Fs(1)h negatively regulates CncC signaling.

<p>(A) The <i>Drosophila fs(1)h</i> gene encodes two protein isoforms: the 120kD Fs(1)h-S and the 210kD Fs(1)h-L. Both isoforms contain two bromodomains (BD) and an extraterminal (ET) domain. In addition, Fs(1)h-L carries a unique C-terminal motif (CTM). (B) dsRNA-mediated knock down of Fs(1)h (3.6 fold, P<0.05), like Keap1 knock down (3.9 fold, P<0.01), increases the activity of a transiently transfected ARE-fluc reporter in S2 cells. In both cases, this stimulatory effect is suppressed by CncC knock down. (C) RT-qPCR experiments show that the CncC target genes <i>gstD1</i> (P<0.0001), <i>gclC</i> (P<0.001) and <i>keap1</i> (P<0.05) are activated upon RU486-induced-knock down of Fs(1)h under the control of tub-GS-Gal4 driver. Measurements of transcript abundance levels were normalized to <i>act5c</i> transcript levels. Fold activation relative to the mRNA levels in mock treated flies is shown. The error bars indicate standard deviation of 3 biological replicates (flies collected from separate vials). (D) Knock down of Fs(1)h-L alone using a specific dsRNA targeting the CTM region was sufficient to induce ARE-fluc activity in S2 cells (3 fold, P<0.01). However, knock down of Fs(1)h-S alone with dsRNA targeting its 3’ UTR did not induce ARE-fluc activity. Error bars in panels B, C and D signify standard deviation of 3 biological replicates.</p

Simultaneous inhibition of Keap1 and BET proteins impart enhanced oxidative stress tolerance.

<p>(A) 5 day old female <i>w</i>^<i>1118</i> flies were placed on food containing 0.4mM oltipraz and/or 0.1mM JQ1 for 4 days and then were exposed to 20μM DEM. Survivorship was assessed. Mantel-Cox log-rank test showed that pre-treatment with either oltipraz or JQ1 significantly increased oxidative stress tolerance (P value <0.005 for control/oltipraz comparison and P value <0.001 for control/JQ1 comparison). It was also found that pre-treatment with both oltipraz and JQ1 extended survival after DEM exposure significantly more than pre-treatment with either drug alone (P value <0.001 for olt/combined comparison and P value <0.005 for JQ1/combined comparison). Qualitatively identical results were found when males were used (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s003" target="_blank">S3D Fig</a>). (B) Proposed model for synergistic activation of Nrf2 signaling by oltipraz and JQ1. CncC is regulated independently by Keap1 and Fs(1)h. Treatment of cells with the Keap1 inhibitor oltipraz relieves a different mechanism of CncC inhibition from that relieved by the BET protein inhibitor JQ1. Combining both manipulations, therefore, has a stronger effect than either treatment by itself and causes a cooperative activation of CncC target genes.</p

Bromodomains mediate the inhibition of CncC by Fs(1)h.

<p>(A) S2 cells transfected with the ARE-fluc reporter plasmid were treated with 1μM JQ1 for 24 hrs, which stimulated ARE-fluc activity (6.5 fold, P<0.001). This increase was strongly diminished after knock down of CncC (JQ1 mediated induction is 3.2 fold, P<0.001) or MafS (JQ1 mediated induction is 4.2 fold, P<0.002). Combined knock down of MafS and CncC almost completely eliminates the JQ1 effect (JQ1 mediated induction is 1.6 fold, P>0.05). The diagram shows the fold change in luciferase activity relative to controls. Error bars indicate standard deviation of 3 biological replicates. (B) 5 day old adult ARE-GFP flies were maintained on food containing 0.25 mM JQ1 for 2 days. Fluorescence images of adult flies showed a strong induction of ARE-GFP reporter activity by JQ1. Two randomly chosen JQ1-treated and two solvent-treated female flies are shown. (C) Co-immuno-precipitation of endogenous Fs(1)h-L with over-expressed CncC-Flag in S2 cells. S2 cells were transfected with either actin-Gal4 plasmid alone (lane 1) or with actin-Gal4 and UAS-CncC-Flag plasmids (lanes 2–5). The cells over-expressing CncC-Flag were treated either with the HDAC inhibitor LBH589 (500nM) and/or JQ1 (10μM) as indicated, or with 0.01% DMSO (vehicle) for 6 hours before they were processed for immuno-precipitation. 10μM JQ1 was also added to the lysate from cells treated with JQ1 to assess the effect of JQ1 on Fs(1)h-CncC interaction. Immuno-precipitation was performed using anti-Flag antibody followed by immuno-blotting with anti-Fs(1)h-L antibody. The acetylation status of CncC in the same immuno-precipitates was examined in western blots using an antibody against acetylated lysine.</p

Fs(1)h-L cell-autonomously inhibits CncC activity and affects oxidative stress resistance in adult flies.

