| Heterochromatic or silent DNA domains are a conserved feature of eukaryotic chromosomes and play important roles in the regulation of gene expression and maintenance of chromosome stability. In the fission yeast, Schizosaccharomyces pombe, epigenetic inheritance of pericentromeric heterochromatin involves processing noncoding RNAs into small interfering RNAs (siRNAs) by the RNA interference (RNAi) machinery. It is unclear precisely how RNAi targets Clr4, the enzyme responsible for methylating histone H3 on lysine 9 (H3K9). Here we investigate the regulation of this RNA-dependent targeting mechanism.;Through the biochemical purification of Rik1, previously shown to interact with Clr4, we identified a complex, CLRC. Upon deletion of any CLRC subunit, we observe a loss of silencing at all heterochromatic regions, accumulation of centromeric noncoding RNAs, and a loss of centromeric siRNAs, indicating that the entire complex plays a role in RNAi-mediated heterochromatin assembly. We then found that CLRC physically interacts with the RNAi complexes. Moreover, artificially tethering Rik1 directly to nascent transcripts results in silencing that depends on RNAi. Remarkably, this silencing is independent of Clr4 and CLRC, suggesting Rik1 is sufficient to target RNAi to transcripts, directly linking CLRC and RNAi.;In cells expressing catalytically inactive Clr4, which cannot methylate H3K9, we noticed decreased association of CLRC with RNAi as well as diminished levels of siRNAs generated from the centromeric dg repeats. Surprisingly, despite the requirement for Clr4 activity, cells carrying H3K9 mutations have relatively high levels of dg siRNAs. Together, these results indicate that Clr4 activity is not only required to mediate the interactions between CLRC and RNAi, but also is required for dg siRNA generation. Furthermore, the latter requirement reflects the activity of Clr4 towards a target(s) other than H3K9.;Our investigations revealed that Clr4 resides in a complex (CLRC), which physically interacts with the RNAi machinery. This interaction is bridged by the Rik1 subunit of CLRC and depends on the enzymatic activity of Clr4 likely towards a target(s) other than H3K9. Overall, these results have provided insights into the mechanism of RNAi-dependent recruitment of the H3K9 histone methyltransferase to chromatin and suggest an unexpected chromatin-independent role for Clr4 in regulating RNAi. |
