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Heterochromatin that depends on histone H3 lysine 9 methylation (H3K9me) renders embedded genes transcriptionally silent 1 – 3 . In the fission yeast Schizosaccharomyces pombe , H3K9me heterochromatin can be transmitted through cell division provided the counteracting demethylase Epe1 is absent 4 , 5 . Heterochromatin heritability might allow wild-type cells under certain conditions to acquire epimutations, which could influence phenotype through unstable gene silencing rather than DNA change 6 , 7 . Here we show that heterochromatin-dependent epimutants resistant to caffeine arise in fission yeast grown with threshold levels of caffeine. Isolates with unstable resistance have distinct heterochromatin islands with reduced expression of embedded genes, including some whose mutation confers caffeine resistance. Forced heterochromatin formation at implicated loci confirms that resistance results from heterochromatin-mediated silencing. Our analyses reveal that epigenetic processes promote phenotypic plasticity, letting wild-type cells adapt to unfavourable environments without genetic alteration. In some isolates, subsequent or coincident gene-amplification events augment resistance. Caffeine affects two anti-silencing factors: Epe1 is downregulated, reducing its chromatin association, and a shortened isoform of Mst2 histone acetyltransferase is expressed. Thus, heterochromatin-dependent epimutation provides a bet-hedging strategy allowing cells to adapt transiently to insults while remaining genetically wild type. Isolates with unstable caffeine resistance show cross-resistance to antifungal agents, suggesting that related heterochromatin-dependent processes may contribute to resistance of plant and human fungal pathogens to such agents. Fission yeast grown in sublethal levels of caffeine develop heterochromatin-dependent epimutations conferring unstable heritable gene silencing that conveys resistance to caffeine, while remaining genetically wild type.

Heterochromatin formation involves histone H3 methylation, with H3K9me2 defining a distinct heterochromatin state that is transcriptionally permissive and can couple with RNAi, and the transition to non-permissive H3K9me3 required for the epigenetic heritability of heterochromatin. A transcriptionally permissive heterochromatin state Heterochromatin silences transcription of repetitive DNA elements and transposons, yet its formation involves a co-transcriptional mechanism that paradoxically promotes small RNA generation to initiate histone H3K9 methylation. Here, Danesh Moazed and colleagues show that, in fission yeast, H3K9me2 defines a distinct heterochromatin state that is transcriptionally permissive. The transition from H3K9me2 to the silent state marked by H3K9me3 seems to be required for the epigenetic heritability of heterochromatin. Given the conservation of H3K9 methylation in heterochromatin, a similar mechanism is likely to be used in other organisms. Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 . H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA 11 , 15 , 18 . Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.

Non-thermal plasma (NTP) generates a complex mixture of reactive oxygen and nitrogen species (RONS) that can impose strong oxidative stress on eukaryotic cells. While the antimicrobial potential of NTP has been widely explored, much less is known about how eukaryotic cells respond to prolonged NTP-induced stress at the cellular and molecular level. Here, we investigated the cellular effects of extended NTP exposure using the fission yeast Schizosaccharomyces pombe as a non-pathogenic eukaryotic model. Our results indicate that extended exposure to NTP significantly reduces cell viability and is associated with increased oxidative stress, as evidenced by increased levels of intracellular RONS and mitochondrial superoxide. These oxidative changes were accompanied by pronounced cellular responses including tubulin depolymerisation, cell cycle arrest, and impaired cell division. In contrast, no significant changes were detected in the expression of genes involved in oxidative stress response and DNA repair. The observed effects are based on cellular, phenotypic, and transcriptomic analyses, while direct identification of oxidatively modified proteins remains to be addressed in future studies. Key points • NTP increases intracellular RONS and mitochondrial superoxide levels • NTP causes tubulin depolymerisation, which is associated with cell cycle arrest • NTP alters the expression of genes involved in post-transcriptional regulation Graphical Abstract

Acrylamide (AA) poses a significant risk to living organisms as it is linked to serious health concerns. AA exposure triggers oxidative stress in cells through elevated ROS and modulation of antioxidant enzymes activities and expression of genes encoding antioxidant enzymes. AA-induced cell proliferation defects are linked to affected cell cycle regulation demonstrated by changes in the expression of genes encoding the major cell cycle regulators cdc2 , cdc13 , and cdc25 , Additionally, cell division defects can be linked to changes in the expression of ark1 and cdc15 , and AA-induced errors in chromosome segregation. The stress response involves signaling pathways like MAPKs (Mitogen-activated protein kinases) or the target of rapamycin (TOR) constituting two complexes TORC 1 and 2. As TORC2 manages the cell response to various stresses, its involvement in AA-mediated stress has been demonstrated by changes in the expression of tor1 , wat1 , ste20 , sin1 , bit61 encoding TORC2 members, and gad8 encoding a direct Tor1substrate, Gad8. To our surprise, AA has not affected the expression of sty1 , which encodes the major stress-regulating kinase of the MAPK pathway in S. pombe . In the presented study we demonstrate, for the first time, that exposure to AA disrupts cellular homeostasis by altering TORC2 signaling and cell cycle regulation ultimately leading to carcinogenesis.

