Kenichi Sajiki

Genetic control of cellular quiescence in S. pombe

Transition from proliferation to quiescence brings about extensive changes in cellular behavior and structure. However, the genes that are crucial for establishing and/or maintaining quiescence are largely unknown. The fission yeast Schizosaccharomyces pombe is an excellent model in which to study this problem, because it becomes quiescent under nitrogen starvation. Here, we characterize 610 temperature-sensitive mutants, and identify 33 genes that are required for entry into and maintenance of quiescence. These genes cover a broad range of cellular functions in the cytoplasm, membrane and nucleus. They encode proteins for stress-responsive and cell-cycle kinase signaling pathways, for actin-bound and osmo-controlling endosome formation, for RNA transcription, splicing and ribosome biogenesis, for chromatin silencing, for biosynthesis of lipids and ATP, for cell-wall and membrane morphogenesis, and for protein trafficking and vesicle fusion. We specifically highlight Fcp1, a CTD phosphatase of RNA polymerase II, which differentially affects the transcription of genes that are involved in quiescence and proliferation. We propose that the transcriptional role of Fcp1 is central in differentiating quiescence from proliferation.

Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases

In dividing fission yeast Schizosaccharomyces pombe cells, the balance between Wee1 kinase and Cdc25 phosphatase which control the cyclin-dependent kinase (CDK) at the G2–M transition determines the rod-shaped cell length. Under nitrogen source starvation or glucose limitation, however, cell size determination is considerably modulated, and cell size shortening occurs for wild-type cells. For several mutants of kinases or phosphatases, including CDK, target of rapamycin complex (TORC) 1 and 2, stress-responsive mitogen-activated protein kinase (MAPK) Sty1/Spc1, MAPK kinase Wis1, calcium- and calmodulin-dependent protein kinase kinase-like Ssp1, and type 2A and 2A-related phosphatases inhibitor Sds23, this cell shortening does not normally occur. In tor1 and ssp1 mutants, cell elongation is observed. Sds23 that binds to and inhibits 2A and 2A-related phosphatases is synergistic with Ssp1 in the cell size determination and survival under low glucose and nitrogen source. Tor2 (TORC1) is required for growth, whereas Tor1 (TORC2) is needed for determining division size according to different nutrient conditions. Surprisingly, in growth-diminished tor2 mutant or rapamycin-treated cells, the requirement of separase/Cut1-securin/Cut2 essential for chromosome segregation is greatly alleviated. By contrast, defects of tor1 with secruin/cut2 or overproduction of Cut1 are additive. While Tor1 and Tor2 are opposite in their apparent functions, both may actually coordinate cell division with growth in response to the changes in nutrients.

Metabolomic Analysis of Fission Yeast at the Onset of Nitrogen Starvation

Microorganisms naturally respond to changes in nutritional conditions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively studied. Here, we report time course metabolomic analysis during one hour immediately after nitrogen starvation, prior to any visible changes in cell morphology except for a tiny increase of cell length per division cycle. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min, when trehalose, 2-oxoglutarate, and succinate increased, while purine biosynthesis intermediates rapidly diminished. At 30–60 min, free amino acids decreased, although several modified amino acids—including hercynylcysteine sulfoxide, a precursor to ergothioneine—accumulated. Most high-energy metabolites such as ATP, S-adenosyl-methionine or NAD+ remained stable during the whole time course. Very rapid metabolic changes such as the shut-off of purine biosynthesis and the rise of 2-oxoglutarate and succinate can be explained by the depletion of NH4Cl. The changes in the levels of key metabolites, particularly 2-oxoglutarate, might represent an important mechanistic step to trigger subsequent cellular regulations.

RNA pol II transcript abundance controls condensin accumulation at mitotically up‐regulated and heat‐shock‐inducible genes in fission yeast

Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP‐seq). We found that condensin binds to RNA polymerase I‐, II‐ and III‐transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat‐shock‐inducible genes, we show that pol II‐mediated transcripts cause condensin accumulation. First, condensins enrichment on mitotically activated genes was abolished by deleting the sep1+ gene that encodes an M‐phase‐specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat‐shock protein genes in interphase and even during mid‐mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II‐mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.

Schizosaccharomyces pombe centromere protein Mis19 links Mis16 and Mis18 to recruit CENP-A through interacting with NMD factors and the SWI/SNF complex.

CENP‐A is a centromere‐specific variant of histone H3 that is required for accurate chromosome segregation. The fission yeast Schizosaccharomyces pombe and mammalian Mis16 and Mis18 form a complex essential for CENP‐A recruitment to centromeres. It is unclear, however, how the Mis16‐Mis18 complex achieves this function. Here, we identified, by mass spectrometry, novel fission yeast centromere proteins Mis19 and Mis20 that directly interact with Mis16 and Mis18. Like Mis18, Mis19 and Mis20 are localized at the centromeres during interphase, but not in mitosis. Inactivation of Mis19 in a newly isolated temperature‐sensitive mutant resulted in CENP‐A delocalization and massive chromosome missegregation, whereas Mis20 was dispensable for proper chromosome segregation. Mis19 might be a bridge component for Mis16 and Mis18. We isolated extragenic suppressor mutants for temperature‐sensitive mis18 and mis19 mutants and used whole‐genome sequencing to determine the mutated sites. We identified two groups of loss‐of‐function suppressor mutations in non‐sense‐mediated mRNA decay factors (upf2 and ebs1), and in SWI/SNF chromatin‐remodeling components (snf5, snf22 and sol1). Our results suggest that the Mis16‐Mis18‐Mis19‐Mis20 CENP‐A‐recruiting complex, which is functional in the G1‐S phase, may be counteracted by the SWI/SNF chromatin‐remodeling complex and non‐sense‐mediated mRNA decay, which may prevent CENP‐A deposition at the centromere.

