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Summary Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational inheritance (TEI) of gene silencing. How the nuclear activities of RNAi are physiologically controlled remains poorly understood. Here, we identify and characterize a family of nuclear Argonaute-interacting proteins (NIPs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. NIP-1 and NIP-2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, NIP-1 and NIP-2 are negative regulators of nuclear RNAi triggered from exogenous sources (exo-RNAi). Loss of NIP-3, an unstable protein mostly expressed in the male germline, misdirects the RNAi response to transposable elements and impairs TEI.Our data support a model wherein NIPs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes in both somatic and germline tissues.

Summary This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization). For complete details on the use and execution of this protocol, please refer to Sawh et al. (2020)., Graphical Abstract, Highlights • An optimized FISH protocol to map chromosome conformation in C. elegans embryos • Removes limitations on the maximum number of targets visualized in a single sample • Customizable to interrogate conformation from the kb to Mb to whole-genome scale • Unsupervised clustering of single chromosomes can define similar populations in vivo, This protocol describes a high-throughput and multiplexed DNA fluorescence in situ hybridization method to trace chromosome conformation in Caenorhabditis elegans embryos. This approach generates single-cell and single-chromosome localization data that can be used to determine chromosome conformation and assess the heterogeneity of structures that exist in vivo. This strategy is flexible through modifications to the probe design steps to interrogate chromosome structure at the desired genomic scale (small-scale loops to whole-chromosome organization).

Current models suggest that chromosome domains segregate into either an active (A) or inactive (B) compartment. B-compartment chromatin is physically separated from the A compartment and compacted by the nuclear lamina. To examine these models in the developmental context of C. elegans embryogenesis, we undertook chromosome tracing to map the trajectories of entire autosomes. Early embryonic chromosomes organized into an unconventional barbell-like configuration, with two densely folded B compartments separated by a central A compartment. Upon gastrulation, this conformation matured into conventional A/B compartments. We used unsupervised clustering to uncover subpopulations with differing folding properties and variable positioning of compartment boundaries. These conformations relied on tethering to the lamina to stretch the chromosome; detachment from the lamina compacted, and allowed intermingling between, A/B compartments. These findings reveal the diverse conformations of early embryonic chromosomes and uncover a previously unappreciated role for the lamina in systemic chromosome stretching.

Summary The RNase III enzyme Dicer is responsible for key steps in the biogenesis of small RNA species in multiple RNA interference pathways. Here, we show that, in the adult C. elegans soma, half of the total DCR-1 protein is expressed as a truncated, stable C-terminal fragment named small DCR-1 (sDCR-1). sDCR-1 operates independently of full-length DCR-1 in two distinct RNAi pathways; it enhances exogenous RNAi (exoRNAi) and concurrently acts as a negative regulator of microRNA (miRNA) biogenesis. Enhancement of exoRNAi relies on sDCR-1 catalytic activity, whereas impinging on miRNA processing does not. Instead, sDCR-1 competes with pre-miRNA processing by interacting with the miRNA-dedicated Argonautes ALG-1 and ALG-2. Finally, triggering a strong exoRNAi response in the presence of elevated levels of sDCR-1 exacerbates the miRNA processing defect. Our results unveil a surprising role for a truncated form of DCR-1 in the modulation of multiple RNAi activities and in the regulation of mechanistic boundaries between pathways.