EGO-1, a Putative RNA-Dependent RNA Polymerase, Is Required for Heterochromatin Assembly on Unpaired DNA during C. elegans Meiosis

During meiosis in C. elegans, unpaired chromosomes and chromosomal regions accumulate high levels of histone H3 lysine 9 dimethylation (H3K9me2), a modification associated with facultative heterochromatin assembly and the resulting transcriptional silencing [1, 2]. Meiotic silencing of unpaired DNA may be a widely conserved genome defense mechanism [3–5]. The mechanisms of meiotic silencing remain unclear, although both transcriptional and posttranscriptional processes are implicated [3–5]. Cellular RNA-dependent RNA polymerases (RdRPs) function in development and RNA-mediated silencing in many species [3, 6, 7] and in heterochromatin assembly in S. pombe [3, 8]. There are four C. elegans RdRPs, including two with known germline functions. EGO-1 is required for fertility and robust germline RNAi [9–11]. RRF-3 acts genetically to repress RNAi and is required for normal meiosis and spermatogenesis at elevated temperatures [12] (S. L’Hernault, personal communication). Among C. elegans RdRPs, we find that only EGO-1 is required for H3K9me2 enrichment on unpaired chromosomal regions during meiosis. This H3K9me2 enrichment does not require Dicer or Drosha nuclease or any of several other proteins required for RNAi. ego-1 interacts genetically with him-17, another regulator of chromatin and meiosis [13], to promote germline development. We conclude that EGO-1 is an essential component of meiotic silencing in C. elegans

Regulation of Heterochromatin Assembly on Unpaired Chromosomes during Caenorhabditis elegans Meiosis by Components of a Small RNA-Mediated Pathway

Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome) become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2). This enrichment requires activity of the cellular RNA-directed RNA polymerase, EGO-1. Here we use genetic mutation, RNA interference, immunofluorescence microscopy, fluorescence in situ hybridization, and molecular cloning methods to identify and analyze three additional regulators of meiotic H3K9me2 distribution: CSR-1 (a Piwi/PAZ/Argonaute protein), EKL-1 (a Tudor domain protein), and DRH-3 (a DEAH/D-box helicase). In csr-1, ekl-1, and drh-3 mutant males, we observed a reduction in H3K9me2 accumulation on the unpaired X chromosome and an increase in H3K9me2 accumulation on paired autosomes relative to controls. We observed a similar shift in H3K9me2 pattern in hermaphrodites that carry unpaired chromosomes. Based on several assays, we conclude that ectopic H3K9me2 accumulates on paired and synapsed chromosomes in these mutants. We propose alternative models for how a small RNA-mediated pathway may regulate H3K9me2 accumulation during meiosis. We also describe the germline phenotypes of csr-1, ekl-1, and drh-3 mutants. Our genetic data suggest that these factors, together with EGO-1, participate in a regulatory network to promote diverse aspects of development

Sophisticated regulation of histone H3 lysine 9 dimethylation accumulation during meiosis in Caenorhabditis elegans

Meiotic silencing of unpaired chromatin (MSUC) is a process that has been proposed to play an essential role in defending against genetic parasites and maintaining gamete quality during sexual reproduction. Our lab uses the model organism, Caenorhabditis elegans, to study this phenomenon. In C. elegans, MSUC is thought to limit transcription of unpaired chromatin by labeling it with a silencing epigenetic marker, histone H3 lysine 9 dimethylation (H3K9me2), and inducing heterochromatin formation. A germline specific RNA-directed RNA polymerase (RdRP), EGO-1, has been shown to be required for the accumulation of H3K9me2. This study, along with the research of several other labs, has also revealed additional regulators that affect the H3K9me2 pattern during meiosis. Two of them function in small RNA pathways: RNA Helicase A (RHA-1), and another RdRP (RRF-3). These results strongly suggest a connection between the small RNAs and MSUC. In this study, I discovered five genes that are involved in regulating the H3K9me2 accumulation pattern during MSUC. Specifically, removal of CSR-1, DRH-3 and EKL-1 function causes an abnormally high level of H3K9me2 on paired chromatin; removal of SIN-3 function leads to absence of H3K9me2 on unpaired chromatin regions that may be highly histone-acetylated; removal of ERI-1 function delays removal of H3K9me2 from developing sperm cells, suggesting that ERI-1, like RRF-3, is a negative regulator of MSUC. The majority of my dissertation focuses on studying the functions of CSR-1, DRH-3 and EKL-1. My results suggest that CSR-1, DRH-3, EKL-1, and EGO-1 may represent a functional pathway in MSUC. I also provide additional information about the functions of SIN-3, HIM-17, RHA-1 and ERI-1. Based on these results, alternative models of the H3K9me2 modification pathway in C. elegans are described

