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Rapid progress in our understanding of chromatin regulation has fueled considerable interest in epigenetic mechanisms governing the stable inheritance of chromatin states. Findings from several systems reveal small RNAs of the RNAi pathway as critical determinants of epigenetic gene silencing. Notably, recent investigations into the mechanisms of RNAi-mediated heterochromatin assembly in the fission yeast Schizosaccharomyces pombe have yielded new insights regarding the roles of RNAi in chromatin regulation and epigenetic inheritance.

Heterochromatin is dynamically regulated during the cell cycle and in response to developmental signals. Recent findings from diverse systems suggest an extensive role for transcription in the assembly of heterochromatin, highlighting the emerging theme that transcription and noncoding RNAs can provide the initial scaffold for the formation of heterochromatin, which serves as a versatile recruiting platform for diverse factors involved in many cellular processes.

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.

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.

International audience; 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.

Histone modifiers play essential roles in controlling transcription and organizing eukaryotic genomes into functional domains. Here, we show that Set1, the catalytic subunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4 methylation (H3K4me), behaves as a repressor of the transcriptome largely independent of Set1C and H3K4me in the fission yeast Schizosaccharomyces pombe. Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1 binding levels do not generally correlate with the levels of transcription. We show that Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci and promoters of stress-response genes. Moreover, we demonstrate that Set1 coordinates with the class II histone deacetylase Clr3 in heterochromatin assembly at prominent chromosomal landmarks and repression of the transcriptome that includes Tf2 retrotransposons, noncoding RNAs, and regulators of development and stress-responses. Our study delineates a molecular framework for elucidating the functional links between transcriptome control and chromatin organization. DOI: http://dx.doi.org/10.7554/eLife.04506.001, eLife digest Genes can be turned on or off at different times in an organism's life. In humans, yeast and other eukaryotes, this is mainly controlled by the way DNA is packaged with proteins—known as histones—in a structure called chromatin. Genes that are switched on, or only temporarily switched off, are associated with areas of the genome where the chromatin is loosely packed. In contrast, genes that remain switched off for long periods of time are found in regions—known as heterochromatin—where the chromatin is tightly packed. There are many enzymes that can modify histones to change the structure of chromatin. One enzyme—called Set1—adds a methyl tag to chromatin, which is known to be associated with genes being switched on. However, Lorenz et al. found that Set1 also has other roles in modifying chromatin in the yeast Schizosaccharomyces pombe. The experiments found that Set1 helps to keep genes switched off and that this role is largely independent of its ability to add the methyl tag to chromatin. Set1 is recruited to many sites across the genome by another protein called Atf1, which is involved in the cell's response to environmental stresses. Lorenz et al. believe that this helps to put these genes in a ‘poised’ off state so that they are ready to be switched on rapidly if needed. Set1 also works with another protein that removes acetyl tags—which encourage chromatin to be less tightly packed—from histones. Together, both proteins contribute to the assembly of heterochromatin and keep genes involved in development and stress responses switched off when they are not required. Collectively, these experiments reveal unexpected and important insights into how Set1—which plays critical roles in many aspects of human health including aging and cancer—works in cells. DOI: http://dx.doi.org/10.7554/eLife.04506.002

In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. Previously, we have shown that RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. Here, we investigate the role of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. We find that Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. We show that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation.

Summary Transcriptional gene silencing (TGS) is the mechanism generally thought by which heterochromatin effects silencing. However, recent discovery in fission yeast of a cis -acting posttranscriptional gene-silencing ( cis -PTGS) pathway operated by the RNAi machinery at heterochromatin challenges the role of TGS in heterochromatic silencing. Here, we describe a multienzyme effector complex (termed SHREC) that mediates heterochromatic TGS in fission yeast. SHREC consists of a core quartet of proteins—Clr1, Clr2, Clr3, and Mit1—which distribute throughout all major heterochromatin domains to effect TGS via distinct activities associated with the histone deacetylase Clr3 and the SNF2 chromatin-remodeling factor homolog Mit1. SHREC is also recruited to the telomeres by multiple independent mechanisms involving telomere binding protein Ccq1 cooperating with Taz1 and the RNAi machinery, and to euchromatic sites, via mechanism(s) distinct from its heterochromatin localization aided by Swi6/HP1. Our analyses suggest that SHREC regulates nucleosome positioning to assemble higher-order chromatin structures critical for heterochromatin functions.