Tools to study the rules for licensing expression and piRNA mediated epigenetic inheritance of silencing in the C. elegans germline

  • Monika Priyadarshini

Student thesis: Doctoral Thesis


In C. elegans, the germline is a tightly regulated tissue where silencing pathways regulate genes, allowing expression of “self” while silencing “non-self.” Doublestranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and piwi-associated RNAs (piRNAs) can transmit this regulation across generations via transgenerational epigenetic inheritance (TEI) mechanisms (Bošković and Rando, 2018). Analogously, some pathways can counteract gene silencing to allow sustained expression in the germline. One such example is a non-coding DNA structure called Periodic An/Tn clusters that can prevent the silencing of transgenes in the germline (Frøkjær-Jensen et al., 2016). In this thesis, I developed a novel piRNA-based tool called piRNA interference (piRNAi), where target-specific short “guide” piRNAs (sg-piRNAs) can robustly silence endogenous genes and transgenes. I have used piRNAi to understand the rules for licensing gene expression and transgenerational epigenetic inheritance in the C. elegans germline. Initially, I describe design rules for generating transgenes with PATC-rich introns that resist germline silencing and are robustly expressed from extrachromosomal arrays. PATC-rich transgenes showed more accurate gene expression patterns and did not prevent germline regulation by 3’ untranslated regions (3’ UTRs). Next, I developed the piRNAi technique to understand the role of PATCs in licensing transgene expression and the rules for how endogenous genes can be targeted for piRNA-mediated silencing and TEI. I demonstrate that a PATC-rich gfp transgene and endogenous genes are not resistant to piRNA-mediated silencing. Finally, I used piRNAi to define rules for TEI: 1. I identified two new endogenous targets for TEI (him-5 and him-8) that can inherit silencing for four and six generations respectively, after transient exposure to sg-piRNAs. 2. I demonstrate that an endogenous gene (him-5) can be semi-permanently silenced in the absence of the piRNA/PRG-1 pathway. 3. The duration of TEI was significantly shortened in a transgene that contained PATC-rich introns. Altogether, my thesis shows that an endogenous small RNA pathway can be reprogrammed to silence endogenous genes and transgenes in the germline, which enables novel experimental paradigms for studying inherited and semipermanent silencing.
Date of AwardNov 2021
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Sciences and Engineering
SupervisorChristian Froekjaer Jensen (Supervisor)


  • piRNAs
  • epigenetics
  • inheritance
  • c. elegans
  • PATCs
  • Transgenes

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