Sam oping Gu

A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, 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 Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

Amplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint

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 dsRNA-targeted chromatin 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 histone H3 lysine 9 trimethylation (H3K9me3), with modification footprints extending several kilobases from the site of dsRNA homology and with locus specificity sufficient to distinguish the targeted locus from the other 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 H3K9me3, 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.

Chromatin-associated periodicity in genetic variation downstream of transcriptional start sites

Might DNA sequence variation reflect germline genetic activity and underlying chromatin structure? We investigated this question using medaka (Japanese killifish, Oryzias latipes), by comparing the genomic sequences of two strains (Hd-rR and HNI) and by mapping ∼37.3 million nucleosome cores from Hd-rR blastulae and 11,654 representative transcription start sites from six embryonic stages. We observed a distinctive ∼200–base pair (bp) periodic pattern of genetic variation downstream of transcription start sites; the rate of insertions and deletions longer than 1 bp peaked at positions of approximately +200, +400, and +600 bp, whereas the point mutation rate showed corresponding valleys. This ∼200-bp periodicity was correlated with the chromatin structure, with nucleosome occupancy minimized at positions 0, +200, +400, and +600 bp. These data exemplify the potential for genetic activity (transcription) and chromatin structure to contribute to molding the DNA sequence on an evolutionary time scale.

Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls

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.

A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference

Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.

A transgenerational role of the germline nuclear RNAi pathway in repressing heat stress-induced transcriptional activation in C. elegans

AbstractBackgroundEnvironmental stress-induced transgenerational epigenetic effects have been observed in various model organisms and human. The capacity and mechanism of such phenomena are poorly understood. In C. elegans, siRNA mediates transgenerational gene silencing through the germline nuclear RNAi pathway. This pathway is also required to maintain the germline immortality when C. elegans is under heat stress. However, the underlying molecular mechanism is unknown. In this study, we investigated the impact of heat stress on chromatin, transcription, and siRNAs at the whole-genome level, and whether any of the heat-induced effects is transgenerationally heritable in either the wild-type or the germline nuclear RNAi mutant animals.ResultsWe performed 12-generation temperature-shift experiments using the wild-type C. elegans and a mutant strain that lacks the germline-specific nuclear Argonaute protein HRDE-1/WAGO-9. By examining the mRNA, small RNA, RNA polymerase II, and H3K9 trimethylation profiles at the whole-genome level, we revealed an epigenetic role of HRDE-1 in repressing heat stress-induced transcriptional activation of over 280 genes. Many of these genes are in or near LTR (long-terminal repeat) retrotransposons. Strikingly, for some of these genes, the heat stress-induced transcriptional activation in the hrde-1 mutant intensifies in the late generations under the heat stress and is heritable for at least two generations after the mutant animals are shifted back to lower temperature. hrde-1 mutation also leads to siRNA expression changes of many genes. This effect on siRNA is dependent on both the temperature and generation.ConclusionsOur study demonstrated that a large number of the endogenous targets of the germline nuclear RNAi pathway in C. elegans are sensitive to heat-induced transcriptional activation. This effect at certain genomic loci including LTR retrotransposons is transgenerational. Germline nuclear RNAi antagonizes this temperature effect at the transcriptional level and therefore may play a key role in heat stress response in C. elegans.

Complex coding of endogenous siRNA, transcriptional silencing and H3K9 methylation on native targets of germline nuclear RNAi in C. elegans

BackgroundSmall RNA-guided transcriptional silencing (nuclear RNAi) is fundamental to genome integrity and epigenetic inheritance. Despite recent progress in identifying the capability and genetic requirements for nuclear RNAi in Caenorhabditis elegans, the natural targets and cellular functions of nuclear RNAi remain elusive.MethodsTo resolve this gap, we coordinately examined the genome-wide profiles of transcription, histone H3 lysine 9 methylation (H3K9me) and endogenous siRNAs of a germline nuclear Argonaute (hrde-1/wago-9) mutant and identified regions on which transcription activity is markedly increased and/or H3K9me level is markedly decreased relative to wild type animals.ResultsOur data revealed a distinct set of native targets of germline nuclear RNAi, with the H3K9me response exhibiting both overlapping and non-overlapping distribution with the transcriptional silencing response. Interestingly LTR retrotransposons, but not DNA transposons, are highly enriched in the targets of germline nuclear RNAi. The genomic distribution of the native targets is highly constrained, with >99% of the identified targets present in five autosomes but not in the sex chromosome. By contrast, HRDE-1-associated small RNAs correspond to all chromosomes. In addition, we found that the piRNA pathway is not required for germline nuclear RNAi activity on native targets.ConclusionGermline nuclear RNAi in C. elegans is required to silence retrotransposons but not DNA transposon. Transcriptional silencing and H3K9me can occur independently of each other on the native targets of nuclear RNAi in C. elegans. Our results rule out a simple model in which nuclear Argonaute protein-associated-small RNAs are sufficient to trigger germline nuclear RNAi responses. In addition, the piRNA pathway and germline nuclear RNAi are specialized to target different types of foreign genetic elements for genome surveillance in C. elegans.

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

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.

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

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.

The zinc transporter ZIPT-7.1 regulates sperm activation in nematodes

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process.