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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-05, Vol.112 (20), p.E2667-E2676
• Main Authors: Leopold, Luciana E, Heestand, Bree N, Seong, Soobin, Shtessel, Ludmila, Ahmed, Shawn
• Format: Article
• Language: English
• Subjects: Animals; Argonaute Proteins - genetics; Argonaute Proteins - metabolism; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Biological Sciences; Caenorhabditis elegans; Caenorhabditis elegans - genetics; Caenorhabditis elegans - physiology; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Chromosomes; DNA Primers - genetics; Epigenetics; Gene Silencing - physiology; Genomics; Green Fluorescent Proteins - genetics; Luminescent Proteins - genetics; Meiosis - physiology; Microscopy, Fluorescence; Microscopy, Interference; Nematodes; PNAS Plus; Polymorphism; Red Fluorescent Protein; Ribonucleic acid; RNA; RNA, Small Interfering - genetics; Species diversity; Transgenes - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1501979112

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. Significance 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 ex