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.     

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.     

SUMO modification is involved in the maintenance of heterochromatin stability in fission yeast

Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.     

Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.     

Chromodomain-mediated oligomerization of HP1 suggests a nucleosome-bridging mechanism for heterochromatin assembly

HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9-methylated chromatin in vitro relies on an interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl mark recognition is highly sensitive to internucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.