Within a eukaryotic cell, both lipid homeostasis and faithful cell cycle progression are meticulously orchestrated. The fission yeast Schizosaccharomyces pombe provides a powerful platform to study the intricate regulatory mechanisms governing these fundamental processes. In S. pombe, the Cbf11 and Mga2 proteins are transcriptional activators of non-sterol lipid metabolism genes, with Cbf11 also known as a cell cycle regulator. Despite sharing a common set of target genes, little was known about their functional relationship. This study reveals that Cbf11 and Mga2 function together in the same regulatory pathway, critical for both lipid metabolism and mitotic fidelity. Deletion of either gene results in a similar array of defects, including slow growth, dysregulated lipid homeostasis, impaired cell cycle progression (cut phenotype), abnormal cell morphology, perturbed transcriptomic and proteomic profiles, and compromised response to the stressors camptothecin and thiabendazole. Remarkably, the double deletion mutant does not exhibit a more severe phenotype compared to the single mutants. In addition, ChIP-nexus analysis reveals that both Cbf11 and Mga2 bind to nearly identical positions within the promoter regions of target genes. Interestingly, Mga2 binding appears to be dependent on the presence of Cbf11 and Cbf11 likely acts as a tether to DNA, while Mga2 is needed to activate the target genes. In addition, the study explores the distribution of Cbf11 and Mga2 homologs across fungi. The presence of both Cbf11 and Mga2 homologs in Basidiomycota contrasts with Ascomycota, which mostly lack Cbf11 but retain Mga2. This suggests an evolutionary rewiring of the regulatory circuitry governing lipid metabolism and mitotic fidelity. In conclusion, this study offers compelling support for Cbf11 and Mga2 functioning jointly to regulate lipid metabolism and mitotic fidelity in fission yeast.

Department of Cell Biology Faculty of Science Charles University Prague Czechia

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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Hapala I, Griač P, Holič R. Metabolism of Storage Lipids and the Role of Lipid Droplets in the Yeast Schizosaccharomyces pombe. Lipids. 2020;55: 513–535. doi: 10.1002/lipd.12275 PubMed DOI

Holič R, Pokorná L, Griač P. Metabolism of phospholipids in the yeast Schizosaccharomyces pombe. Yeast. 2020;37: 73–92. doi: 10.1002/yea.3451 PubMed DOI

Meyers A, Del Rio ZP, Beaver RA, Morris RM, Weiskittel TM, Alshibli AK, et al.. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe. Traffic. 2016;17: 657–69. doi: 10.1111/tra.12394 PubMed DOI PMC

Meyers A, Chourey K, Weiskittel TM, Pfiffner S, Dunlap JR, Hettich RL, et al.. The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe. J Microbiol. 2017;55: 112–122. doi: 10.1007/s12275-017-6205-1 PubMed DOI

Makarova M, Gu Y, Chen J-S, Beckley JR, Gould KL, Oliferenko S. Temporal Regulation of Lipin Activity Diverged to Account for Differences in Mitotic Programs. Curr Biol. 2016;26: 237–243. doi: 10.1016/j.cub.2015.11.061 PubMed DOI PMC

Yam C, He Y, Zhang D, Chiam K-H, Oliferenko S. Divergent strategies for controlling the nuclear membrane satisfy geometric constraints during nuclear division. Curr Biol. 2011;21: 1314–9. doi: 10.1016/j.cub.2011.06.052 PubMed DOI

Foo S, Cazenave-Gassiot A, Wenk MR, Oliferenko S. Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis. J Cell Sci. 2023;136. doi: 10.1242/jcs.260568 PubMed DOI

Takemoto A, Kawashima SA, Li J-J, Jeffery L, Yamatsugu K, Elemento O, et al.. Nuclear envelope expansion is crucial for proper chromosomal segregation during a closed mitosis. J Cell Sci. 2016;129: 1250–9. doi: 10.1242/jcs.181560 PubMed DOI PMC

