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H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification., Competing Interests: The authors have declared that no competing interests exist.

Heat-shock protein 90 (Hsp90) was recently identified as a silencing factor required for RNA interference (RNAi)-dependent heterochromatin assembly in the fission yeast Schizosaccharomyces pombe. As Hsp90 is known to contribute to the formation of small RNA-containing effector complexes, it would be expected that Hsp90 is also involved in the RNAi pathway in fission yeast. However, upon investigation, we found it very difficult to determine how Hsp90 modulates RNAi-dependent heterochromatin assembly in the cell. A lack of detectable small interfering RNAs in hsp90 mutant cells prevented us from examining the role of Hsp90 in the siRNA loading in the cell. In addition, deletion of genes encoding co-chaperones for Hsp90 appears not to affect RNAi-dependent pericentromeric silencing. One possible approach for elucidating the role of Hsp90 in RNAi-dependent heterochromatin assembly is the use of forward genetic screens to identify novel factors linking Hsp90 with other known RNAi factors. Here, we discuss the benefits of conducting further screenings and present some technical hints to help identify new factors.

Background: Heat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. While the RNAi process is coupled with heterochromatin assembly in several model organisms, it remains unclear whether the Hsps contribute to epigenetic gene regulation. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type-I Hsp40 protein) in RNAi-dependent heterochromatin assembly., Results: Using a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing factors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi-dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial reduction in the level of dimethylation of histone H3 at Lys9 at the pericentromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA-induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5., Conclusions: Our results suggest that the Hsps Hsp90 and Mas5 contribute to RNAi-dependent heterochromatin assembly. In particular, Mas5 appears to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of the epigenome in other organisms.

Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation., Competing Interests: The authors declare no conflict of interest.

Heterochromatin at the pericentromeric repeats in fission yeast is assembled and spread by an RNAi-dependent mechanism, which is coupled with the transcription of non-coding RNA from the repeats by RNA polymerase II. In addition, Rrp6, a component of the nuclear exosome, also contributes to heterochromatin assembly and is coupled with non-coding RNA transcription. The multi-subunit complex Mediator, which directs initiation of RNA polymerase II-dependent transcription, has recently been suggested to function after initiation in processes such as elongation of transcription and splicing. However, the role of Mediator in the regulation of chromatin structure is not well understood. We investigated the role of Mediator in pericentromeric heterochromatin formation and found that deletion of specific subunits of the head domain of Mediator compromised heterochromatin structure. The Mediator head domain was required for Rrp6-dependent heterochromatin nucleation at the pericentromere and for RNAi-dependent spreading of heterochromatin into the neighboring region. In the latter process, Mediator appeared to contribute to efficient processing of siRNA from transcribed non-coding RNA, which was required for efficient spreading of heterochromatin. Furthermore, the head domain directed efficient transcription in heterochromatin. These results reveal a pivotal role for Mediator in multiple steps of transcription-coupled formation of pericentromeric heterochromatin. This observation further extends the role of Mediator to co-transcriptional chromatin regulation., Competing Interests: The authors have declared that no competing interests exist.

In Schizosaccharomyces pombe, the RNA interference (RNAi) machinery converts pericentromeric transcripts into small interfering RNAs (siRNAs) and is required for the assembly of pericentromeric heterochromatin. Here we describe a mutation in the second largest subunit of RNA polymerase II (RNAPII). Both wild-type and mutant RNAPII localized to the pericentromere. However, the mutation resulted in the loss of heterochromatic histone modifications and in the accumulation of pericentromeric transcripts, accompanied by the loss of siRNAs. This phenotype resembles mutants in RNAi and suggests that RNAPII couples pericentromeric transcription with siRNA processing and heterochromatin assembly.

Background: Heat-shock molecular chaperone proteins (Hsps) promote the loading of small interfering RNA (siRNA) onto RNA interference (RNAi) effector complexes. While the RNAi process is coupled with heterochromatin assembly in several model organisms, it remains unclear whether the Hsps contribute to epigenetic gene regulation. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type-I Hsp40 protein) in RNAi-dependent heterochromatin assembly. Results: Using a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing factors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi-dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial reduction in the level of dimethylation of histone H3 at Lys9 at the pericentromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA-induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5. Conclusions: Our results suggest that the Hsps Hsp90 and Mas5 contribute to RNAi-dependent heterochromatin assembly. In particular, Mas5 appears to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of the epigenome in other organisms. [ABSTRACT FROM AUTHOR]