Woodhouse, Rachel M.Frolows, NatalyaMonteiro, Dhruv S.Hawes, Jessica J.Hawdon, AzelleDavies, MichaelWatson, OwenLennox, Victoria S.Ashe, Alyson2026-07-032026-07-030016-6731PubMed:40460278ORCID:/0000-0002-4807-3466/work/219178038https://hdl.handle.net/1885/733812819Transgenerational epigenetic inheritance (TEI) is the transfer of nongenetic information between generations. In Caenorhabditis elegans, RNA interference (RNAi) is a conserved process initiated by double-stranded RNA, which can induce TEI. While many factors have been implicated in TEI, whether they act in establishment or maintenance of the transgenerational signal, and the generation in which they act, has not been defined. Here, we characterize the actions of glh-1, hrde-1, −2, −4, morc-1, nrde-1, −2, −4, set-25, −32, wago-1, −4, znfx-1, pup-1, and emb-4 within RNAi-induced TEI. We show that these genes can be classified into 3 groups: those involved in only establishment or maintenance, or those involved in both. We identify a heterochromatin-based pathway established in the P0 generation by histone methyltransferases and maintained in later generations by MORC-1, upstream of HRDE-1-dependent silencing. By investigating lineage dynamics, we provide evidence that inheritance patterns are partially determined in RNAi-exposed parents, but that variation between offspring also contributes. And finally, we demonstrate that polyUG RNAs broadly correlate with, but do not define, inheritance patterns. Together, this work forms a cohesive model of RNAi-induced TEI.We thank the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis (University of Sydney) for access to microscopes and assistance with imaging and analysis. We acknowledge Protein Production in Sydney Analytical (University of Sydney). We also thank the CGC (University of Minnesota) for strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and Wormbase (Sternberg et al. 2024). RMW, NF, DSM, and JJH were supported by a Research Training Program Scholarship from the Australian Government. NF was additionally supported by a Postgraduate Research Supplementary Scholarship from the Commonwealth Scientific and Industrial Research Organisation (CSIRO). AA was supported by FT180100653, DP200102904, and DP240100725 from the Australian Research Council. We thank the Australian Microscopy and Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis (University of Sydney) for access to microscopes and assistance with imaging and analysis. We acknowledge Protein Production in Sydney Analytical (University of Sydney). We also thank the CGC (University of Minnesota) for strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and Wormbase (). RMW, NF, DSM, and JJH were supported by a Research Training Program Scholarship from the Australian Government. NF was additionally supported by a Postgraduate Research Supplementary Scholarship from the Commonwealth Scientific and Industrial Research Organisation (CSIRO). AA was supported by FT180100653, DP200102904, and DP240100725 from the Australian Research Council.enPublisher Copyright: © The Author(s) 2025.C. eleganschromatinepigenetic inheritanceepigeneticssmall RNAWormBase2025-08-0110.1093/genetics/iyaf106105013138146