Yanagida M. Fission yeast cut mutations revisited: control of anaphase. Trends Cell Biol. 1998;8: 144–9. doi: 10.1016/s0962-8924(98)01236-7 PubMed DOI

Zach R, Převorovský M. The phenomenon of lipid metabolism “cut” mutants. Yeast. 2018;35: 631–637. doi: 10.1002/yea.3358 PubMed DOI

Hughes AL, Todd BL, Espenshade PJ. SREBP pathway responds to sterols and functions as an oxygen sensor in fission yeast. Cell. 2005;120: 831–42. doi: 10.1016/j.cell.2005.01.012 PubMed DOI

Lee C-YS, Yeh T-L, Hughes BT, Espenshade PJ. Regulation of the Sre1 hypoxic transcription factor by oxygen-dependent control of DNA binding. Mol Cell. 2011;44: 225–34. doi: 10.1016/j.molcel.2011.08.031 PubMed DOI PMC

Burr R, Stewart E V., Shao W, Zhao S, Hannibal-Bach HK, Ejsing CS, et al.. Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast. J Biol Chem. 2016;291: 12171–83. doi: 10.1074/jbc.M116.723650 PubMed DOI PMC

Chellappa R, Kandasamy P, Oh CS, Jiang Y, Vemula M, Martin CE. The membrane proteins, Spt23p and Mga2p, play distinct roles in the activation of Saccharomyces cerevisiae OLE1 gene expression. J Biol Chem. 2001;276: 43548–56. doi: 10.1074/jbc.M107845200 PubMed DOI

Převorovský M, Groušl T, Staňurová J, Ryneš J, Nellen W, Půta F, et al.. Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division. Exp Cell Res. 2009;315: 1533–47. doi: 10.1016/j.yexcr.2008.12.001 PubMed DOI

Převorovský M, Oravcová M, Tvarůžková J, Zach R, Folk P, Půta F, et al.. Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles. PLoS One. 2015;10: e0137820. doi: 10.1371/journal.pone.0137820 PubMed DOI PMC

Převorovský M, Půta F, Folk P. Fungal CSL transcription factors. BMC Genomics. 2007;8: 233. doi: 10.1186/1471-2164-8-233 PubMed DOI PMC

Burr R, Stewart E V., Espenshade PJ. Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors. J Biol Chem. 2017;292: 5311–5324. doi: 10.1074/jbc.M117.778209 PubMed DOI PMC

Princová J, Salat-Canela C, Daněk P, Marešová A, de Cubas L, Bähler J, et al.. Perturbed fatty-acid metabolism is linked to localized chromatin hyperacetylation, increased stress-response gene expression and resistance to oxidative stress. PLoS Genet. 2023;19: e1010582. doi: 10.1371/journal.pgen.1010582 PubMed DOI PMC

Zach R, Tvarůžková J, Schätz M, Ťupa O, Grallert B, Převorovský M. Mitotic defects in fission yeast lipid metabolism “cut” mutants are suppressed by ammonium chloride. FEMS Yeast Res. 2018;18: 1–7. doi: 10.1093/femsyr/foy064 PubMed DOI PMC

Marešová A, Oravcová M, Rodríguez-López M, Hradilová M, Zemlianski V, Häsler R, et al.. Critical importance of DNA binding for CSL protein functions in fission yeast. J Cell Sci. 2024;137. doi: 10.1242/jcs.261568 PubMed DOI

Převorovský M, Oravcová M, Zach R, Jordáková A, Bähler J, Půta F, et al.. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast. Cell Cycle. 2016;15: 3082–3093. doi: 10.1080/15384101.2016.1235100 PubMed DOI PMC

Vishwanatha A, Princová J, Hohoš P, Zach R, Převorovský M. Altered cohesin dynamics and H3K9 modifications contribute to mitotic defects in the cbf11Δ lipid metabolism mutant. J Cell Sci. 2023;136. doi: 10.1242/jcs.261265 PubMed DOI

He Q, Johnston J, Zeitlinger J. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat Biotechnol. 2015;33: 395–401. doi: 10.1038/nbt.3121 PubMed DOI PMC

