RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

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.

The NDT80 gene of Saccharomyces cerevisiae, which encodes a global activator of transcription of middle sporulation-specific genes, is first expressed after the activation of early meiotic genes but prior to activation of middle sporulation-specific genes. Both upstream repression sequence 1 (URS1) and mid-sporulation element (MSE) sites are present in the promoter region of the NDT80 gene; these elements have been shown previously to contribute to the regulation of expression of early and middle sporulation-specific genes, respectively, by mediating repression in growing cells and activation at specific times during sporulation. In this study, we have shown that the overlapping windows of URS1-and MSE-mediated repression and activation are responsible for the distinctive premiddle expression pattern of the NDT80 gene. Our data suggest that a Sum1-associated repression complex bound at the NDT80 MSE sites prevents Ime1 tethered at the NDT80 URS1 sites from activating transcription of the NDT80 gene at the time that Ime1-dependent activation of early URS1-regulated meiotic genes is occurring. We propose that a decrease in the efficiency of Sum1-mediated repression as cells progress through the early events of the sporulation program allows the previously inactive Ime1 tethered at the URS1 NDT80 sites to promote a low level of expression of the NDT80 gene. This initial phase of URS1-dependent NDT80 expression is followed by Ndt80-dependent upregulation of its own expression, which requires the MSE NDT80 sites and occurs concomitantly with Ndt80-dependent activation of a set of middle MSE-regulated sporulation-specific genes. Mutation of IME2 prevents expression of NDT80 in sporulating cells. We show in this study that NDT80 is expressed and that middle genes are activated in cells of an ⌬ime2/⌬ime2 ⌬sum1/⌬sum1 strain in sporulation medium. This suggests that Ime2 activates expression of NDT80 by eliminating Sum1-mediated repression.The sporulation program of the yeast Saccharomyces cerevisiae provides a simple model system to study the temporal control of gene expression during development. On entry into the sporulation program, which is triggered by starvation, a diploid a/␣ cell completes one round of premeiotic DNA replication and then progresses through a lengthy prophase during which a high level of recombination occurs and homologous chromosomes pair. Cells then undergo the reductional and equational meiotic divisions, which result in a single four-lobed nucleus that contains the four haploid complements of chromosomes. The nuclear lobes are engulfed and ultimately pinched off by the prospore membranes that extend from the spindle pole bodies. The deposition of spore wall material within the prospore membrane completes the sporulation process, generating four mature spores arranged tetrahedrally within the ascus.Studies aimed at identifying genes that are differentially expressed as cells progress through sporulation or that serve sporulation-specific roles have defined four temporally distinct classes of sp...

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.Propagation of viable cells is ensured by surveillance mechanisms that monitor successful completion of key events at various points throughout the cell cycle, referred to as checkpoints (16). If a defect is sensed, a signal transduction pathway becomes active, leading to an arrest or delay in the cell cycle so that the defect can be corrected. Yeast has proven to be a useful organism to study how a checkpoint signal is established, the mechanisms that detect and relay these signals to downstream targets, and the manner by which these targets lead to cell-cycle arrest (reviewed in references 9, 11, 28, and 33). As cells of Saccharomyces cerevisiae progress through the mitotic cell cycle, checkpoint mechanisms monitor such events as DNA replication, spindle assembly, bud morphogenesis, and nuclear migration. Defects in these events, as well as externally promoted perturbations, such as DNA damage, trigger checkpoint-mediated cell-cycle arrest. An additional checkpoint, the meiotic recombination checkpoint, operates during meiosis in response to defects in meiotic recombination and chromosome synapsis...

SUMMARY The effectiveness of RNA interference (RNAi) in many organisms is potentiated through the signal-amplifying activity of a targeted RNA directed RNA polymerase (RdRP) system that can convert a small population of exogenously-encountered dsRNA fragments into an abundant internal pool of small interfering RNA (siRNA). As for any biological amplification system, we expect an underlying architecture that will limit the ability of a randomly encountered trigger to produce an uncontrolled and self-escalating response. Investigating such limits in C. elegans, we find that feed-forward amplification is limited by a critical biosynthetic and structural distinction at the RNA level between (i) triggers that can produce amplification and (ii) siRNA products of the amplification reaction. By assuring that initial (primary) siRNAs can act as triggers but not templates for activation, and that the resulting (secondary) siRNAs can enforce gene silencing on additional targets without unbridled trigger amplification, the system achieves substantial but fundamentally limited signal amplification.