<p>(A) Ubiquitous knock down of Fs(1)h (in the v51227 RNAi line) in adult flies, using the RU486-inducible tub-GS-Gal4 driver stimulates ARE-GFP reporter activity in most tissues. Two RU486-treated and two mock-treated females are shown in this panel and in panel C. The same flies are shown under UV-illumination to visualize GFP fluorescence and under white light. (B) Knock down of Fs(1)h in actin-flipout-Gal4 clones increased ARE-GFP activity in a cell-autonomous manner in the crop of adult <i>Drosophila</i> gut. In the cells in which Fs(1)h expression is knocked down (marked by the expression of RFP, red) ARE-GFP reporter activity (green) is increased. (C) RU486-induced over-expression of Fs(1)h from the EP-fs(1)h allele reduces oltipraz-stimulated ARE-GFP reporter activity in the whole body. Similar effects are seen after ubiquitous over-expression of Fs(1)h-L from a UAS construct (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s002" target="_blank">S2 Fig</a>). (D) Fs(1)h-L over-expression in actin-flipout-Gal4 clones (labeled by RFP, red), cell- autonomously reduces ARE-GFP activity in the ejaculatory bulb of adult males. (E) Ubiquitous Fs(1)h knock-down in adult <i>Drosophila</i> by inducible expression of a UAS-Fs(1)h^RNAi transgene under the control of the tub-GS-Gal4 driver increases oxidative stress resistance. Survival after exposure to 20μM DEM was recorded and the data were analyzed by Mantel-Cox log-rank test. Female flies incubated on RU486 food, showed significantly increased resistance to DEM (P value <0.001) compared to those on control food. A similar effect was observed in males (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s003" target="_blank">S3 Fig</a>). The standard deviations of percent survival among biological replicates in this and other stress sensitivity assays are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s006" target="_blank">S1 Document</a>. (F) Fs(1)h over-expression increases stress sensitivity. Fs(1)h was over-expressed from the EP-fs(1)h allele in female flies by exposing them to food containing 300μM RU486 for 4 days. Lethality after exposure to 20μM DEM was recorded and analyzed by Mantel-Cox log-rank test. The flies that were kept on RU486 food, showed significantly increased sensitivity to DEM (P value < 0.0001) compared to those on control food. The same experiment was also conducted with males and produced qualitatively the same result (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.s003" target="_blank">S3 Fig</a>).</p

Stimulation of CncC target gene expression is independent of <i>de novo</i> protein synthesis.

<p>(A) S2 cells, transfected with the ARE fluc reporter were pre-treated with cycloheximide (CHX) or mock-treated for 4 hours and then exposed to 1 μM JQ1 or DMSO as a solvent control for 8 hrs. as indicated in the figure. In the absence of CHX luciferase activity was easily detectable and was strongly responsive to JQ1 treatment. No luciferase activity was detectable in CHX-treated cells, both in basal and JQ1-treated conditions. The absence of luciferase indicates that inhibition of <i>de novo</i> protein synthesis in this experiment is efficient. The averages of ARE-luciferase activity in biological replicates are shown here. (B and C) The expression of two CncC-regulated mRNAs (<i>gstD1</i> and <i>keap1</i>) remained JQ1 inducible in in the absence (P<0.05 and P<0.01 respectively for <i>gstD1</i> and <i>keap1</i>) and the presence of CHX (P<0.01 and P<0.02 respectively for <i>gstD1</i> and <i>keap1</i>). Note that <i>gstD1</i> expression, like that of many other stress-inducible genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.ref035" target="_blank">35</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006072#pgen.1006072.ref036" target="_blank">36</a>] increases in the presence of CHX. Nevertheless, the JQ1 response remains robust under CHX treatment also in this case. The mRNA levels were normalized to <i>actin5C</i> transcript levels. The error bars indicate standard deviation of 3 biological replicates.</p

Knock-down of the screened splicing factors does not lead to a major decrease in overall splicing efficiency.

