Project description:Here, we report the high-throughput profiling of histone modification (H3K9me2) in fission yeast Schizosaccharomyces pombe. We generated genome-wide H3K9me2 maps of fission yeast mutants in swo1-26 (temperature sensitive, ts) cells at 25℃ and 37℃. We find that H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects.
Project description:High-resolution time-course analysis of the fission yeast (S. pombe) cell cycle transcriptional program following synchronization by cdc25 (ts) block and release
Project description:Cellular nutrient states control whether cells proliferate, or whether they enter or exit quiescence. Here, we report characterizations of fission yeast temperature-sensitive (ts) mutants of the evolutionarily conserved transmembrane protein, Cwh43, and explore its relevance to utilization of glucose, nitrogen-source, and lipids. GFP-tagged Cwh43 localizes at ER associated with the nuclear envelope and the plasma membrane, as in budding yeast. We found that cwh43 mutants failed to divide in low glucose and lost viability during quiescence under nitrogen starvation. In cwh43 mutant, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 apparently consumed glucose in the culture media. Furthermore, we found that cwh43 mutant had elevated levels of triacylglycerols (TGs) and coenzyme A, and that it accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen-sources, as well as storage lipid metabolism. These results may fit to a notion developed in budding yeast that Cwh43 conjugates ceramide to GPI (glycosylphosphatidylinositol)-anchored proteins and maintains integrity of membrane organization. </br></br> Lipidome assay data is reported in the current study MTBLS578. </br> Metabolome assay data associated to this study is reported in MTBLS577. </br><br/> Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS577' target='_blank'><span class='label label-success'>MTBLS577</span></a>
Project description:Cellular nutrient states control whether cells proliferate, or whether they enter or exit quiescence. Here, we report characterizations of fission yeast temperature-sensitive (ts) mutants of the evolutionarily conserved transmembrane protein, Cwh43, and explore its relevance to utilization of glucose, nitrogen-source, and lipids. GFP-tagged Cwh43 localizes at ER associated with the nuclear envelope and the plasma membrane, as in budding yeast. We found that cwh43 mutants failed to divide in low glucose and lost viability during quiescence under nitrogen starvation. In cwh43 mutant, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 apparently consumed glucose in the culture media. Furthermore, we found that cwh43 mutant had elevated levels of triacylglycerols (TGs) and coenzyme A, and that it accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen-sources, as well as storage lipid metabolism. These results may fit to a notion developed in budding yeast that Cwh43 conjugates ceramide to GPI (glycosylphosphatidylinositol)-anchored proteins and maintains integrity of membrane organization. </br></br> Metabolome assay data is reported in the current study MTBLS577. </br> Lipidome assay data associated to this study is reported in MTBLS578. </br><br/> Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS578' target='_blank'><span class='label label-success'>MTBLS578</span></a>
Project description:We report the high-throughput profiling of histone modification (H3K9me2) in fission yeast Schizosaccharomyces pombe. By obtaining 1-10 ng immunoprecipitated DNA, we generated genome-wide H3K9me2 maps of fission yeast mutants with either deletions of non-essential kinetochore genes or conditional inactivation (temperature sensitive, ts) mutations in essential ones. We find that the impairment of the kinetochore componnets cause various levels (from no to prominent) of heterochroamatin spreading into centromeric core regions. Hence, we conclude that the integrity of the inner kinetochore is required to maintain normal centromeric chromatin organization as well as distinct centromere identity.
Project description:Transition from proliferation to quiescence brings about extensive changes in cellular behavior and structure. However, genes critical for establishing and/or for maintaining quiescence are largely unknown. The fission yeast S. pombe is found as an excellent model for studying this problem, because it becomes quiescent under nitrogen starvation. Here we characterize 610 temperature-sensitive (ts) mutants, and identify 33 genes required for entry into and the maintenance of quiescence. These genes cover a broad range of cellular functions in the cytoplasm, membrane and the nucleus, encoding proteins for stress-responsive and cell cycle kinase signaling pathway, actin-bound and osmo-controlling endosome formation, RNA transcription, splicing and ribosome biogenesis, chromatin silencing, biosynthesis of lipid and ATP, cell wall and membrane morphogenesis, protein trafficking and vesicle fusion. We specifically highlight Fcp1, CTD phosphatase of RNA polymerase II, which differentially affects transcription of genes involved in quiescence and proliferation. We propose that the transcriptional role of Fcp1 is central to differentiate quiescence from proliferation. Experiment Overall Design: Gene expression profile under nitrogen rich or starved condition at 26ºC or 37ºC in WT (L972) or fcp1-452 ts mutant strain of fission yeast.
Project description:Alternative splicing is prevalent in plants, but little is known about its regulation in the context of developmental and signaling pathways. We describe here a new factor that influences pre-mRNA splicing and is essential for embryonic development in Arabidopsis thaliana. This factor was retrieved in a genetic screen that identified mutants impaired in expression of an alternatively spliced GFP reporter gene. In addition to the known spliceosomal component PRP8, the screen retrieved a previously uncharacterized protein containing a Replication termination factor2 (Rtf2) domain defined by a C2HC2 zinc finger. The Rtf2 protein was discovered in fission yeast, where it stabilizes paused DNA replication forks by an unknown mechanism. When homozygous, a null mutation in Arabidopsis RTF2 (AtRTF2) is embryo-lethal, indicating that it encodes an essential protein. As revealed by quantitative RT-PCR, impaired expression of GFP in atrtf2 and prp8 mutants is attributable to inefficient splicing of the GFP pre-mRNA. A genome-wide analysis using RNA-seq demonstrated that 12% of total introns display a significant degree of retention in atrtf2 mutants. Intron-retaining transcripts are enriched from genes encoding proteins involved in signaling pathways and membrane transport. Affinity purification of AtRTF2 followed by mass spectrometry identified several known and predicted splicing proteins. In a yeast two-hybrid screen, AtRTF2 interacted with Exo70B1, a peripheral subunit of the exocyst, which is involved in vesicle trafficking. Considering these results and previous suggestions that Rtf2 constitutes an ubiquitin-related domain, we discuss possible roles of AtRTF2 in ubiquitin-based regulation of pre-mRNA splicing and membrane signaling to the spliceosome. Rtf2 is SDR1 (= AtRTF2) and was discovered in a genetic suppressor screen using the dms4 mutant. DMS4 was described in Kanno et al (2010) EMBO Rep. 11:65-71. Examination of whole-genome DNA methylation status in transgenic Arabidopsis plants
Project description:RNA-seq analysis compares gene expression of the fission yeast (Schizossacharomyces pombe) ecl123∆ mutant at various times after phosphate starvation (4, 8, 12, 24, 36 and 48h) to the ecl123∆ mutant fission yeast strain grown in phosphate replete conditions (0h, prior to phosphate starvation).
Project description:Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases required for polarized growth and cell shape. Kin1, the sole MARK/PAR-1 family kinase in fission yeast, regulates cell polarity and cytokinesis through unknown mechanisms. We performed a large-scale phosphoproteomics screen to identify and characterize Kin1 substrates in cells.