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.

Maintaining cells in an undifferentiated totipotent state is essential for initiating developmental programs that lead to a fully formed organism in each generation and for maintaining immortal germ cells across generations. Caenorhabditis elegans is a powerful genetic model organism to study early germ cell development due to the animal’s transparency and the ability to screen for mutant phenotypes. However, our ability to use standard techniques to study gene expression using fluorescent reporter genes has been limited due to germline-specific silencing mechanisms that repress transgenes. Therefore, we lack even basic knowledge of how expression is regulated in C. elegans germ cells. In this study, we develop methods to overcome these silencing mechanisms by using a class of noncoding DNA, called Periodic An/Tn Clusters (PATCs), to prevent transgene silencing in the germline. We use these improved tools to test the proposed role of putative germline-specific regulatory DNA motifs and the role a periodic TT signal within germline promoters. We fused GFP to the promoter of a germline expressed gene (pcn-1), which is enriched for PATCs and contains a germline-specific motif (TTAAAG). Our results show that despite enrichment and phylogenetic conservation, the TTAAAG motif is not required for germline expression. To test additional motifs and periodic TTs, we have designed a system that will allow us to test synthetic gene fragments for bi-directional germline expression. These tools will allow us to rapidly test motif redundancy, motif spacing, and TT periodicity using gfp and rfp signals in the germline and will enable experiments aimed at understanding the role of germline regulatory elements.