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.     

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.     

Distinct roles for Sir2 and RNAi in centromeric heterochromatin nucleation,spreading and maintenance

Epigenetically regulated heterochromatin domains govern essential cellular activities. A key feature of heterochromatin domains is the presence of hypoacetylated nucleosomes, which are methylated on lysine 9 of histone H3 (H3K9me). Here, we investigate the requirements for establishment, spreading and maintenance of heterochromatin using fission yeast centromeres as a paradigm. We show that establishment of heterochromatin on centromeric repeats is initiated at modular ‘nucleation sites’ by RNA interference (RNAi), ensuring the mitotic stability of centromere‐bearing minichromosomes. We demonstrate that the histone deacetylases Sir2 and Clr3 and the chromodomain protein Swi6^HP1 are required for H3K9me spreading from nucleation sites, thus allowing formation of extended heterochromatin domains. We discovered that RNAi and Sir2 along with Swi6^HP1 operate in two independent pathways to maintain heterochromatin. Finally, we demonstrate that tethering of Sir2 is pivotal to the maintenance of heterochromatin at an ectopic locus in the absence of RNAi. These analyses reveal that Sir2, together with RNAi, are sufficient to ensure heterochromatin integrity and provide evidence for sequential establishment, spreading and maintenance steps in the assembly of centromeric heterochromatin.     

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.     

Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation

Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.     

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres.     

Panspecies Small-Molecule Disruptors of Heterochromatin-Mediated Transcriptional Gene Silencing

Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.     

Centromeres are specialized replication domains in heterochromatin

The properties that define centromeres in complex eukaryotes are poorly understood because the underlying DNA is normally repetitive and indistinguishable from surrounding noncentromeric sequences. However, centromeric chromatin contains variant H3-like histones that may specify centromeric regions. Nucleosomes are normally assembled during DNA replication; therefore, we examined replication and chromatin assembly at centromeres in Drosophila cells. DNA in pericentric heterochromatin replicates late in S phase, and so centromeres are also thought to replicate late. In contrast to expectation, we show that centromeres replicate as isolated domains early in S phase. These domains do not appear to assemble conventional H3-containing nucleosomes, and deposition of the Cid centromeric H3-like variant proceeds by a replication-independent pathway. We suggest that late-replicating pericentric heterochromatin helps to maintain embedded centromeres by blocking conventional nucleosome assembly early in S phase, thereby allowing the deposition of centromeric histones.