PROJECT SUMMARY: Nuclear RNAi is an evolutionarily conserved pathway in which small interfering RNAs (siRNAs) guide chromatin modifications and transcriptional repression. It protects the host genome by epigenetically silencing transposons and other “non-self” DNA. Since its discovery, nuclear RNAi has provided a powerful paradigm to study RNA-mediated chromatin regulation and transgenerational epigenetic inheritance. In C. elegans, nuclear RNAi and heritable silencing can be conveniently triggered by feeding worms with exogenous dsRNA targeting a native gene. This approach is complemented by investigating molecular events occurring at the endogenous targets in the C. elegans genome. Using this uniquely tractable system, our published works done in the past funding cycle have made the following discoveries, which also raised new questions: (1) The heterochromatin mark H3K9me3 can be functionally decoupled from transcriptional silencing during the maintenance phase. What is the H3K9me3-independent transcriptional repression mechanism? (2) The heterochromatin enzyme SET-32 is required for the establishment phase but dispensable for the maintenance phase of RNAi. How does SET-32 promote silencing establishment? (3) Endogenous targets are transiently expressed in a subset of germ cells at the proliferation and early meiotic stage in wild type adult animals. What is the mechanism of the developmental regulation of the low level transcription, and how does it contribute to the reinforcement of epigenetic silencing memory? (4) When we de-silence endogenous targets of nuclear RNAi, their transcripts are enriched in germline nuclei. What is the significance of this nuclear localization in triggering RNAi? In the proposed new funding cycle, we will take both novel and established genetic, biochemical, cell biology, and computational approaches to answer these questions by achieving the following aims. (1) Investigate the transgenerational epigenetic mechanisms of silencing establishment. (2) Characterize a novel histone modification, H3K23me3, and its role in germline nuclear RNAi. (3) Determine the triggering mechanisms at the endogenous target of nuclear RNAi. The proposed studies, which explore fundamental yet unmapped territory of modern biology, will advance our understanding of RNA-chromatin interaction, inheritance of epigenetic states, and genome surveillance, which are all relevant to human development and disease.