Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.

The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse genetic and functional genomic approaches. Proteomic analyses of DNA damage-induced apoptosis have not been shown because of a limited number of cells undergoing apoptosis. We applied mass spectrometry-based quantitative proteomics to evaluate protein changes induced by ionizing radiation (IR) in isolated <i>C. elegans</i> germlines. For this purpose, we used isobaric peptide termini labeling (IPTL) combined with the data analysis tool IsobariQ, which utilizes MS/MS spectra for relative quantification of peak pairs formed during fragmentation. Using stringent statistical critera, we identified 48 proteins to be significantly up- or down-regulated, most of which are part of a highly interconnected protein–protein interaction network dominated by proteins involved in translational control. RNA-mediated depletion of a selection of the IR-regulated proteins revealed that the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel negative regulator of DNA-damage induced apoptosis. Finally, a central role of nucleolar proteins in orchestrating these responses was confirmed as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis in the <i>C. elegans</i> germline

Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.

The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse genetic and functional genomic approaches. Proteomic analyses of DNA damage-induced apoptosis have not been shown because of a limited number of cells undergoing apoptosis. We applied mass spectrometry-based quantitative proteomics to evaluate protein changes induced by ionizing radiation (IR) in isolated <i>C. elegans</i> germlines. For this purpose, we used isobaric peptide termini labeling (IPTL) combined with the data analysis tool IsobariQ, which utilizes MS/MS spectra for relative quantification of peak pairs formed during fragmentation. Using stringent statistical critera, we identified 48 proteins to be significantly up- or down-regulated, most of which are part of a highly interconnected protein–protein interaction network dominated by proteins involved in translational control. RNA-mediated depletion of a selection of the IR-regulated proteins revealed that the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel negative regulator of DNA-damage induced apoptosis. Finally, a central role of nucleolar proteins in orchestrating these responses was confirmed as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis in the <i>C. elegans</i> germline

Quantitative Proteome Analysis Reveals RNA Processing Factors As Modulators of Ionizing Radiation-Induced Apoptosis in the <i>C. elegans</i> Germline.

The nematode <i>Caenorhabditis elegans</i> is an organism most recognized for forward and reverse genetic and functional genomic approaches. Proteomic analyses of DNA damage-induced apoptosis have not been shown because of a limited number of cells undergoing apoptosis. We applied mass spectrometry-based quantitative proteomics to evaluate protein changes induced by ionizing radiation (IR) in isolated <i>C. elegans</i> germlines. For this purpose, we used isobaric peptide termini labeling (IPTL) combined with the data analysis tool IsobariQ, which utilizes MS/MS spectra for relative quantification of peak pairs formed during fragmentation. Using stringent statistical critera, we identified 48 proteins to be significantly up- or down-regulated, most of which are part of a highly interconnected protein–protein interaction network dominated by proteins involved in translational control. RNA-mediated depletion of a selection of the IR-regulated proteins revealed that the conserved CAR-1/CGH-1/CEY-3 germline RNP complex acts as a novel negative regulator of DNA-damage induced apoptosis. Finally, a central role of nucleolar proteins in orchestrating these responses was confirmed as the H/ACA snRNP protein GAR-1 was required for IR-induced apoptosis in the <i>C. elegans</i> germline

VPA-synthetic lethal or -sensitizer genes identified from the chromatin library screen in <i>C. elegans</i>.

*<p>No corresponding human homolog.</p

Conserved pathways contribute to VPA resistance.

