Altered cohesin dynamics and H3K9 modifications contribute to mitotic defects in the cbf11Δ lipid metabolism mutant.

Mitotic fidelity is crucial for the faithful distribution of genetic information into the daughter cells. Many fungal species, including the fission yeast Schizosaccharomyces pombe, undergo a closed form of mitosis, during which the nuclear envelope does not break down. In S. pombe, numerous processes have been identified that contribute to successful completion of mitosis. Notably, perturbations of lipid metabolism can lead to catastrophic mitosis and the 'cut' phenotype. It has been suggested that these mitotic defects are caused by insufficient membrane phospholipid supply during the anaphase nuclear expansion. However, it is not clear whether additional factors are involved. In this study, we characterized in detail mitosis in an S. pombe mutant lacking the Cbf11 transcription factor, which regulates lipid metabolism genes. We show that in cbf11Δ cells mitotic defects have already appeared prior to anaphase, before the nuclear expansion begins. Moreover, we identify altered cohesin dynamics and centromeric chromatin structure as additional factors affecting mitotic fidelity in cells with disrupted lipid homeostasis, providing new insights into this fundamental biological process.

The phenomenon of lipid metabolism "cut" mutants.

Every cell cycle iteration culminates with the resolution of a mitotic nucleus into a pair of daughter nuclei, which are distributed between the two daughter cells. In the fission yeast Schizosaccharomyces pombe, the faithful division of a mitotic nucleus depends on unperturbed lipogenesis. Upon genetically or chemically induced perturbation of lipid anabolism, S. pombe cells fail to separate the two daughter nuclei and subsequently initiate lethal cytokinesis resulting in the so-called "cut" terminal phenotype. Evidence supporting a critical role of lipid biogenesis in successful mitosis in S. pombe has been accumulating for almost two decades, but the exact mechanism explaining the reported observations had been elusive. Recently, several studies established a functional link between biosynthesis of structural phospholipids, nuclear membrane growth, and the fidelity of "closed" mitosis in S. pombe. These novel insights suggest a mechanistic explanation for the mitotic defects characteristic for some S. pombe mutants deficient in lipid anabolism and extend our knowledge of metabolic modulation within the context of the cell cycle. In this review, we cover the essential role of lipogenesis in "closed" mitosis, focusing mainly on S. pombe as a model system.

Mitotic defects in fission yeast lipid metabolism 'cut' mutants are suppressed by ammonium chloride.

Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.

Correction: Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles.

[This corrects the article DOI: 10.1371/journal.pone.0137820.].

Increased expression of Polδ does not alter the canonical replication program in vivo.

Background: In vitro experiments utilising the reconstituted Saccharomyces cerevisiae eukaryotic replisome indicated that the efficiency of the leading strand replication is impaired by a moderate increase in Polδ concentration. It was hypothesised that the slower rate of the leading strand synthesis characteristic for reactions containing two-fold and four-fold increased concentration of Polδ represented a consequence of a relatively rare event, during which Polδ stochastically outcompeted Polε and, in an inefficient manner, temporarily facilitated extension of the leading strand. Inspired by this observation, we aimed to determine whether similarly increased Polδ levels influence replication dynamics in vivo using the fission yeast Schizosaccharomyces pombe as a model system. Methods: To generate S. pombe strains over-expressing Polδ, we utilised Cre-Lox mediated cassette exchange and integrated one or three extra genomic copies of all four Polδ genes. To estimate expression of respective Polδ genes in Polδ-overexpressing mutants, we measured relative transcript levels of cdc1 + , cdc6 + (or cdc6 L591G ), cdc27 + and cdm1 + by reverse transcription followed by quantitative PCR (RT-qPCR). To assess the impact of Polδ over-expression on cell physiology and replication dynamics, we used standard cell biology techniques and polymerase usage sequencing. Results: We provide an evidence that two-fold and four-fold over-production of Polδ does not significantly alter growth rate, cellular morphology and S-phase duration. Polymerase usage sequencing analysis further indicates that increased Polδ expression does not change activities of Polδ, Polε and Polα at replication initiation sites and across replication termination zones. Additionally, we show that mutants over-expressing Polδ preserve WT-like distribution of replication origin efficiencies. Conclusions: Our experiments do not disprove the existence of opportunistic polymerase switches; however, the data indicate that, if stochastic replacement of Polε for Polδ does occur i n vivo, it represents a rare phenomenon that does not significantly influence canonical replication program.

Activation of Gcn2 in response to different stresses.

All organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation. This pathway requires GCN1, which ensures delivery of the uncharged tRNA onto GCN2. However, Gcn2 is activated by a number of other stresses which do not obviously involve accumulation of uncharged tRNAs, raising the question how Gcn2 is activated under these conditions. Here we investigate the requirement for ongoing translation and tRNA binding for Gcn2 activation after different stresses in fission yeast. We find that mutating the tRNA-binding site on Gcn2 or deleting Gcn1 abolishes Gcn2 activation under all the investigated conditions. These results suggest that tRNA binding to Gcn2 is required for Gcn2 activation not only in response to starvation but also after UV irradiation and oxidative stress.

CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles.

BACKGROUND: Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. CONCLUSIONS/SIGNIFICANCE: Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast.