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To initiate a discussion of the relative merits of various cell systems in acute toxicity testing we have considered the suitability of seven systems in testing three types of toxicity i.e. systemic toxicity in general, systemic toxicity to specific target organs, and local toxicity Available data indicate that systemic toxicity can be predicted by simple systems with non-differentiated cell lines, as well as by primary cultures of hepatocytes. The combined use of both cell systems is probably the method of choice. Specific, cellular, target organ toxicity is often tested in primary cultures of various specific cells. Such use of these cells seems to be irrational, and ought to be replaced by a parallel use of specific and non-specific cell cultures. An analysis of the differential cytotoxicity to both cell types would then point out specific target organ toxicity. It is uncertain whether non-specific cell lines or primary cultures of target-specific cells should be used in tests of local toxicity. Future parallel use of both cell types to produce data on differential cytotoxicity would settle the question.

SummaryCentromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms, including the fission yeast, Schizosaccharomyces pombe, the RNAi pathway contributes to the formation of silent chromatin in pericentromeric regions. Little is known about how chromatin-remodeling factors contribute to heterochromatic integrity and centromere function. Here we show that the histone chaperone and remodeling complex FACT is required for centromeric-heterochromatin integrity and accurate chromosome segregation. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for pob3+ are viable and alleviate gene silencing at centromeric repeats and at the silent mating-type locus. Importantly, like heterochromatin and RNAi pathway mutants, Pob3 null strains exhibit lagging chromosomes on anaphase spindles. Whereas the processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, Swi6-association with the centromere is reduced. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in heterochromatin integrity and in centromere function.

In Schizosaccharomyces pombe the mating-type information is stored at two transcriptionally silent loci (mat2 and mat3). The region between these sites (K region) is inert for meiotic crossing over. The mating-type genes (M or P) are expressed only when present at a third, active locus (mat1). We have earlier shown that the positional regulation of P genes is based on repression at the silent site, caused by elements in the flanking DNA sequences. In this study we have mutagenized a sterile mat1 deleted strain and selected for cells that are able to conjugate. Recessive mutations of this type should define genes encoding trans-acting factors involved in repression of the silent mating-type loci. Before this work mutations in two genes, clr1 and swi6, had been shown to allow both expression of the silent loci and recombination in the K region. The sensitivity of the present selection is demonstrated by the isolation of new mutations that derepress one or both of the silent loci (M-mating or bi-mating). The frequency of M-mating mutants was almost two orders of magnitude higher than that of bi-mating mutants and in all mutants analyzed mat3-M expression was significantly higher than mat2-P expression. The mutations define three new genes, clr2, clr3 and clr4. In addition we show that the rik1 mutant previously known to allow recombination in the K region also depresses the silent loci.

International audience; Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown also to affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localise with the three silent chromosomal regions. Here the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated in further detail. Fluorescence in situ hybridisation (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalised from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents which destabilise microtubules and show a synergistic interaction with a mutation in the beta-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres which is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation.

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.

Cohesin is a highly conserved ring-shaped protein complex made of four essential subunits: Psm1, Psm3, Rad21 and Psc3. By its ability to capture DNA molecules within its ring-like structure, cohesion plays a key role in many cellular processes such as chromosome segregation, DNA damage signalling and repair, transcriptional gene regulation and nuclear organization. To ensure all of its biological functions, cohesin must be tightly regulated in space and time. This regulation relies in part on the control of cohesin binding to chromatin (DNA capture). Cohesin recruitment to chromatin requires the action of a “loading complex” made of two conserved and essential proteins named Mis4 and Ssl3 in the fission yeast. How this complex regulates where and when DNA capture by the cohesin ring must occur remains poorly understood. To identify regulators of cohesin binding to chromatin we have performed a genetic screen for suppressors of the thermosensitive mutation mis4-367. This genetic screen has led to the identification of the cyclin-dependent-kinase Pef1 that acts as a negative regulator of sister chromatids cohesion may be bynegatively controlling cohesin binding to chromatin. Strong experimental evidences indicate that Pef1 exerts its function at least in part by directly phosphorylating the Rad21 subunit of the cohesin complex. Interestingly, a genetic screen made in parallel identified the Pph3/Psy2 phosphatase as implicated in the establishment of sister chromatid cohesion by regulating Rad21 dephosphorylation. Strikingly, the control of Rad21 phosphorylation status appears central to the cohesion process in the fission yeast S. pombe., Le complexe cohésine est un complexe protéique en forme d'anneau composé de quatre sous-unités essentielles très conservées: Smc1, Smc3, Rad21 et Scc3. Par sa capacité à encercler les molécules d’ADN, les cohésines participent à de nombreux processus cellulaires tels que la ségrégation des chromosomes, la signalisation et la réparation des dommages à l’ADN, la régulation de la transcription et l'organisation du génome. Pour assurer ces différentes fonctions biologiques les cohésines doivent être finement régulées à la fois dans le temps et l’espace. Ces régulations reposent en partie sur le contrôle de leur association à la chromatine (capture de l’ADN). Cela nécessite l'action d'un «facteur de chargement » composé de deux protéines conservées et essentielles, Mis4 et Ssl3 chez la levure S. pombe. Comment ce complexe régule la capture de l’ADN par l’anneau de cohésine dans l'espace et le temps demeure à ce jour très mal compris. Afin d’identifier des régulateurs de l’association des cohésines à la chromatine, nous avons réalisé un crible génétique visant à rechercher des suppresseurs de la mutation thermosensible mis4-367. Ce crible a conduit à l’identification de la Cyclin-Dependent Kinase Pef1 qui agit comme un régulateur négatif de la cohésion des chromatides soeurs en contrôlant vraisemblablement négativement l’association des cohésines à la chromatine. De forts arguments expérimentaux indiquent que Pef1 exerce sa fonction en régulant directement la phosphorylation de la sous-unité Rad21 du complexe cohésine. De façon intéressante, via un autre crible génétique, nous avons identifié la phosphatase Pph3/Psy2 qui joue un rôle dans l’établissement de la cohésion des chromatides soeurs en contrôlant la déphosphorylation de Rad21.Ensemble, ces données suggèrent que le contrôle de l’état de phosphorylation de la sous-unité Rad21 du complexe cohésine joue un rôle central dans le processus de cohésion chez la levure S. Pombe.