Wnt/Frizzled Signaling Requires dPRR, the Drosophila Homolog of the Prorenin Receptor

SummaryWnt/Wg signaling pathways are of key importance during development and disease [1–4]. Canonical and noncanonical Wnt/Frizzled (Fz) pathways share a limited number of signaling components that are part of the membrane proximal signaling complex. In Drosophila, Fz [5–7] and Dishevelled (Dsh) [8, 9] are the only two components known to be involved in both Wnt/β-catenin and planar cell polarity (PCP) signaling. PCP signaling is required for the planar polarization of epithelial cells [10, 11], which occurs, for instance, during hair orientation and gastrulation in vertebrates [12]. Both pathways have been studied intensively in the past years. However, it still remains unresolved whether additional components are required at the receptor complex. Here we identify the Drosophila homolog of the mammalian prorenin receptor (dPRR) as a conserved modulator of canonical Wnt/β-cat and Fz/PCP signaling. We show that dPRR depletion affects Wg target genes in cultured cells and in vivo. PRR is required for epithelial planar polarity in Drosophila and for convergent extension movements in Xenopus gastrulae. Furthermore, dPRR binds to Fz and Fz2 receptors. In summary, our data suggest that dPRR has an evolutionarily conserved role at the receptor level for activation of canonical and noncanonical Wnt/Fz signaling pathways

Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation

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

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

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