Cellular robustness conferred by genetic crosstalk underlies resistance against chemotherapeutic drug doxorubicin in fission yeast.

Doxorubicin is an anthracycline antibiotic that is among one of the most commonly used chemotherapeutic agents in the clinical setting. The usage of doxorubicin is faced with many problems including severe side effects and chemoresistance. To overcome these challenges, it is important to gain an understanding of the underlying molecular mechanisms with regards to the mode of action of doxorubicin. To facilitate this aim, we identified the genes that are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe. We further demonstrated interplay between factors controlling various aspects of chromosome metabolism, mitochondrial respiration and membrane transport. In the nucleus we observed that the subunits of the Ino80, RSC, and SAGA complexes function in the similar epistatic group that shares significant overlap with the homologous recombination genes. However, these factors generally act in synergistic manner with the chromosome segregation regulator DASH complex proteins, possibly forming two major arms for regulating doxorubicin resistance in the nucleus. Simultaneous disruption of genes function in membrane efflux transport or the mitochondrial respiratory chain integrity in the mutants defective in either Ino80 or HR function resulted in cumulative upregulation of drug-specific growth defects, suggesting a rewiring of pathways that synergize only when the cells is exposed to the cytotoxic stress. Taken together, our work not only identified factors that are required for survival of the cells in the presence of doxorubicin but has further demonstrated that an extensive molecular crosstalk exists between these factors to robustly confer doxorubicin resistance.

Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione-related and other defense gene deletions

Living organisms have evolved multiple sophisticated mechanisms to deal with reactive oxygen species. We constructed a collection of twelve single‐gene deletion strains of the fission yeast Schizosaccharomyces pombe designed for the study of oxidative and heavy metal stress responses. This collection contains deletions of biosynthetic enzymes of glutathione (Δgcs1 and Δgsa1), phytochelatin (Δpcs2), ubiquinone (Δabc1) and ergothioneine (Δegt1), as well as catalase (Δctt1), thioredoxins (Δtrx1 and Δtrx2), Cu/Zn‐ and Mn‐ superoxide dismutases (SODs; Δsod1 and Δsod2), sulfiredoxin (Δsrx1) and sulfide‐quinone oxidoreductase (Δhmt2). First, we employed metabolomic analysis to examine the mutants of the glutathione biosynthetic pathway. We found that ophthalmic acid was produced by the same enzymes as glutathione in S. pombe. The identical genetic background of the strains allowed us to assess the severity of the individual gene knockouts by treating the deletion strains with oxidative agents. Among other results, we found that glutathione deletion strains were not particularly sensitive to peroxide or superoxide, but highly sensitive to cadmium stress. Our results show the astonishing diversity in cellular adaptation mechanisms to various types of oxidative and metal stress and provide a useful tool for further research into stress responses.

Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

Genetic regulation of mitotic competence in G0 quiescent cells

Among 85 genes that maintain mitotic competence in quiescent eukaryotic cells, Nem1 protects nuclei from autophagy by regulating lipin. Quiescent (G0 phase) cells must maintain mitotic competence (MC) to restart the cell cycle. This is essential for reproduction in unicellular organisms and also for development and cell replacement in higher organisms. Recently, suppression of MC has gained attention as a possible therapeutic strategy for cancer. Using a Schizosaccharomyces pombe deletion-mutant library, we identified 85 genes required to maintain MC during the G0 phase induced by nitrogen deprivation. G0 cells must recycle proteins and RNA, governed by anabolism, catabolism, transport, and availability of small molecules such as antioxidants. Protein phosphatases are also essential to maintain MC. In particular, Nem1-Spo7 protects the nucleus from autophagy by regulating Ned1, a lipin. These genes, designated GZE (G-Zero Essential) genes, reveal the landscape of genetic regulation of MC.

The putative ceramide-conjugation protein Cwh43 regulates G0 quiescence, nutrient metabolism and lipid homeostasis in fission yeast

ABSTRACT 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 mutants, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 cells apparently consumed glucose in the culture medium. Furthermore, we found that cwh43 mutant cells had elevated levels of triacylglycerols (TGs) and coenzyme A, and that they accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in the cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen sources, as well as storage lipid metabolism. These results may fit a notion developed in budding yeast stating that Cwh43 conjugates ceramide to glycosylphosphatidylinositol (GPI)-anchored proteins and maintains integrity of membrane organization. Summary: A characterization of fission yeast temperature-sensitive mutants of the putative ceramide-conjugation protein Cwh43, is presented in order to explore its relevance to utilization of glucose, nitrogen source and lipids.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. Jo ur na l o f C el l S ci en ce • A cc ep te d m an us cr ip t