Concept: Schizosaccharomyces pombe
Over the last decade, the genome-scale metabolic models have been playing increasingly important roles in elucidating metabolic characteristics of biological systems for a wide range of applications including, but not limited to, system-wide identification of drug targets and production of high value biochemical compounds. However, these genome-scale metabolic models must be able to first predict known in vivo phenotypes before it is applied towards these applications with high confidence. One benchmark for measuring the in silico capability in predicting in vivo phenotypes is the use of single-gene mutant libraries to measure the accuracy of knockout simulations in predicting mutant growth phenotypes.
Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.
Drug-induced haploinsufficiency (DIH) in yeast has been considered a valuable tool for drug target identification. A plant metabolite, plumbagin, has potent anticancer activity via reactive oxygen species (ROS) generation. However, the detailed molecular targets of plumbagin for ROS generation are not understood. Here, using DIH and heterozygous deletion mutants of the fission yeast Schizosaccharomyces pombe, we identified 1, 4-phopshatidylinositol 5-kinase (PI5K) its3 as a new molecular target of plumbagin for ROS generation. Plumbagin showed potent anti-proliferative activity (GI(50); 10 µM) and induced cell elongation and septum formation in wild-type S. pombe. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a crucial role in the anti-proliferative activity in S. pombe. Interestingly, significant DIH was observed in an its3-deleted heterozygous mutant, in which ROS generation by plumbagin was higher than that in wild-type cells, implying that its3 contributes to ROS generation by plumbagin in this yeast. In MCF7 human breast cancer cells, plumbagin significantly decreased the level of a human ortholog, 1, 4-phopshatidylinositol 5-kinase (PI5K)-1B, of yeast its3, and knockdown of PI5K-1B using siPI5K-1B increased the ROS level and decreased cell viability. Taken together, these results clearly show that PI5K-1B plays a crucial role in ROS generation as a new molecular target of plumbagin. Moreover, drug target screening using DIH in S. pombe deletion mutants is a valuable tool for identifying molecular targets of anticancer agents.
In the fission yeast Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP-chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation (Rfl1, Adn2, Adn3, Sre2, and Yox1), in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of mbx2(+) and deletion of rfl1(+) resulted in strong flocculation and transcriptional upregulation of gsf2(+)/pfl1(+) and several other putative flocculin genes (pfl2(+)-pfl9(+)). Overexpression of the pfl(+) genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double pfl(+) overexpression. Among the pfl1(+) genes, only loss of gsf2(+) abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of adn2(+) or adn3(+) overexpression was likely mediated by the transcriptional activation of cell wall-remodeling genes including gas2(+), psu1(+), and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed gsf2(+) expression in an inhibitory feed-forward loop involving mbx2(+). These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall-remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.
BACKGROUND: Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. RESULTS: We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. CONCLUSIONS: We provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.
Regulation of polarised cell growth is essential for many cellular processes including spatial coordination of cell morphology changes during the division cycle. We present a mathematical model of the core mechanism responsible for the regulation of polarised growth dynamics during the fission yeast cell cycle. The model is based on the competition of growth zones localised at the cell tips for a common substrate distributed uniformly in the cytosol. We analyse the bifurcations in this model as the cell length increases, and show that the growth activation dynamics provides an explanation for the new-end take-off (NETO) as a saddle-node bifurcation at which the cell sharply switches from monopolar to bipolar growth. We study the parameter sensitivity of the bifurcation diagram and relate qualitative changes of the growth pattern, e.g. delayed or absent NETO, to previously observed mutant phenotypes. We investigate the effects of imperfect asymmetric cell division, and show that this leads to distinct growth patterns that provide experimentally testable predictions for validating the presented competitive growth zone activation model. Finally we discuss extension of the model for describing mutant cells with more than two growth zones.
BACKGROUND: Replication and transcription, the two key functions of DNA, require unwinding of the DNA double helix. It has been shown that replication origins in the budding yeast, Saccharomyces cerevisiae contain an easily unwound stretch of DNA. We have used a recently developed method for determining the locations and degrees of stress-induced duplex destabilization (SIDD) for all the reported replication origins in the genome of the fission yeast, Schizosaccharomyces pombe. RESULTS: We have found that the origins are more susceptible to SIDD as compared to the non-origin intergenic regions (NOIRs) and genes. SIDD analysis of many known origins in other eukaryotes suggests that SIDD is a common property of replication origins. Interestingly, the previously shown deletion-dependent changes in the activities of the origins of the ura4 origin region on chromosome 3 are paralleled by changes in SIDD properties, suggesting SIDD’s role in origin activity. SIDD profiling following in silico deletions of some origins suggests that many of the closely spaced S. pombe origins could be clusters of two or three weak origins, similar to the ura4 origin region. CONCLUSION: SIDD appears to be a highly conserved, functionally important property of replication origins in S. pombe and other organisms. The distinctly low SIDD scores of origins and the long range effects of genetic alterations on SIDD properties provide a unique predictive potential to the SIDD analysis. This could be used in exploring different aspects of structural and functional organization of origins including interactions between closely spaced origins.
The final step in post-translational processing of Ras and Rho GTPases involves methylation of the prenylated cysteine residue by an isoprenylcysteine-O-carboxyl methyltransferase (ICMT). ICMT activity is essential for cell growth and development in higher eukaryotes, and inhibition of GTPase methylation has become an attractive target in cancer therapy to inactivate prenylated oncoproteins. However, the specificity and dynamics of the GTPase methylation process remain to be fully clarified. Notably, cells lacking Mam4, the ICMT ortholog in the fission yeast Schizosaccharomyces pombe, are viable. We have exploited this feature to analyze the role of methylation on GTPase localization and function. We show that methylation differentially affects GTPase membrane localization, being particularly relevant for plasma membrane tethering and downstream signaling of palmitoylated and farnesylated GTPases Ras1 and Rho2 lacking C-terminal polybasic motifs. Indeed, Ras1 and Rho2 cysteine methylation is required for proper regulation of differentiation elicited by MAPK Spk1 and for stress-dependent activation of the cell integrity pathway (CIP) and its main effector MAPK Pmk1. Further, Mam4 negatively regulates TORC2 signaling by a cross-inhibitory mechanism relying on Rho GTPase methylation. These results highlight the requirement for a tight control of GTPase methylation in vivo to allow adequate GTPase function.
We investigated D-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E. coli displayed oxidase activity to neutral and basic D-amino acids, but not to an L-amino acid or acidic D-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without D-amino acid, and was approximately doubled by adding D-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. L-Alanine also induced the activity, but only by about half of that induced by D-alanine. The induction by D-alanine reached a maximum level at 2 h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was D-alanine, followed by D-proline and then D-serine. Not effective were N-carbamoyl-D,L-alanine (a better inducer of DAO than D-alanine in the yeast Trigonopsis variabilis), and both basic and acidic D-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.
The conserved PIF helicase family appears to function in replication to ensure termination and passage through regions that slow or arrest replication fork movement. Findings in fission yeast extend evidence from budding yeast, and argue for universal mechanisms that ensure replication integrity.