Preparation of Functionalized Magnetic Polystyrene Microspheres and Their Application in Food Safety Detection

Based on the specific binding of sulfonic acid groups to melamine, β-agonists and other compounds, Fe3O4 nano-magnetic beads were coated with polystyrene using an improved micro-suspension emulsion polymerization method, thus forming core–shell magnetic polystyrene microspheres (Fe3O4@PS) with Fe3O4 as the core and polystyrene as the shell. These functionalized microspheres, which can be used as magnetic solid-phase extraction (MSPE) adsorbent, were prepared after further sulfonation. These microspheres were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analysis and saturation magnetization measurement. The results showed that these sulfonated magnetic polystyrene microspheres had favorable sphericity. The particle size of these microspheres ranged from 1 μm to 10 μm. Additionally, these microspheres had good dispersion and magnetic responses in both inorganic and organic solvents. Moreover, these functionalized magnetic polystyrene microspheres were tested and evaluated by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The results indicated that these sulfonated magnetic polystyrene microspheres (Fe3O4@SPS) could effectively adsorb such illegal additives as β-agonists and melamine in the food matrix

Correction: Intestinal Autophagy Improves Healthspan and Longevity in C. elegans During Dietary Restriction.

[This corrects the article DOI: 10.1371/journal.pgen.1006135.]

Intestinal Autophagy Improves Healthspan and Longevity in <i>C</i>. <i>elegans</i> during Dietary Restriction

<div><p>Dietary restriction (DR) is a dietary regimen that extends lifespan in many organisms. One mechanism contributing to the conserved effect of DR on longevity is the cellular recycling process autophagy, which is induced in response to nutrient scarcity and increases sequestration of cytosolic material into double-membrane autophagosomes for degradation in the lysosome. Although autophagy plays a direct role in DR-mediated lifespan extension in the nematode <i>Caenorhabditis elegans</i>, the contribution of autophagy in individual tissues remains unclear. In this study, we show a critical role for autophagy in the intestine, a major metabolic tissue, to ensure lifespan extension of dietary-restricted <i>eat-2</i> mutants. The intestine of <i>eat-2</i> mutants has an enlarged lysosomal compartment and flux assays indicate increased turnover of autophagosomes, consistent with an induction of autophagy in this tissue. This increase in intestinal autophagy may underlie the improved intestinal integrity we observe in <i>eat-2</i> mutants, since whole-body and intestinal-specific inhibition of autophagy in <i>eat-2</i> mutants greatly impairs the intestinal barrier function. Interestingly, intestinal-specific inhibition of autophagy in <i>eat-2</i> mutants leads to a decrease in motility with age, alluding to a potential cell non-autonomous role for autophagy in the intestine. Collectively, these results highlight important functions for autophagy in the intestine of dietary-restricted <i>C</i>. <i>elegans</i>.</p></div

Autophagy gene knockdown decreases the motility of wild-type and dietary-restricted <i>eat-2</i> animals.

<p>Quantification of body bends in wild-type (WT, N2) animals (<b>A</b>) and <i>eat-2(ad1116)</i> mutants (<b>B</b>) fed from Day 1 of adulthood with bacteria containing empty vector (control) or expressing <i>atg-18/Wipi</i>-targeting dsRNA. Data are the mean ± SEM of 6 animals per time point. For WT animals, *<i>P</i> = 0.03 on Day 11, **<i>P</i> = 0.008 on Day 13, and *<i>P</i> = 0.014 on Day 15, Student’s <i>t</i>-test. For <i>eat-2(ad1116)</i> animals, **<i>P</i> = 0.004 on Day 11 and **<i>P</i> = 0.002 on Day 15, Student’s <i>t</i>-test. The experiments were repeated three times with WT animals or eleven times with <i>eat-2(ad1116)</i> with similar results. (<b>C, D</b>) Quantification of body bends in <i>eat-2(ad1116)</i>; <i>sid-1(qt9); myo-3p</i>::<i>sid-1</i> (<b>C</b>) and <i>eat-2(ad1116)</i>; <i>rde-1(ne219); nhx-2p</i>::<i>rde-1</i> (<b>D</b>) transgenic animals fed from Day 1 of adulthood with bacteria containing empty vector (control) or expressing <i>atg-18/Wipi</i>-targeting dsRNA. Data are the mean ± SEM of 10–12 animals per time point. For (<b>C</b>), the differences were insignificant by Student’s <i>t</i>-test on all days; however, whole-body RNAi controls were carried out in parallel and showed effects, confirming RNAi clone efficiency. For (<b>D</b>), *<i>P</i> = 0.021 on Day 5, *<i>P</i> = 0.012 on Day 7, ****<i>P</i><0.0001 on Day 13 and 15, Student’s <i>t</i>-test. Similar motility phenotypes were observed in animals subjected to the same RNAi treatments on solid media plates. Experiments were repeated three times with similar results.</p

The age-related breakdown of intestinal barrier function is reduced in dietary-restricted <i>eat-2</i> mutants.

