The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions-centromeres, the mating-type region, and telomeres-and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1 þ gene causes centromerespecific decreases in Swi6 localization and histone H3-K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating-type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1-mediated establishment steps differently than do other heterochromatic regions.

The RNA-induced transcriptional silencing (RITS) complex, containing Ago1, Chp1, Tas3 and centromeric small interfering RNAs (siRNAs), is required for heterochromatic gene silencing at centromeres. Here, we identify a second fission yeast Argonaute complex (Argonaute siRNA chaperone, ARC), which contains, in addition to Ago1, two previously uncharacterized proteins, Arb1 and Arb2, both of which are required for histone H3 Lys9 (H3-K9) methylation, heterochromatin assembly and siRNA generation. Furthermore, whereas siRNAs in the RITS complex are mostly single-stranded, siRNAs associated with ARC are mostly double-stranded, indicating that Arb1 and Arb2 inhibit the release of the siRNA passenger strand from Ago1. Consistent with this observation, purified Arb1 inhibits the slicer activity of Ago1 in vitro, and purified catalytically inactive Ago1 contains only double-stranded siRNA. Finally, we show that slicer activity is required for the siRNA-dependent association of Ago1 with chromatin and for the spreading of histone H3-K9 methylation.

Heterochromatic gene silencing at the pericentromeric DNA repeats in fission yeast requires the RNA interference (RNAi) machinery. The RNA-induced transcriptional silencing (RITS) complex mediates histone H3 lysine 9 (H3K9) methylation and recruits the RNA-dependent RNA polymerase complex (RDRC) to promote double-stranded RNA (dsRNA) synthesis and siRNA generation. Here we show that ectopic expression of a long hairpin RNA bypasses the requirement for chromatin-dependent steps in siRNA generation. The ability of hairpin-produced siRNAs to silence homologous sequences in trans is subject to local chromatin structure, requires HP1, and correlates with antisense transcription at the target locus. Furthermore, although hairpin siRNAs can be produced in the absence of RDRC, trans-silencing of reporter genes by hairpin-produced siRNAs is completely dependent on the dsRNA synthesis activity of RDRC. These results provide insights into the regulation of siRNA action and reveal roles for cis-dsRNA synthesis and HP1 in siRNA-mediated heterochromatin assembly.

Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase (Pol ) or lacking one of the nonessential and histone fold motif-containing subunits of Pol , Dpb3 and Dpb4. We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, we demonstrate that without Pol and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol and ISW2/yCHRAC function independently: Pol operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.The eukaryotic genome is packaged into either euchromatin or heterochromatin. Heterochromatin is defined cytologically as the fraction of the genome that is constitutively condensed throughout the cell cycle (25) and corresponds to regions of highly repeated DNA such as centromeres or telomeres (13). In this region, DNA-mediated metabolisms, such as transcription, recombination, and replication, are generally inactive. In the case of chromosomal DNA replication, heterochromatin replicates in late S phase, while euchromatic regions replicate in early S phase (27).In a simple eukaryote, Saccharomyces cerevisiae, although its genome is too small to define heterochromatin cytologically, the HMR and HML cryptic mating-type loci, ribosomal DNA (rDNA) repeats, and telomeres are loci affected by silencing (22,26,52). Like heterochromatin in higher eukaryotes, HM loci and telomeric regions are replicated in late S phase (17,47), and these silenced regions are associated with histone hypoacetylation (5,8,23,39). The silencing process has three phases, establishment or assembly of a silent chromatin, its maintenance through the cell cycle, and its inheritance to daughter cells, as described by Lau et al. (35). Factors that are involved in these phases of silencing are identified from studies of mutant strains that lose silencing. Mutations in SIR2, SIR3, and SIR4 eliminate the silencing at HM loci, whereas mutations in SIR1 partially reduce it, because Sir1 is required only for establishment, whereas Sir2, Sir3, and Sir4 are required for both establishment and maintenance (45). Sir2, Sir3, and Sir4 form a Sir complex at HM loci (40). The Sir complex does not directly bind DNA but rather interacts with DNA-binding factors, including Rap1, Orc, and Abf1, that bind to silencer DNA elements ...

Highlights d rDNA copy loss associates with SIR2 repression and rDNA copy recovery d UAF, an activator complex for RNA polymerase I, monitors rDNA copy loss d UAF binds to the SIR2 promoter to repress and induce rDNA copy recovery d The amount of UAF determines the rDNA copy number