<p><b>A</b>) The human homologs/orthologs of data from all screens were combined in an <i>in silico</i> approach to extract and predict common functionalities and components. Proteins identified in our datasets (<i>red</i>), as well as direct interactors and indirect interactors (<i>black</i>) mediated via one neighbor extracted by the FunCoup browser, were imported into Cytoscape which returned five clusters of enriched process which were manually grouped (grey shade) to illustrate that “APC-dependent proteasomal ubiquitin-dependent protein degradation” and “Response to unfolded protein” are the major conserved processes responding to VPA. In addition, “Regulation of TGFβ receptor signaling” and “Transport” are also functionally important in providing resistance to VPA. The scale depicts color representation of significance by Benjamini-Hochberg correction, where white nodes are not significant, yellow <i>p</i><0.05 and orange <i>p</i><7×10⁻⁷. <b>B</b>) Protein interaction network reveal conserved hubs that promote VPA resistance. Data from all screens were combined <i>in silico</i> and the functional interaction network among the common proteins (diamonds) were extracted using FunCoup. ACTB, HSC70, and TUBA1B (<i>red</i>) were in the primary list whereas MAPKAPK2, HSP90AA2 and HSP90AB1 (<i>black</i>) were identified in all approaches as interactors. ACTB, HSC70, HSP90AA2, HSP90AB1 and TUBA1B represent evolutionary conserved nodes providing resistance to VPA. <b>C</b>) VPA (1 mM) was combined with inhibitors of tubulin (vincristine (VCR), 1 nM) or HSP90 (geldanamycin (GA), 5 nM) in MOLM-13 AML cells and investigated for effects on apoptosis measured by Hoechst staining after 48 hours of treatment. Arrows indicate fragmented and condensed nuclei. Scale bar = 10 µm. Combinations of 1 mM VPA and 1 nM vincristine (<b>D</b>), 2 mM VPA and inhibitor of actin polymerization cytochalasin B (<b>E</b>), 1 mM VPA and 5 nM geldanamycin (<b>F</b>), and 0.2 mM SAHA and 5 nM geldanamycin (<b>G</b>) all show statistically significant synergism of drug interaction, two-way ANOVA, * <i>p</i><0.05, ** <i>p</i><0.001, *** <i>p</i><0.0001. Error bars represent standard error of mean (SEM).</p

Valproic acid-modulated phosphoproteins from BNML rat leukemia progressive disease.

<p>Positive values of ratios between VPA-treated and control animals indicate proteins with elevated expressed level, negative values are proteins with reduced expression in VPA treated animals.</p>*<p>RNAi was not performed.</p>**<p>Synthetic lethality could not be assessed because of severe developmental arrest by RNAi alone.</p

UTX-1 is required for VPA action in <i>C. elegans</i> embryos and in human cells.

<p><b>A</b>) Two <i>UTX</i> wild type cell lines (MV4-11 and NB4) as well as the <i>UTX</i> mutant cell line THP-1 were treated with 1 mM VPA for 48 hours and analyzed for H3K27me3 and H2BK120ac expression. The mean intensity on one representative Western blot was calculated and normalized to beta-actin. The numbers shown are in arbitrary units compared to MV4-11 control. Blots show the increase of H3K27 trimethylation as well as an increase in the degree of H2BK120 acetylation except THP-1, where the level of methylation is unchanged and level of acetylation is decreased. <b>B</b>) AML cell lines with mutated <i>UTX</i> are resistant to VPA. MV4-11 and NB4 as well as two <i>UTX</i> mutant cell lines (MONO-MAC-1 and THP-1) were subjected to 2 mM VPA for 48 hours and scored for abnormal nuclei by Hoechst staining. * indicates t-test versus MV4-11, x indicates versus NB4. ***/xxx <i>p</i><0.0004. Error bars represent SEM. <b>C</b>) The mutant cell lines show decreased apoptosis, determined by Hoechst staining compared to the wild type cells. Arrows indicate fragmented and condensed nuclei. Scale bar = 10 µm. <b>D</b>) MV4-11 or NB4 cells were subjected to 600 nM UTX siRNA or negative control siRNA for 18 hours prior to 48 hours treatment with 2 mM VPA. Cells were scored for abnormal nuclei by Hoechst staining, showing UTX siRNA to reduce the effect of VPA on cell death. Values are normalized against untreated cells. ** indicates t-test of UTX siRNA versus negative control siRNA, <i>p</i> = 0.014. Error bars represent SEM. <b>E</b>) Western blot of MV4-11 or NB4 cells treated with 600 nM negative control siRNA or UTX siRNA for 24 hours. Numbers shown are arbitrary units compared to negative control siRNA. The blot shows a 60% decrease in UTX by siRNA treatment, confirming the efficacy of transfection. <b>F</b>) The <i>C. elegans</i> strain AZ212 was fed the empty vector L4440 or <i>utx-1</i> (RNAi), and the <i>utx-1(3136)</i> mutant was fed <i>E. coli</i> OP50 and exposed to 15 mM VPA at L4 larval stage for 24 hours at 20°C. The embryos were immunostained using antibodies recognizing H4K8ac. In the 100-cell stage embryos, baseline levels of this activating acetylation mark were observed in untreated control worms, whereas the <i>utx-1</i> (RNAi) and <i>utx-1(3136)</i> mutant worms showed highly increased acetylation levels (100% and 76% acetylation, respectively). Error bars represent SEM. <b>G</b>) Worms were treated as in F) and stained with a H3K36me2 antibody. At the 100-cell stage, control embryos showed 73% methylation while both <i>utx-1</i> (RNAi) and the <i>utx-1(3136)</i> mutant showed baseline methylation. By VPA treatment, control worms show baseline methylation whereas both <i>utx-1</i> (RNAi) and <i>utx-1(3136)</i> mutant shows 86% and 67% methylation respectively. Error bars represent SEM.</p

