RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.

The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.

RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. Here we show that RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, our analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance.Heterochromatic chromosomal domains occur in a wide range of eukaryotes and have a crucial role in regulation of gene expression, sister chromatid cohesion and maintenance of genomic stability 1,2 . The genome of the fission yeast Schizosaccharomyces pombe contains large blocks of heterochromatin associated with pericentromeric repeats, telomeres and the silent mating-type region (mat) 2 . Heterochromatin assembly at these loci involves a conserved array of histone modifications that includes histone deacetylation and the Clr4-mediated methylation of histone H3 at Lys9 (H3-Lys9), which is essential for the recruitment of chromodomain protein Swi6, a homolog of the fruit fly and mammalian HP1 proteins [3][4][5] .The mechanisms that specify particular chromosomal regions as sites of heterochromatin assembly are not known. It is well known, however, that the main targets of heterochromatin assembly are DNA repetitive elements such as transposons and satellite repeats present at pericentric regions in large eukaryotic genomes 2,6,7 . The RNA interference (RNAi) pathway 8,9 might be involved in heterochromatin nucleation at repeat loci 10-15 . In S. pombe, deletion of components of the RNAi pathway such as Argonaute (Ago1), Dicer (Dcr1) or RNA-dependent RNA polymerase (Rdp1) disrupts heterochromatinmediated silencing that correlates with loss of H3-Lys9 methylation and Swi6 association with heterochromatic loci 14,15 . In addition, siRNAs corresponding to centromeric repeats have been identified 16 , and the centromere-homologous cenH sequence (96% similar to dg and dh centromeric repeats 17 ) found at the mat locus is an RNAidependent heterochromatin nucleation center 14 . An RNAi effector complex (RITS) containing Chp1, Tas3 and Ago1 is involved in heterochromatin assembly 18 . RITS contains siRNAs that are believed to serve as specificity determinants for targeting RNAi effector complexes to homologous sequences 8,[...