Caenorhabditis elegansheterochromatin factor SET-32 plays an essential role in transgenerational establishment of nuclear RNAi-mediated epigenetic silencing

Abstract

Epigenetic inheritance contributes fundamentally to transgenerational physiology and fitness. Mechanistic understanding of RNA-mediated chromatin modification and transgenerational epigenetic inheritance, which in C. elegans can be triggered by exogenous double-stranded RNA (exo-dsRNA) or facilitated by endogenous small interfering RNAs (endo-siRNAs), has mainly been limited to the post-initiation phases of silencing. Indeed, the dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here we found that the onset of exo-dsRNA-induced nuclear RNAi is a transgenerational process, and that establishment requires SET-32, one of the three putative histone methyltransferases (HMTs) that are required for H3K9me3 deposition at the nuclear RNAi targets. We also performed multigenerational whole-genome analyses to examine the establishment of silencing at endogenous targets of germline nuclear RNAi. The nuclear Argonaute protein HRDE-1 is essential for the maintenance of nuclear RNAi. Repairing a loss-of-function mutation in hrde-1 by CRISPR restored the silencing of endogenous targets in animals carrying wild type set-32. However, for numerous endogenous targets, repairing the hrde-1 mutation in a set-32;hrde-1 double mutant failed to restore their silencing states in up to 20 generations after the hrde-1 repair, using a similar genome editing approach. We found that despite a prominent role in the establishment of silencing, however, set-32 is completely dispensable for the maintenance of silencing once HRDE-1-dependent gene repression is established. Our study indicates that: 1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements; and 2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process. In addition, our study reveals a novel paradigm in which a heterochromatin factor primarily functions to promote the establishment of transgenerational silencing, expanding mechanistic understanding of the well-recognized role of heterochromatin in epigenetic maintenance.

Germ Granules Govern Small RNA Inheritance

“…Typically, dsRNA-triggered heritable responses persist for 3-5 generations [6,7], owing to a dedicated pathway that regulates the duration of each heritable response [7,8]. Maintenance of RNAi over generations involves specialized argonaute proteins [9][10][11], amplification of the small RNAs by RNA-dependent RNA polymerases [12,13] (RdRPs), and chromatin modifiers [8,[14][15][16]. Many of the factors that participate in transgenerational small RNA inheritance reside in germline-specific ribonucleoprotein complexes termed germ granules [17][18][19][20][21][22][23].…”

Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

“…Typically, dsRNA-triggered heritable responses persist for 3-5 generations 6,7 , owing to a dedicated pathway which regulates the duration of each heritable response 7,8 . Maintenance of RNAi interference (RNAi) over generations involves specialized argonaute proteins [9][10][11] , amplification of the small RNAs by RNA-dependent RNA Polymerases 12,13 (RdRPs), and chromatin modifiers 8,[14][15][16] .…”