Oravcová M, Teska M, Půta F, Folk P, Převorovský M. Fission yeast CSL proteins function as transcription factors. PLoS One. 2013;8: e59435. doi: 10.1371/journal.pone.0059435 PubMed DOI PMC

Pancaldi V, Saraç OS, Rallis C, McLean JR, Převorovský M, Gould K, et al.. Predicting the fission yeast protein interaction network. G3 (Bethesda). 2012;2: 453–67. doi: 10.1534/g3.111.001560 PubMed DOI PMC

Carpy A, Krug K, Graf S, Koch A, Popic S, Hauf S, et al.. Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast). Mol Cell Proteomics. 2014;13: 1925–36. doi: 10.1074/mcp.M113.035824 PubMed DOI PMC

Marguerat S, Schmidt A, Codlin S, Chen W, Aebersold R, Bähler J. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell. 2012;151: 671–83. doi: 10.1016/j.cell.2012.09.019 PubMed DOI PMC

Převorovský M, Atkinson SR, Ptáčková M, McLean JR, Gould K, Folk P, et al.. N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast. PLoS One. 2011;6: e23650. doi: 10.1371/journal.pone.0023650 PubMed DOI PMC

Larochelle M, Bergeron D, Arcand B, Bachand F. Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast. J Cell Sci. 2019;132. doi: 10.1242/jcs.232249 PubMed DOI

Skruzny M, Pohl E, Abella M. FRET Microscopy in Yeast. Biosensors (Basel). 2019;9: 1–17. doi: 10.3390/bios9040122 PubMed DOI PMC

Selicky T, Jurcik M, Mikolaskova B, Pitelova A, Mayerova N, Kretova M, et al.. Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe. Int J Mol Sci. 2022;23. doi: 10.3390/ijms232112800 PubMed DOI PMC

Zemlianski V, Marešová A, Princová J, Holič R, Häsler R, Ramos Del Río MJ, et al.. Nitrogen availability is important for preventing catastrophic mitosis in fission yeast. J Cell Sci. 2024;137. doi: 10.1242/jcs.262196 PubMed DOI

Hartmuth S, Petersen J. Fission yeast Tor1 functions as part of TORC1 to control mitotic entry through the stress MAPK pathway following nutrient stress. J Cell Sci. 2009;122: 1737–1746. doi: 10.1242/jcs.049387 PubMed DOI

Kettenbach AN, Deng L, Wu Y, Baldissard S, Adamo ME, Gerber SA, et al.. Quantitative phosphoproteomics reveals pathways for coordination of cell growth and division by the conserved fission yeast kinase pom1. Mol Cell Proteomics. 2015;14: 1275–87. doi: 10.1074/mcp.M114.045245 PubMed DOI PMC

Koch A, Krug K, Pengelley S, Macek B, Hauf S. Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast. Sci Signal. 2011;4: rs6. doi: 10.1126/scisignal.2001588 PubMed DOI

Mak T, Jones AW, Nurse P. The TOR-dependent phosphoproteome and regulation of cellular protein synthesis. EMBO J. 2021;40: e107911. doi: 10.15252/embj.2021107911 PubMed DOI PMC

Køhler JB, Tammsalu T, Jørgensen MM, Steen N, Hay RT, Thon G. Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast. Nat Commun. 2015;6: 8827. doi: 10.1038/ncomms9827 PubMed DOI PMC

Zielinska DF, Gnad F, Schropp K, Wiśniewski JR, Mann M. Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core machinery. Mol Cell. 2012;46: 542–8. doi: 10.1016/j.molcel.2012.04.031 PubMed DOI

Beckley JR, Chen J-S, Yang Y, Peng J, Gould KL. A Degenerate Cohort of Yeast Membrane Trafficking DUBs Mediates Cell Polarity and Survival. Mol Cell Proteomics. 2015;14: 3132–41. doi: 10.1074/mcp.M115.050039 PubMed DOI PMC

