Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.

The viscoelastic properties of the cytoplasm of living yeast cells were investigated by studying the motion of lipid granules naturally occurring in the cytoplasm. A large frequency range of observation was obtained by a combination of video-based and laser-based tracking methods. At time scales from 10(-4) to 10(2) s, the granules typically perform subdiffusive motion with characteristics different from previous measurements in living cells. This subdiffusive behavior is thought to be due to the presence of polymer networks and membranous structures in the cytoplasm. Consistent with this hypothesis, we observe that the motion becomes less subdiffusive upon actin disruption.

Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Histone deacetylases (HDACs) are important for gene regulation and the maintenance of heterochromatin in eukaryotes. Schizosaccharomyces pombe was used as a model system to investigate the functional divergence within this conserved enzyme family. S. pombe has three HDACs encoded by the hda1 ؉ , clr3 ؉ , and clr6 ؉ genes. Strains mutated in these genes have previously been shown to display strikingly different phenotypes when assayed for viability, chromosome loss, and silencing. Here, conserved differences in the substrate binding pocket identify Clr6 and Hda1 as class I HDACs, while Clr3 belongs in the class II family. Furthermore, these HDACs were shown to have strikingly different subcellular localization patterns. Hda1 was localized to the cytoplasm, while most of Clr3 resided throughout the nucleus. Finally, Clr6 was localized exclusively on the chromosomes in a spotted pattern. Interestingly, Clr3, the only HDAC present in the nucleolus, was required for ribosomal DNA (rDNA) silencing. Clr3 presumably acts directly on heterochromatin, since it colocalized with the centromere, mating-type region, and rDNA as visualized by in situ hybridization. In addition, Clr3 could be cross-linked to mat3 in chromatin immunoprecipitation experiments. Western analysis of bulk histone preparations indicated that Hda1 (class I) had a generally low level of activity in vivo and Clr6 (class I) had a high level of activity and broad in vivo substrate specificity, whereas Clr3 (class II) displayed its main activity on acetylated lysine 14 of histone H3. Thus, the distinct functions of the S. pombe HDACs are likely explained by their distinct cellular localization and their different in vivo specificities.

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.