<p><b>(A, B)</b> DIC images of wild-type (WT, N2) animals (<b>A</b>) or <i>eat-2(ad1116)</i> animals (<b>B</b>) after soaking in blue food dye for 3 h on Day 7 (left) and Day 13 (right) of adulthood. Black arrows point to areas where blue dye has leaked from the intestinal lumen into the body cavity, giving rise to the Smurf phenotype. Scale bars, 200 μm (left panels) and 50 μm (right panels, showing higher magnifications of the boxed regions in the left panels). (<b>C)</b> Quantification of body-cavity leakage in WT and <i>eat-2(ad1116)</i> animals over time. Animals were examined after soaking in dye for 3 h on the indicated days of adulthood (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006135#sec010" target="_blank">Methods</a>). Data are the mean ± SEM of three biological repeats per time point, each with 8–10 animals. *<i>P</i><0.03, Student’s <i>t-</i>test.</p

Autophagy-gene knockdown reduces the intestinal integrity of dietary-restricted <i>eat-2</i> mutants.

<p><b>(A)</b> DIC images of <i>eat-2(ad1116)</i> animals fed from Day 1 of adulthood with bacteria containing empty vector (control) or expressing <i>atg-18/Wipi</i>-targeting dsRNA, and then soaked in blue food dye for 3 h on Day 15 of adulthood. Scale bars, 200 μm. (<b>B</b>) Quantification of body-cavity leakage in <i>eat-2(ad1116)</i> animals fed from Day 1 of adulthood with bacteria containing empty vector (control) or expressing <i>atg-18/Wipi</i>-targeting dsRNA. Data are the mean ± SEM of three biological repeats, each with 8–10 animals per time point. **<i>P</i> = 0.005, Student’s <i>t</i>-test. (<b>C</b>) Quantification of body-cavity leakage in 15-day-old <i>eat-2(ad1116)</i>; <i>rde-1(ne219); nhx-2p</i>::<i>rde-1</i> transgenic animals fed from Day 1 of adulthood with bacteria containing empty vector (control) or expressing <i>atg-18/Wipi</i>-targeting dsRNA. Data are the mean ± SEM of three biological repeats, each with 8–10 animals. *<i>P</i> = 0.025, Student’s <i>t</i>-test.</p

Analysis of the intestine of dietary-restricted <i>eat-2</i> mutants indicate enlarged lysosomal compartment and increased autophagic flux.

<p>(A) Quantification of GFP::LGG-1 punctae in the anterior intestine of WT and <i>eat-2(ad1116)</i> mutants fed from the L4 larval stage to Day 4 of adulthood with bacteria containing empty vector or expressing <i>vha15</i>/V-ATPase-targeting dsDNA. Data are the mean ± SEM of 7–21 animals per condition. ***<i>P</i> = 0.0002 for RNAi control <i>eat-2(ad1116)</i> versus RNAi for <i>vha-15</i>/V-ATPase by two-way ANOVA. The experiment was repeated four times with similar results. (B) Q-PCR analysis of putative autophagy-related and lysosomal gene expression in wild-type (WT) and <i>eat-2(ad1116)</i> animals on Days 1 and 5 of adulthood. Data are the mean ± SEM of biological triplicates, each with three technical replicates, and are normalized to expression in the WT (N2) animals. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, and ****<i>P</i><0.0001, one-way ANOVA. (C) Representative electron micrographs of intestinal cross-sections of WT and <i>eat-2(ad1116)</i> animals on Day 3 of adulthood. Arrows indicate autolysosome-like structures. (D) Quantification of autolysosome-like structures. Data are the mean ± SEM of 14–20 micrographs. *<i>P</i> = 0.0051, Student’s <i>t</i>-test. (E) Representative images of overlaid fluorescence and differential interference contrast (DIC) channels showing the intestine of 5-day-old WT and <i>eat-2(ad1116)</i> adult animals labeled with LysoTracker Red DND-99. (F). Quantification of fluorescence intensity in the anterior intestine. Data are the mean ± SEM of 10 animals. *<i>P</i><0.0001, Student’s <i>t-</i>test. The experiment was repeated six times with similar results.</p