RNA interference (RNAi) in C. elegans is remarkably potent and efficient. It is potent because minuscule amounts of RNA silencing signals can be amplified and it is efficient because the silencing signals are mobile, resulting in silencing in all tissues in the treated animal and even its progeny. How do RNA-based silencing signals travel between cells and generations, and could the mechanism that enables inherited RNAi also enable inheritance of other epigenetic information? To begin to address these kinds of questions my lab performed a genetic screen to identify genes that support the systemic spreading of RNAi silencing signals. The first gene we identified and characterized was sid-1, which encodes a dsRNA channel protein. Among the first experiments we did was to fuse sid-1 sequences upstream of the start codon to GFP to learn that sid-1 was expressed in all tissues sensitive to systemic RNAi. The experiment completed, the transgenic lines were frozen and stored away.
Recently, in response to some anomalous results, Olga Minkina thawed these strains for a control experiment. She found that the wild-type worms expressing GFP under the control of the sid-1 promoter were defective for RNAi. This was a startling result. Even more startling, the non-green progeny, that is the worms that did not inherit the extrachromosomal reporter, were also RNAi defective. This RNAi defective state persisted in the progeny of these non-green worms for a variable number of generations but each line always reverted to RNAi sensitivity. Olga then showed that resistance and sensitivity correlated with sid-1 mRNA levels. Thus the sid-1 promoter transgene induced an epigenetic meta-stable silenced state at the sid-1 locus.
The remarkable stability of sid-1 silencing, which is transmitted at 100% penetrance without selection for up to 13 generations, allowed Olga to use genetic, molecular, and genomic techniques to characterize the silencing and inheritance mechanisms (Molecular Cell (PDF) 2017). She discovered that small RNA silencing pathways are essential for silencing and heritance of silencing and that the levels of sid-1 antisense small RNAs correlate with the silenced state. She also showed that the silenced state was transmitted by mothers, not fathers, and that the silenced state could be transmitted in the absence of the silenced locus – thus transmission of the silenced state is not dependent on epigenetic marks on the chromatin. She then obtained the first solid genetic evidence for RNA mediated inheritance, showing that the small RNAs embody the information transmitted between generations. Finally, her discoveries revealed that two histone methyltransferases previously associated with inherited silencing are not required for inheritance of the silencing, but are required to execute silencing at the sid-1 locus in somatic cells. These results suggest that the mechanism of epigenetic silencing in the germline and soma are distinct.
RNAi silencing pathways are readily investigated in C. elegans, however, for ease of discovery and characterization, most of the analysis has focused on artificial reporter gene sensors. Olga’s analysis identified numerous differences between transgene silencing and silencing at the endogenous sid-1 locus. The remarkably persistent silencing and reliable re-expression of the endogenous sid-1 locus coupled to its easily scored function provides a terrific model for investigating whether and how “natural” transgenerational epigenetic silencing is initiated and to begin to characterize the importance of “Lamarkian” inheritance.
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