNAs under the control of <i>yolk-GAL4</i> (green) undergo a large induction of AMPs at 8–24 hours post-infection, flies expressing dCAP-D3 or RBF1 dsRNA in the fat body fail to exhibit maximal, sustained induction. B) qRT-PCR analyses for transcript levels of the <i>Diptericin</i> AMP gene in these flies show that while flies expressing GFP or RBF1 dsRNAs under the control of <i>yolk-GAL4</i> (green) undergo a large induction of AMPs at 8–24 hours post-infection, flies expressing dCAP-D3 dsRNA in the fat body fail to exhibit maximal, sustained induction. The inset boxes in the upper right corner of each graph are a larger representation of the 0 hour timepoint and depict basal transcription levels. Asterisks emphasize statistical significance (p≤0.05) as determined by a students paired t-test. Three independent experiments are shown and results for each experiment are the average of three sets of five infected adults per genotype, per timepoint.</p

RBF1 and dCAP-D3 regulate many of the same transcripts in the fly.

<p>RNA was isolated from <i>rbf1</i> mutant and <i>dCap-D3</i> mutant female third instar larvae and adult flies. cDNA was hybridized to Nimblegen 385 k whole genome arrays. A) Venn diagrams show the numbers of RBF1, dCAP-D3 or RBF1/dCAP-D3 shared target genes which exhibited at least a 2 fold log change in expression with a p value of ≤0.15. Genes significantly upregulated in the mutant flies are shown in red while genes significantly downregulated are shown in green. B) P values for shared RBF1 and dCAP-D3 target genes indicate that RBF1 and dCAP-D3 regulate a significant number of the same genes in both adults and larvae. The numbers above the diagonal represent p-values for upregulated shared subsets and are colored red while the numbers below the diagonal represent p-values for downregulated shared genes and are colored green. C) qRT-PCR analyses of 12 E2F targets shows that the majority of RBF1/dCAP-D3 shared targets are not E2F targets. The one target that was significantly upregulated in dCAP-D3 and RBF1 mutant flies, <i>CG5250</i>, is highlighted in red. Results are the average of three independent experiments involving 10 female flies per genotype. D) Significant (p≤0.05) Gene Ontology (GO) groupings for shared target genes include defense response genes in the adult fly. The top box lists GO categories for upregulated shared genes in mutant larvae only, and the bottom box lists selected GO categories for downregulated shared genes in adults only. There were no significant GO groupings for upregulated shared target genes in adults or for downregulated shared target genes in the larvae.</p