Heterochromatin in eukaryotic genomes regulates diverse chromosomal processes including transcriptional silencing. However, in Schizosaccharomyces pombe RNA polymerase II (RNAPII) transcription of centromeric repeats is essential for RNA-interference-mediated heterochromatin assembly. Here we study heterochromatin dynamics during the cell cycle and its effect on RNAPII transcription. We describe a brief period during the S phase of the cell cycle in which RNAPII preferentially transcribes centromeric repeats. This period is enforced by heterochromatin, which restricts RNAPII accessibility at centromeric repeats for most of the cell cycle. RNAPII transcription during S phase is linked to loading of RNA interference and heterochromatin factors such as the Ago1 subunit of the RITS complex and the Clr4 methyltransferase complex subunit Rik1 (ref. 7). Moreover, Set2, an RNAPII-associated methyltransferase that methylates histone H3 lysine 36 at repeat loci during S phase, acts in a pathway parallel to Clr4 to promote heterochromatin assembly. We also show that phosphorylation of histone H3 serine 10 alters heterochromatin during mitosis, correlating with recruitment of condensin that affects silencing of centromeric repeats. Our analyses suggest at least two distinct modes of heterochromatin targeting to centromeric repeats, whereby RNAPII transcription of repeats and chromodomain proteins bound to methylated histone H3 lysine 9 mediate recruitment of silencing factors. Together, these processes probably facilitate heterochromatin maintenance through successive cell divisions.

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,[...

An epistasis map (E-MAP) was constructed in the fission yeast, Schizosaccharomyces pombe, by systematically measuring the phenotypes associated with pairs of mutations. This high-density, quantitative genetic interaction map focused on various aspects of chromosome function, including transcription regulation and DNA repair/replication. The E-MAP uncovered a previously unidentified component of the RNA interference (RNAi) machinery (rsh1) and linked the RNAi pathway to several other biological processes. Comparison of the S. pombe E-MAP to an analogous genetic map from the budding yeast revealed that, whereas negative interactions were conserved between genes involved in similar biological processes, positive interactions and overall genetic profiles between pairs of genes coding for physically associated proteins were even more conserved. Hence, conservation occurs at the level of the functional module (protein complex), but the genetic cross talk between modules can differ substantially.Genetic interactions report on the extent to which the function of one gene depends on the presence of a second. This phenomenon, known as epistasis, can be used for defining functional relationships between genes and the pathways in which the corresponding proteins function. Two main categories of genetic interactions exist: (i) negative (e.g., synthetic sickness/ lethality) and (ii) positive (e.g., suppression). We have developed a quantitative approach, termed epistasis map (E-MAP), allowing us to measure the whole spectrum of genetic interactions, both positive and negative (1,2). In budding yeast, Saccharomyces cerevisiae, it has been demonstrated that positive genetic interactions can identify pairs of genes whose products are physically associated and/or function in the same pathway (1,2), whereas negative interactions exist between genes acting on parallel pathways (3,4).We developed the Pombe Epistasis Mapper (PEM) approach (5) that allows high-throughput generation of double mutants in the fission yeast, Schizosaccharomyces pombe. Fission yeast is more similar to metazoans than is S. cerevisiae, owing to its large complex centromere structure, the restriction of spindle construction to mitotic entry, gene regulation by histone methylation and chromodomain heterochromatin proteins, gene and transposon regulation by the RNA interference (RNAi) pathway, and the widespread presence of introns in genes. To further study these processes and to try to understand how genetic interaction networks have evolved (6), we generated an E-MAP in S. pombe that focuses on nuclear function, designed to be analogous to one we created in budding yeast (2). An E-MAP in S. pombeUsing our PEM system (5), we generated a quantitative genetic interaction map in S. pombe, comprising ~118,000 distinct double mutant combinations among 550 genes involved in various aspects of chromosome function ( Fig. 1A and tables S1 and S4) (7). The genes on the map were chosen on the basis of a previous budding yeast E-MAP (1,2) and also included factor...