Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans

Transgenes can be permanently silenced in a single generation via a previously described small RNA-induced epigenetic silencing (RNAe) mechanism, which is promoted by the presence of a perfect Piwi-interacting RNA (piRNA) target site. In this study, we identify a previously unidentified mechanism capable of silencing single-copy transgenes that lack perfect piRNA target sites and that is triggered by a lack of chromosomal pairing during meiosis for multiple generations. Multigenerational RNAe can lead to reversible or permanent transgene silencing and may provide insight into variability in the expression of single-copy transgenes or single-copy genomic insertions, which are commonly used in experimental biology. Our analysis of “multigenerational RNAe” also offers new insights into potentially common epigenetic silencing events relevant to genome expression in the germline and embryo

Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans

Single-copy transgenes in Caenorhabditis elegans can be subjected to a potent, irreversible silencing process termed small RNA-induced epigenetic silencing (RNAe). RNAe is promoted by the Piwi Argonaute protein PRG-1 and associated Piwi-interacting RNAs (piRNAs), as well as by proteins that promote and respond to secondary small interfering RNA (siRNA) production. Here we define a related siRNA-mediated silencing process, termed “multigenerational RNAe,” which can occur for transgenes that are maintained in a hemizygous state for several generations. We found that transgenes that contain either GFP or mCherry epitope tags can be silenced via multigenerational RNAe, whereas a transgene that possesses GFP and a perfect piRNA target site can be rapidly and permanently silenced via RNAe. Although previous studies have shown that PRG-1 is typically dispensable for maintenance of RNAe, we found that both initiation and maintenance of multigenerational RNAe requires PRG-1 and the secondary siRNA biogenesis protein RDE-2. Although silencing via RNAe is irreversible, we found that transgene expression can be restored when hemizygous transgenes that were silenced via multigenerational RNAe become homozygous. Furthermore, multigenerational RNAe was accelerated when meiotic pairing of the chromosome possessing the transgene was abolished. We propose that persistent lack of pairing during meiosis elicits a reversible multigenerational silencing response, which can lead to permanent transgene silencing. Multigenerational RNAe may be broadly relevant to single-copy transgenes used in experimental biology and to shaping the epigenomic landscape of diverse species, where genomic polymorphisms between homologous chromosomes commonly result in unpaired DNA during meiosis

<i>nhr-62</i> modulates DR-induced fat metabolism.

<p>(A) Representative pictures of Oil Red O stained worms. Scale bar = 20 µM. (B) Oil Red O intensity is increased relative to wild-type (N2) or <i>eat-2(ad465)</i> controls by mutation of <i>nhr-62</i> (combined data from 3 independent experiments). (C) Total triglycerides of day 1 adults relative to wild-type (N2) (combined data from 16 independent experiments). (D) Fatty acid profiles of day 1 adult animals (combined data from 11 independent experiments). (E) Total saturated, monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) calculated from (D). <i>eat-2;nhr-62</i> had increased saturated fat compared to wild-type (N2) and decreased MUFAs and PUFAs compared to <i>eat-2(ad465)</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003651#pgen.1003651.s003" target="_blank">Figure S3</a> for physiologic traits of <i>nhr-62</i> mutation. *p<0.05, **p<0.01, ***p<0.001 by single factor ANOVA with Tukey test. Mean (Center Line) ± SD (Box) with bars representing an outlier coefficient of 1.5, or Mean±SEM.</p

<i>nhr-62</i> specifically regulates DR-induced longevity.

<p>(A) Survivorship curves for wild-type (N2), <i>eat-2(ad465)</i>, <i>nhr-62(tm1818)</i>, <i>eat-2;nhr-62</i>, and <i>eat-2;nhr-62(dhEx627)</i>. Extra chromosomal array <i>dhEx627</i> (carrying a wild-type <i>nhr-62</i>) in <i>eat-2;nhr-62</i> resulted in a significant increase in lifespan compared to both <i>nhr-62(tm1818)</i> and <i>eat-2;nhr-62</i> mutants (p<0.001). (B) Wild-type (N2) worms with <i>dhEx627</i> exhibited a significant increase in lifespan compared to wild-type (p<0.001). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003651#pgen.1003651.s002" target="_blank">Figure S2</a> for physiologic traits of overexpression. (C,D) Both wild-type (N2) and <i>nhr-62(tm1818)</i> worms fed <i>daf-2</i> RNAi or <i>cco-1</i> RNAi had a significant increase in lifespan when compared to controls (p<0.001). (E) BDR curve for wild-type (N2) and <i>nhr-62(tm1818)</i> worms. The lifespan of wild-type and <i>nhr-62(tm1818)</i> were not different at the three most concentrated food dilutions, but were significantly different at the remaining 7 dilutions (p<0.001). (F) Survivorship curves for wild-type (N2) and <i>nhr-62(tm1818)</i> fed either 6.45×10⁸ CFU/ml or 3.23×10⁸ CFU/ml. p-values calculated by the log-rank test.</p

Expression pattern of <i>nhr-62</i>::<i>gfp.</i>

<p>A full length fusion of <i>nhr-62::gfp</i> resides in the nuclei of (A) pharyngeal cells (pha), (B) sensory neurons (ne) (C) intestinal cells (int) (D) spermatheca (spe) (E) hypodermis (hyp) (F) excretory cell (exc). Scale bar is 10 µM.</p