Strachan J, Leidecker O, Spanos C, Le Coz C, Chapman E, Arsenijevic A, et al.. SUMOylation regulates Lem2 function in centromere clustering and silencing. J Cell Sci. 2023;136. doi: 10.1242/jcs.260868 PubMed DOI PMC

Bhattacharya S, Shcherbik N, Vasilescu J, Smith JC, Figeys D, Haines DS. Identification of Lysines within Membrane-Anchored Mga2p120 that Are Targets of Rsp5p Ubiquitination and Mediate Mobilization of Tethered Mga2p90. J Mol Biol. 2009;385: 718–725. doi: 10.1016/j.jmb.2008.11.018 PubMed DOI PMC

Vasconcelles MJ, Jiang Y, McDaid K, Gilooly L, Wretzel S, Porter DL, et al.. Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. Implications for the conservation of oxygen sensing in eukaryotes. J Biol Chem. 2001;276: 14374–84. doi: 10.1074/jbc.M009546200 PubMed DOI

Zhang S, Skalsky Y, Garfinkel DJ. MGA2 or SPT23 is required for transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae. Genetics. 1999;151: 473–83. Available: http://www.ncbi.nlm.nih.gov/pubmed/9927444 PubMed PMC

Zhang S, Burkett TJ, Yamashita I, Garfinkel DJ. Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1997;17: 4718–29. doi: 10.1128/MCB.17.8.4718 PubMed DOI PMC

Jiang Y, Vasconcelles MJ, Wretzel S, Light A, Martin CE, Goldberg MA. MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae. Mol Cell Biol. 2001;21: 6161–9. doi: 10.1128/MCB.21.18.6161–6169.2001 PubMed DOI PMC

Kwon EG, Laderoute A, Chatfield-Reed K, Vachon L, Karagiannis J, Chua G. Deciphering the transcriptional-regulatory network of flocculation in Schizosaccharomyces pombe. PLoS Genet. 2012;8: e1003104. doi: 10.1371/journal.pgen.1003104 PubMed DOI PMC

Torres-Garcia S, Di Pompeo L, Eivers L, Gaborieau B, White SA, Pidoux AL, et al.. SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast. Wellcome Open Res. 2020;5: 274. doi: 10.12688/wellcomeopenres.16405.1 PubMed DOI PMC

Petersen J, Russell P. Growth and the Environment of Schizosaccharomyces pombe. Cold Spring Harb Protoc. 2016;2016: pdb.top079764. doi: 10.1101/pdb.top079764 PubMed DOI PMC

Rodríguez-López M, Cotobal C, Fernández-Sánchez O, Borbarán Bravo N, Oktriani R, Abendroth H, et al.. A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast. Wellcome Open Res. 2016;1: 19. doi: 10.12688/wellcomeopenres.10038.3 PubMed DOI PMC

Gregan J, Rabitsch PK, Rumpf C, Novatchkova M, Schleiffer A, Nasmyth K. High-throughput knockout screen in fission yeast. Nat Protoc. 2006;1: 2457–64. doi: 10.1038/nprot.2006.385 PubMed DOI PMC

Princová J, Schätz M, Ťupa O, Převorovský M. Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing. J Vis Exp. 2019; 1–9. doi: 10.3791/59889 PubMed DOI

Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc. 2019;14: 68–85. doi: 10.1038/s41596-018-0082-x PubMed DOI

Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc. 2007;2: 1896–906. doi: 10.1038/nprot.2007.261 PubMed DOI

Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, et al.. The one hour yeast proteome. Mol Cell Proteomics. 2014;13: 339–47. doi: 10.1074/mcp.M113.034769 PubMed DOI PMC

Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26: 1367–72. doi: 10.1038/nbt.1511 PubMed DOI

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014;13: 2513–26. doi: 10.1074/mcp.M113.031591 PubMed DOI PMC

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al.. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods. 2016;13: 731–40. doi: 10.1038/nmeth.3901 PubMed DOI

Conway JR, Lex A, Gehlenborg N. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics. 2017;33: 2938–2940. doi: 10.1093/bioinformatics/btx364 PubMed DOI PMC