A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures

Histone H2A.Z Cooperates with RNAi and Heterochromatin Factors to Suppress Antisense RNAs

Eukaryotic transcriptomes are characterized by widespread transcription of noncoding and antisense RNAs, which is linked to key chromosomal processes, such as chromatin remodelling, gene regulation and heterochromatin assembly. However, these transcripts can be deleterious, and their accumulation is suppressed by several mechanisms including degradation by the nuclear exosome. The mechanisms by which cells differentiate coding RNAs from transcripts targeted for degradation are not clear. Here we show that the variant histone H2A.Z, which is loaded preferentially at the 5\u27 ends of genes by the Swr1 complex containing a JmjC domain protein, mediates suppression of antisense transcripts in the fission yeast Schizosaccharomyces pombe genome. H2A.Z is partially redundant in this regard with the Clr4 (known as SUV39H in mammals)-containing heterochromatin silencing complex that is also distributed at euchromatic loci, and with RNA interference component Argonaute (Ago1). Loss of Clr4 or Ago1 alone has little effect on antisense transcript levels, but cells lacking either of these factors and H2A.Z show markedly increased levels of antisense RNAs that are normally degraded by the exosome. These analyses suggest that as well as performing other functions, H2A.Z is a component of a genome indexing mechanism that cooperates with heterochromatin and RNAi factors to suppress read-through antisense transcripts

Alp13, an MRG family protein, is a component of fission yeast Clr6 histone deacetylase required for genomic integrity

The post-translational modifications of histones are key to the modulation of chromatin structure. Distinct patterns of modifications established by histone-modifying enzymes control diverse chromosomal processes. Here, we report the purification and molecular characterization of the fission yeast Clr6 histone deacetyl ase involved in higher order chromatin assembly. We show that a chromodomain protein Alp13, which belongs to the conserved MRG protein family linked to cellular senescence in humans, is associated with Clr6. In addition, Clr6 interacts with homologs of the mammalian transcriptional co-repressors Sin3, Pst1 and Pst2, and a WD40 repeat-containing protein, Prw1. Alp13, Pst2 and Prw1 form a stable complex with Clr6 in the nucleus. Deletion of any of these factors causes progressive loss of viability and sensitivity to DNA-damaging agents, and impairs condensation/resolution of chromosomes during mitosis. This is accompanied by hyperacetylation of histones and a reduction in histone H3 Ser10 phosphorylation, which correlates with chromosome condensation during mitosis. These results link the MRG family protein Alp13 to histone deacetylation, and suggest that Clr6 and its associated factors are essential for fundamental chromosomal events

Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin-specific histone tail modifications

Heterochromatin is a functionally important chromosomal component, especially at centromeres. In fission yeast, conserved heterochromatin-specific modifications of the histone H3 tail, involving deacetylation of Lys 9 and Lys 14 and subsequent methylation of Lys 9, promote the recruitment of a heterochromatin protein, Swi6, a homolog of the Drosophila heterochromatin protein 1. However, the primary determinants of the positioning of heterochromatin are still unclear. The fission yeast proteins Abp1, Cbh1, and Cbh2 are homologs of the human protein CENP-B that bind to centromeric α-satellite DNA and associate with centromeric heterochromatin. We show that the CENP-B homologs are functionally redundant at centromeres, and that Abp1 binds specifically to centromeric heterochromatin. In the absence of Abp1 or Cbh1, the centromeric association of Swi6 is diminished, resulting in a decrease in silencing of the region. CENP-B-homolog double disruptants show a synergistic reduction of Swi6 at centromeric heterochromatin, indicating that the three proteins are functionally redundant in the recruitment of Swi6. Furthermore, using chromatin immunoprecipitation assays, we show that disruption of CENP-B homologs causes a decrease in heterochromatin-specific modifications of histone H3. These results indicate that the CENP-B homologs act as site-specific nucleation factors for the formation of centromeric heterochromatin by heterochromatin-specific modifications of histone tails