Centromere silencing and function in fission yeast is governed by the amino terminus of histone H3

BACKGROUND: Centromeric domains often consist of repetitive elements that are assembled in specialized chromatin, characterized by hypoacetylation of histones H3 and H4 and methylation of lysine 9 of histone H3 (K9-MeH3). Perturbation of this underacetylated state by transient treatment with histone deacetylase inhibitors leads to defective centromere function, correlating with delocalization of the heterochromatin protein Swi6/HP1. Likewise, deletion of the K9-MeH3 methyltransferase Clr4/Suvar39 causes defective chromosome segregation. Here, we create fission yeast strains retaining one histone H3 and H4 gene; the creation of these strains allows mutation of specific N-terminal tail residues and their role in centromeric silencing and chromosome stability to be investigated. RESULTS: Reduction of H3/H4 gene dosage to one-third does not affect cell viability or heterochromatin formation. Mutation of lysines 9 or 14 or serine 10 within the amino terminus of histone H3 impairs centromere function, leading to defective chromosome segregation and Swi6 delocalization. Surprisingly, silent centromeric chromatin does not require the conserved lysine 8 and 16 residues of histone H4. CONCLUSIONS: To date, mutation of conserved N-terminal residues in endogenous histone genes has only been performed in budding yeast, which lacks the Clr4/Suvar39 histone methyltransferase and Swi6/HP1. We demonstrate the importance of conserved residues within the histone H3 N terminus for the maintenance of centromeric heterochromatin in fission yeast. In sharp contrast, mutation of two conserved lysines within the histone H4 tail has no impact on the integrity of centromeric heterochromatin. Our data highlight the striking divergence between the histone tail requirements for the fission yeast and budding yeast silencing pathways.     

cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site

BACKGROUND: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established. RESULTS: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9. CONCLUSIONS: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.     

Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin

The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.     

Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4‐mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4‐mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi‐deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.     

Role of Swi6/HP1 self-association-mediated recruitment of Clr4/Suv39 in establishment and maintenance of heterochromatin in fission yeast

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.     

A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission yeast

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(+) gene causes centromere-specific 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.