Histone methylation capacity affects basal histone acetylation in <i>C. elegans</i> embryos.

<p>VPA-treatment induced global acetylation in 100-cell stage <i>C. elegans</i> embryos. The strain AZ212, expressing GFP in fusion with H2B, was fed the empty vector L4440 (RNAi), the VPA-sensitizers <i>lex-1</i> (RNAi), <i>utx-1</i> (RNAi) (histone demethylase), and <i>set-12</i> (RNAi) (histone methyltransferase), and exposed to 15 mM VPA at L4 larval stage for 24 hours at 20°C. The embryos were fixed with acetone and methanol prior to staining with an Acetyl-Histone H4 (Lys8) antibody. At the 100-cell stage, baseline levels of acetylation were seen in untreated control worms. Global hyperacetylation was observed after treatment with VPA by all. Depletion of <i>lex-1</i>, <i>utx-1</i> and <i>set-12</i> gives increased baseline acetylation in the absence of VPA. Scale bar = 10 µm.</p

Survival of leukemic BN rats without and with valproic acid treatment.

<p><b>A</b>) BN rats were injected with 10×10⁶ BNML cells on day 0 before treatment started on day 10. Animals were treated with vehicle or VPA intra peritoneal (400 mg/kg) for 5 days, with 2 days off, in total for four weeks. Animals did not display symptoms for leukemia until termination of treatment, representing a responsive VPA-model. <b>B</b>) BN rats were injected with 5×10⁶ BNML cells on day 0 before treatment (170 mg/kg VPA, per orally) started at day 16. Animals were treated successively with 170 mg/kg VPA <i>b.i.d.</i> from day 17. Animals treated with VPA showed significantly increased survival compared with control rats (median survival increased from 30 to 32.5 days, <i>p</i> = 0.0037). However, the disease progressed and animals displayed high leukemic burden upon sacrifice at humane endpoint, representing a VPA-resistant model. Red marks represent days of treatment, grey marks represent no treatment.</p

Gene expression analysis, phosphoproteomics and <i>C. elegans</i> chemical-genetic screen identify conserved responses to valproic acid.

<p><b>A</b>) Human primary AML blasts were treated with 600 µM valproic acid (VPA), resulting in the discrimination of responsive and non-responsive cells to VPA by gene array expression studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048992#pone.0048992-Stapnes1" target="_blank">[14]</a>. <b>B</b>) Leukemic BNML rats were treated with vehicle or VPA (170 mg/kg <i>b.i.d.</i>). Phosphorylated proteins were collected from leukemic blasts from the spleen <i>post mortem</i> and separated by DIGE. Differentially represented phosphoproteins in animals treated with VPA were identified by Orbitrap mass spectrometry. <b>C</b>) The Ahringer chromatin-associated gene library was combined with 15 mM VPA for 48 hours and screened for synthetic lethality defined by developmental arrest. <b>A+B+C</b>) Functional validation of targets from all three screens (A, B and C) by combining RNAi with VPA (15 mM) in <i>C. elegans.</i> The effect on acetylation for chosen targets was investigated by immunofluorescense in <i>C. elegans</i> embryos (lower panel).</p