<p>(A) Following knock-down of a selected set of identified splicing factors, the levels of unspliced pre-mRNA were determined with amplicons that contained the intron-exon junction, the spliced messages were specifically amplified with amplicons where one primer spanned the exon-exon junction. An amplicon confined within an exon was used to normalize to the total amount of transcript produced at the locus. The values shown are the mean ± SD of three biological replicates.No increase of pre-mRNA levels was detected at any of the three introns in our CG15098 model locus and the levels of spliced transcript were comparable with the total transcript levels. (B) For the second intron of the <i>tsr</i> gene, we detected an increase of unspliced pre-mRNA after knock-down of <i>l(1)10Bb</i>. Nonetheless, the levels of spliced message were comparable to the total transcript levels, indicating that even in this case the majority of transcripts are still spliced. * <i>p</i> = 0.023, Student’s t-test (two-sided, n = 3 biological replicates) (C) Induction of a downstream DNA break slows CG15098 transcript maturation. We induced cleavage at the CG15098 lcous or in the TCTP gene as a control, isolated total RNA and performed qPCR analysis with random primed cDNA. The nascent RNA levels are much lower than those of mature CG15098 (see part A), but calculating the ratio of cut vs. uncut samples displays only the DNA-break induced changes. We detected a ~1.6-fold increase of nascent RNA directly upstream of the break (p<0.01, student’s t-test, 3 biological replicates) and there may be a trend towards slightly increased levels of nascent RNA further upstream at the second intron. We did not observe any changes in the level of mature CG15098 mRNA, which was to be expected given its far greater abundance and presumably slower turnover.</p

Mei-P26 is expressed in the embryonic and larval PGCs.

<p>(A–E) Immunofluorescence staining of embryos (A–C), of a third stage larval testis (D), and of a third stage larval ovary (E). Vasa staining (red) marks the germ cells, Mei-P26 staining (green) indicates the localization of Mei-P26 and Hts staining (blue) labels the fusomes in (D) or the spectrosomes in (E). (A–C) Immunofluorescence images of wild-type embryos. Throughout embryogenesis, Mei-P26 is detectable after formation of the pole cells in the germ line. (A) Lateral view of an embryo at stage 4. (B) Dorsal view of an embryo at stage 11. (C) Lateral view of an embryo at stage 13. Scale bars represent 50 µm. (D) Apical tip of a testis from a third-stage larva. Asterisk indicates the hub cells. Mei-P26 is weekly expressed in the GSCs (arrow) and accumulates in the in the nuclei of differentiating spermatocytes (arrowheads). Scale bar represents 20 µm. (E) Ovary of a wild-type third-stage larva. Mei-P26 accumulates in the germ cells. Anterior is at the top, scale bar represents 20 µm.</p

Splicing stimulates siRNA formation at <i>Drosophila</i> DNA double-strand breaks

<div><p>DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured <i>Drosophila</i> cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-<i>cas9</i> followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of <i>Drosophila melanogaster</i>. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks.</p></div

Model for the sequence of events leading to DNA DSB-induced siRNA formation in <i>Drosophila</i>.

<p>We propose that when the RNA polymerase reaches the DNA double-strand break, the co-transcriptional splicing process is adversely affected. This leads to a signaling event that emanates from the pre-catalytically stalled spliceosome, potentially augmented by formation of an R-loop, or a remodeling/modification of the RNA polymerase complex to enable a “U-turn” move and the synthesis of a long RNA hairpin. In both cases, double-stranded RNA is eventually produced when the normal mRNA transcript base-pairs with the break-induced antisense transcript. This double-stranded RNA is then converted into siRNAs by Dcr-2 and loaded into Ago2.</p

<i>Feo</i> is required for mitosis of larval germ cells.

<p>(A–D) Immunofluorescence images of third stage larval ovaries. Ovaries of larvae injected with <i>feo</i> dsRNA are rudimentary and contain fewer but larger germ cells than the wild type suggesting that the PGCs were unable to undergo mitotic divisions. All ovaries are shown with anterior to the top. Scale bar represents 20 µm. Vasa staining labels the germ cells (red), Tj staining labels the somatic intermingled cells (blue), Fas3 staining labels the anterior somatic cells in (A,B), Hts staining labels the germ-cell specific spherical spectrosomes in (C,D). (E,F) Immunofluorescence images of larval ovaries. (E) Wild-type ovary. (B) Expression of <i>feo</i>-shRNA in the germ line driven by the <i>nos-Gal4-VP16</i> driver induces PGCs with multiple centrosomes. Vasa staining labels the germ cells (red), γ-Tubulin staining labels the centrosomes (green), DAPI marks the nuclei (blue). Arrows indicate the centrosomes. Scale bar represents 20 µm. (G–I) Immunofluorescence images of first-stage larval testes. (G) Wild-type control testis. (H,I) Testes of a larva treated with <i>feo</i> dsRNA (G) and a <i>feo^EA86/Y</i> mutant (I) contain few, abnormally enlarged germ cells. Vasa staining labels the germ cells (red), Tj staining labels the somatic intermingled cells (blue) and Fas3 labels the hub cells (green). Scale bar represents 10 µm.</p