This study was designed to identify the natural product compound produced by the actinomycete strain “S149” which is capable of inducing a strong small cell phenotype, called wee, in the fission yeast Schizosaccharomyces pombe . We purified the bioactive molecule, which, on the basis of mass spectrometry data, was identified as a novel zinc-bound form of the previously published molecule JBIR-141. JBIR-141 was not previously known to be a zincophore and does not possess structural features common to other bacterial zinc-binding natural product compounds. Testing the effect of the Zn 2+ bound form of JBIR-14 against a series of S. pombe deletion mutants which express a wee phenotype suggested its potential target in fission yeast is either Pyp1, which is involved in regulation of the onset of mitosis, an important control in the eukaryotic cell cycle, or an element in the Pyp1 signalling pathway.

Fusarium verticillioides poses a high food safety risk worldwide due to its mycotoxin production. Successful control of Fusaria may rely on promising biocontrol agents, including yeasts. Although the fission yeast Schizosaccharomyces pombe tolerated Fusarium mycotoxins well, including zearalenone, T2, deoxynivalenol, and fumonisins (FUMs), it did not significantly inhibit the growth of F. verticillioides. Meanwhile fumonisin B1 (FB1) supplementation did not decrease S. pombe cell density in submerged liquid cultures, the colony-forming capability of the yeast was reduced. RNA sequencing showed that S. pombe genes involved in cell adhesion and flocculation were downregulated after FB1 exposure. In addition, the expression of several hydrolase genes was also altered. In co-cultures with F. verticillioides , genes encoding oxidoreductases and hydrolases and those linked to purine nucleotide metabolisms were downregulated, while the expression of genes involved in membrane and transport processes was increased. The expression of several F. verticillioides genes also changed after co-cultivation. Oxidoreductase, transmembrane transport, and purine metabolism genes were upregulated under co-culturing; meanwhile, hydrolase genes, together with carbon metabolism and polysaccharide catabolism genes, were downregulated. Co-cultivation also decreased fumonisin production via the downregulation of genes FUM19 , FUM21 , and FvATFA encoding the fumonisin transporter, a local Zn(II)2Cys6-type transcriptional regulator and an important global regulator bZIP-type transcription factor, respectively. Although further experiments should clarify the mechanism of the fission yeast-elicited inhibition of fumonisin production, these results may pave the way for the development and implementation of novel, innovative approaches to control mycotoxin production by F. verticillioides in the feed and food chain. Key points • 0.5 ppm FB1 reduced the colony-forming ability of S. pombe and caused transcriptional changes. • Expression of transport and hydrolase genes changed in yeast during co-cultivation with mold. • Two FUM cluster genes and FvATFA were downregulated in Fusarium co-cultured with S. pombe.

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2 -mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is essential for the correct folding of proteins entering the secretory pathway and forming membranes. However, the precise mechanisms by which disruptions in protein folding within the ER activate proteostatic mechanisms remain to be fully elucidated. In this study, we demonstrate that in Schizosaccharomyces pombe the antineoplastic agent hydroxyurea (HU) induces a transient perinuclear ER expansion, Bip1 accumulation, and the clustering of nuclear pore complexes in a specific region of the nuclear envelope. This striking phenotype is mimicked by diamide (DIA), a specific inducer of thiol stress, and can be prevented or rapidly reversed by dithiothreitol, a reducing agent, suggesting that ER expansion results from disulfide stress. Furthermore, HU or DIA treatments resulted in the accumulation of misfolded proteins in cytoplasmic foci containing Hsp104 disaggregase and Hsp70/Ssa1 chaperones. Our data show that HU impacts redox-dependent protein folding, impairs the secretory pathway, and activates specific proteostatic mechanisms in both the ER and the cytoplasm.

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(2)2G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.

We identify the S UMO‐ T argeted Ub iquitin L igase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO‐like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N‐terminus. STUbL‐mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8‐Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5–Slx8. Cells lacking Slx8‐Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8‐Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8‐Rfp mutants have similar DNA repair defects.