Epigenetic information is frequently erased near the start of each new generation (1). In some cases, however, epigenetic information can be transmitted from parent to progeny (epigenetic inheritance) (2). A particularly striking example of epigenetic inheritance is dsRNA-mediated gene silencing (RNAi) in C. elegans, which can be inherited for more than five generations (3–8). To understand this process we conducted a genetic screen for animals defective for transmitting RNAi silencing signals to future generations. This screen identified the gene heritable RNAi defective (hrde)-1. hrde-1 encodes an Argonaute (Ago) that associates with small interfering (si)RNAs in germ cells of the progeny of animals exposed to dsRNA. In nuclei of these germ cells, HRDE-1 engages the Nrde nuclear RNAi pathway to direct H3K9me3 at RNAi targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed siRNAs, which direct nuclear gene silencing in germ cells. In hrde-1 or nuclear RNAi deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Ago HRDE-1 directs gene-silencing events in germ cell nuclei, which drive multi-generational RNAi inheritance and promote immortality of the germ cell lineage. We propose that C. elegans uses the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

Exogenous double-stranded RNA (dsRNA) has been shown to exert homology-dependent effects at the level of both target mRNA stability and chromatin structure. Using C. elegans undergoing RNAi as an animal model, we have investigated the generality, scope, and longevity of chromatin-targeted dsRNA effects and their dependence on components of the RNAi machinery. Using high-resolution genome-wide chromatin profiling, we found that a diverse set of genes can be induced to acquire locus-specific enrichment of H3K9 trimethylation, with modification footprints extending several kilobases from the site of dsRNA homology and with locus specificity sufficient to distinguish the targeted locus from among all 20,000 genes in the C. elegans genome. Genetic analysis of the response indicated that factors responsible for secondary siRNA production during RNAi were required for effective targeting of chromatin. Temporal analysis revealed that H3K9 methylation, once triggered by dsRNA, can be maintained in the absence of dsRNA for at least two generations before being lost. These results implicate dsRNA-triggered chromatin modification in C. elegans as a programmable and locus-specific response defining a metastable state that can persist through generational boundaries.

BackgroundUltra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run.ResultsWe prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001.ConclusionsThis approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets.

BackgroundGermline 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.ResultsHere 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.ConclusionsNuclear 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-017-0114-8) contains supplementary material, which is available to authorized users.