C. elegans Heterochromatin Factor SET-32 Plays an Essential Role in Transgenerational Establishment of Nuclear RNAi-Mediated Epigenetic Silencing

Abstract

SUMMARY 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 exogenous dsRNA-induced nuclear RNAi in C. elegans is a transgenerational process, and it requires a putative histone methyltransferase (HMT), SET-32. By developing a CRISPR-based genetic approach, we found that silencing establishment at the endogenous targets of germline nuclear RNAi also requires SET-32. Although SET-32 and two H3K9 HMTs, MET-2 and SET-25, are dispensable for the maintenance of silencing, they do contribute to transcriptional repression in mutants that lack the germline nuclear Argonaute protein HRDE-1, suggesting a conditional role of heterochromatin in the maintenance phase. 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 transge-nerational, chromatin-based process.

C. elegansnuclear RNAi factor SET-32 deposits the transgenerational heritable histone modification, H3K23me3

“…Although SET-32 is dispensable for silencing maintenance when in the presence of wild type HRDE-1, set-32 hrde-1 double mutants show enhanced de-silencing when compared to hrde-1 single mutants 15 . These results suggest a conditional requirement of H3K23me3 for silencing maintenance, possibly to function as a failsafe or backup silencing mechanism.…”

“…The involvement of SET-32 in both the maintenance and establishment phases of nuclear RNAi 7,15,21,24 suggests that H3K23me3 functions in these phases as well. During the establishment phase of nuclear RNAi, the host organism encounters a foreign genetic element for the first time and must repress its active transcription.…”

“…Recent studies have shown that SET-32 is required for silencing establishment at both exogenous dsRNA targets and endogenous targets 15,21 . Unlike in the maintenance phase, the requirement of SET-32 in silencing establishment is not conditional on the lack of HRDE-1, suggesting that H3K23me3 plays a more prominent role in the establishment phase than the maintenance phase.…”

“…SET-25 and SET-32, but not MET-2, are required for the silencing of a piRNA-targeted reporter gene 10 . In addition, while MET-2, SET-25, and SET-32 were all dispensable for silencing maintenance in nuclear RNAi, SET-32 and, to a lesser extent, SET-25 are required for the silencing establishment 15,21 . Given SET-32’s unique role in nuclear RNAi, we hypothesized that its biochemical activity may differ from MET-2 and SET-25.…”

“…In current models, HRDE-1 binds secondary siRNAs and recruits nuclear RNAi factors, including chromatin modifying enzymes and remodeling factors, to genomic sites of RNAi 14 . Germline nuclear RNAi-deficient mutants in C. elegans exhibit several phenotypes, including progressive sterility under heat stress (Mrt phenotype) and large-scale de-silencing and chromatin decompaction at the endogenous targets 5-8,10,12,13,15-17 . We and others have observed the intricacies of this chromatin decompaction (explained below and in the discussion), the consequences are, as yet, unknown.…”