Characterizing Epigenetic and RNA-Mediated Regulation of Behavior in Caenorhabditis elegans

Abstract

Chapter I: Transgenerational epigenetic inheritance (TEI) enables organisms to transmit environmentally induced epigenetic traits across. In Caenorhabditis elegans, exposure to the pathogenic bacterium Pseudomonas aeruginosa (PA14) induces learned avoidance behavior that can persist in progeny. Here, we examined the contribution of set-32 and rbr-2 to baseline pathogen preference using backcrossed mutant strains to reduce background genetic variation. Using genotyping, backcrossing, and naïve choice assays, we confirmed mutant strains and minimized background genetic variation to accurately assess behavioral phenotypes. Our lab showed that set-32 mutants exhibit impaired learning despite normal naïve attraction, whereas rbr-2 mutants display an increased naïve avoidance while retaining the ability to learn avoidance. In our choice assays, we found that both mutants exhibited no statistically significant difference in naïve preference compared to wild-type worms. These findings suggest that neither gene may be required for baseline attraction to PA14. Chapter II: RNA interference (RNAi) is a conserved mechanism of gene regulation that depends on a coordinated uptake and intracellular transport of double-stranded RNA (dsRNA). In Caenorhabditis elegans, the transmembrane protein SID-1 plays a central role in mediating systematic RNAi, however its tissue specific function remains unclear. Here, we investigated the role of SID-1 in amphid sheath (AMsh) glial cells, and tested whether its mammalian homolog, SIDT-1, can compensate for the loss of SID-1. Implementing 1% butanone chemotaxis assays, we found that AMsh-specific knockdown of sid-1 produced variable effects on baseline chemotaxis across biological replicates, limiting our ability to draw definitive conclusions about its role in these cells. Additionally, we expressed SIDT-1 in sid-1 mutants. Across replicates, SIDT-1 expression did not consistently restore wild-type chemotaxis behavior, and similar variability was observed with a SIDT-1;SID-1 fusion construct. However, independent data from our lab demonstrate that both SIDT-1 expression and the SIDT-1;SID-1 fusion construct can reproducibly rescue sid-1 phenotypes under optimized conditions. These findings suggest that while our results are variable and inconclusive on their own, SID-1–mediated RNA transport in AMsh glia likely plays an important role for chemotaxis behavior.