Non-membrane-bound organelles such as nucleoli, processing bodies, Cajal bodies and germ granules form by the spontaneous self-assembly of specific proteins and RNAs. How these biomolecular condensates form and interact is poorly understood. Here we identify two proteins, ZNFX-1 and WAGO-4, that localize to Caenorhabditis elegans germ granules (P granules) in early germline blastomeres. Later in germline development, ZNFX-1 and WAGO-4 separate from P granules to define an independent liquid-like condensate that we term the Z granule. In adult germ cells, Z granules assemble into ordered tri-condensate assemblages with P granules and Mutator foci, which we term PZM granules. Finally, we show that one biological function of ZNFX-1 and WAGO-4 is to interact with silencing RNAs in the C. elegans germline to direct transgenerational epigenetic inheritance. We speculate that the temporal and spatial ordering of liquid droplet organelles may help cells to organize and coordinate the complex RNA processing pathways that underlie gene-regulatory systems, such as RNA-directed transgenerational epigenetic inheritance.

Gene silencing mediated by dsRNA (RNAi) can persist for multiple generations in (termed RNAi inheritance). Here we describe the results of a forward genetic screen in that has identified six factors required for RNAi inheritance: GLH-1/VASA, PUP-1/CDE-1, MORC-1, SET-32, and two novel nematode-specific factors that we term here (heritable RNAi defective) HRDE-2 and HRDE-4 The new RNAi inheritance factors exhibit mortal germline (Mrt) phenotypes, which we show is likely caused by epigenetic deregulation in germ cells. We also show that HRDE-2 contributes to RNAi inheritance by facilitating the binding of small RNAs to the inheritance Argonaute (Ago) HRDE-1 Together, our results identify additional components of the RNAi inheritance machinery whose conservation provides insights into the molecular mechanism of RNAi inheritance, further our understanding of how the RNAi inheritance machinery promotes germline immortality, and show that HRDE-2 couples the inheritance Ago HRDE-1 with the small RNAs it needs to direct RNAi inheritance and germline immortality.

Transgenerational epigenetic inheritance (TEI) is the inheritance of epigenetic information for two or more generations. In most cases, TEI is limited to a small number of generations (two to three). The short-term nature of TEI could be set by innate biochemical limitations to TEI or by genetically encoded systems that actively limit TEI. In Caenorhabditis elegans, double-stranded RNA (dsRNA)-mediated gene silencing [RNAi (RNA interference)] can be inherited (termed RNAi inheritance or RNA-directed TEI). To identify systems that might actively limit RNA-directed TEI, we conducted a forward genetic screen for factors whose mutation enhanced RNAi inheritance. This screen identified the gene heritable enhancer of RNAi (heri-1), whose mutation causes RNAi inheritance to last longer (. 20 generations) than normal. heri-1 encodes a protein with a chromodomain, and a kinase homology domain that is expressed in germ cells and localizes to nuclei. In C. elegans, a nuclear branch of the RNAi pathway [termed the nuclear RNAi or NRDE (nuclear RNA defective) pathway] promotes RNAi inheritance. We find that heri-1(2) animals have defects in spermatogenesis that are suppressible by mutations in the nuclear RNAi Argonaute (Ago) HRDE-1, suggesting that HERI-1 might normally act in sperm progenitor cells to limit nuclear RNAi and/or RNAi inheritance. Consistent with this idea, we find that the NRDE nuclear RNAi pathway is hyperresponsive to experimental RNAi treatments in heri-1 mutant animals. Interestingly, HERI-1 binds to genes targeted by RNAi, suggesting that HERI-1 may have a direct role in limiting nuclear RNAi and, therefore, RNAi inheritance. Finally, the recruitment of HERI-1 to chromatin depends upon the same factors that drive cotranscriptional gene silencing, suggesting that the generational perdurance of RNAi inheritance in C. elegans may be set by competing pro-and antisilencing outputs of the nuclear RNAi machinery.