Project Summary Recent epidemiological studies suggest that human health can be influenced by ancestral experience. Ancestral diet and stress have been identified as possible modifiers of health in descendants. However, the mechanisms by which the ancestral experience is transmitted to descendants as gene regulatory information to cause effects is not understood. While RNA and epigenetic modifications have been proposed to correlate with ancestral experience, the complexity of mammalian biology has presented a challenge to establish causal mechanisms. In particular, the molecular agent(s) that transmit the information from one generation to the next and the machinery that promotes changes in gene expression that can be stable for many generations are not well understood. Identifying such markers of ancestral influence can enable the early diagnosis of susceptibility to diseases such as obesity and diabetes. We recently found that RNAs exported from neurons in response to double-stranded RNA (dsRNA) expression can enter germ cells in the nematode C. elegans and silence matching genes in descendants. Intriguingly, the persistence of this silencing depends on as yet unknown features of the gene. Because extracellular RNAs are also found in human circulation, they are candidates for similar transport to the next generation and for triggering subsequent transgenerational effects. In support of this idea, three key aspects appear to be conserved in mammals including humans: use of a dsRNA-selective importer for uptake of extracellular dsRNA, use of Argonaute proteins for gene silencing, and use of histone modifications for stable propagation of the regulatory state of a gene. The goal of this proposal is to understand mechanisms that regulate the ability of extracellular RNA in ancestors to affect genes of matching sequence in descendants. We will determine the features of extracellular RNA that enable it to trigger transgenerational gene silencing using cell biological and genetic approaches. We will determine the factors that make a gene susceptible to transgenerational changes in gene regulation using forward genetic, genome editing, and epigenome editing approaches. Finally, we will identify and validate loci in the C. elegans genome that are susceptible to ancestral influences using insights that we have gained from the silencing of a model gene by dsRNA exported from ancestral neurons. These studies will provide a clear picture of mechanisms that can promote or inhibit gene regulation by inherited RNAs in an animal, which are likely to be extendable to other animals including mammals.