Decoupling the downstream effects of germline nuclear RNAi reveals that H3K9me3 is dispensable for heritable RNAi and the maintenance of endogenous siRNA-mediated transcriptional silencing in Caenorhabditis elegans

Epigenetics Chromatin. 2017 Feb 15;10:6. doi: 10.1186/s13072-017-0114-8. eCollection 2017.


Background: Germline nuclear RNAi in C. elegans is a transgenerational gene-silencing pathway that leads to H3K9 trimethylation (H3K9me3) and transcriptional silencing at the target genes. H3K9me3 induced by either exogenous double-stranded RNA (dsRNA) or endogenous siRNA (endo-siRNA) is highly specific to the target loci and transgenerationally heritable. Despite these features, the role of H3K9me3 in siRNA-mediated transcriptional silencing and inheritance of the silencing state at native target genes is unclear. In this study, we took combined genetic and whole-genome approaches to address this question.

Results: Here we demonstrate that siRNA-mediated H3K9me3 requires combined activities of three H3K9 histone methyltransferases: MET-2, SET-25, and SET-32. set-32 single, met-2 set-25 double, and met-2 set-25;set-32 triple mutant adult animals all exhibit prominent reductions in H3K9me3 throughout the genome, with met-2 set-25;set-32 mutant worms losing all detectable H3K9me3 signals. Surprisingly, loss of high-magnitude H3K9me3 at the native nuclear RNAi targets has no effect on the transcriptional silencing state. In addition, the exogenous dsRNA-induced transcriptional silencing and heritable RNAi at oma-1, a well-established nuclear RNAi reporter gene, are completely resistant to the loss of H3K9me3.

Conclusions: Nuclear RNAi-mediated H3K9me3 in C. elegans requires multiple histone methyltransferases, including MET-2, SET-25, and SET-32. H3K9me3 is not essential for dsRNA-induced heritable RNAi or the maintenance of endo-siRNA-mediated transcriptional silencing in C. elegans. We propose that siRNA-mediated transcriptional silencing in C. elegans can be maintained by an H3K9me3-independent mechanism.

MeSH terms

  • Animals
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans / metabolism
  • Caenorhabditis elegans Proteins / antagonists & inhibitors
  • Caenorhabditis elegans Proteins / genetics
  • Caenorhabditis elegans Proteins / metabolism*
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Chromatin Immunoprecipitation
  • Genome
  • Genomic Instability
  • Germ Cells / metabolism
  • High-Throughput Nucleotide Sequencing
  • Histone Methyltransferases
  • Histone-Lysine N-Methyltransferase / genetics
  • Histone-Lysine N-Methyltransferase / metabolism
  • Histones / genetics
  • Histones / metabolism*
  • Methylation
  • Microscopy, Fluorescence
  • Mutagenesis
  • RNA Interference*
  • RNA, Double-Stranded / metabolism
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Sequence Analysis, RNA
  • Transcription, Genetic


  • Caenorhabditis elegans Proteins
  • Carrier Proteins
  • Histones
  • OMA-1 protein, C elegans
  • RNA, Double-Stranded
  • RNA, Messenger
  • RNA, Small Interfering
  • Histone Methyltransferases
  • Histone-Lysine N-Methyltransferase