Topoisomerase 1 facilitates nucleosome reassembly at stress genes during recovery.

Chromatin remodeling is essential to allow full development of alternative gene expression programs in response to environmental changes. In fission yeast, oxidative stress triggers massive transcriptional changes including the activation of hundreds of genes, with the participation of histone modifying complexes and chromatin remodelers. DNA transcription is associated to alterations in DNA topology, and DNA topoisomerases facilitate elongation along gene bodies. Here, we test whether the DNA topoisomerase Top1 participates in the RNA polymerase II-dependent activation of the cellular response to oxidative stress. Cells lacking Top1 are resistant to H2O2 stress. The transcriptome of Δtop1 strain was not greatly affected in the absence of stress, but activation of the anti-stress gene expression program was more sustained than in wild-type cells. Top1 associated to stress open reading frames. While the nucleosomes of stress genes are partially and transiently evicted during stress, the chromatin configuration remains open for longer times in cells lacking Top1, facilitating RNA polymerase II progression. We propose that, by removing DNA tension arising from transcription, Top1 facilitates nucleosome reassembly and works in synergy with the chromatin remodeler Hrp1 as opposing forces to transcription and to Snf22 / Hrp3 opening remodelers.

Comparing Mitochondrial Activity, Oxidative Stress Tolerance, and Longevity of Thirteen Ascomycota Yeast Species.

Aging is characterized by a number of hallmarks including loss of mitochondrial homeostasis and decay in stress tolerance, among others. Unicellular eukaryotes have been widely used to study chronological aging. As a general trait, calorie restriction and activation of mitochondrial respiration has been proposed to contribute to an elongated lifespan. Most aging-related studies have been conducted with the Crabtree-positive yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and with deletion collections deriving from these conventional yeast models. We have performed an unbiased characterization of longevity using thirteen fungi species, including S. cerevisiae and S. pombe, covering a wide range of the Ascomycota clade. We have determined their mitochondrial activity by oxygen consumption, complex IV activity, and mitochondrial redox potential, and the results derived from these three methodologies are highly overlapping. We have phenotypically compared the lifespans of the thirteen species and their capacity to tolerate oxidative stress. Longevity and elevated tolerance to hydrogen peroxide are correlated in some but not all yeasts. Mitochondrial activity per se cannot anticipate the length of the lifespan. We have classified the strains in four groups, with members of group 1 (Kluyveromyces lactis, Saccharomyces bayanus and Lodderomyces elongisporus) displaying high mitochondrial activity, elevated resistance to oxidative stress, and elongated lifespan.

Formation of Transient Protein Aggregate-like Centers Is a General Strategy Postponing Degradation of Misfolded Intermediates.

When misfolded intermediates accumulate during heat shock, the protein quality control system promotes cellular adaptation strategies. In Schizosaccharomyces pombe, thermo-sensitive proteins assemble upon stress into protein aggregate-like centers, PACs, to escape from degradation. The role of this protein deposition strategy has been elusive due to the use of different model systems and reporters, and to the addition of artificial inhibitors, which made interpretation of the results difficult. Here, we compare fission and budding yeast model systems, expressing the same misfolding reporters in experiments lacking proteasome or translation inhibitors. We demonstrate that mild heat shock triggers reversible PAC formation, with the collapse of both reporters and chaperones in a process largely mediated by chaperones. This assembly postpones proteasomal degradation of the misfolding reporters, and their Hsp104-dependent disassembly occurs during stress recovery. Severe heat shock induces formation of cytosolic PACs, but also of nuclear structures resembling nucleolar rings, NuRs, presumably to halt nuclear functions. Our study demonstrates that these distantly related yeasts use very similar strategies to adapt and survive to mild and severe heat shock and that aggregate-like formation is a general cellular scheme to postpone protein degradation and facilitate exit from stress.

Expression of the H2O2 Biosensor roGFP-Tpx1.C160S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals.

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

Perturbed fatty-acid metabolism is linked to localized chromatin hyperacetylation, increased stress-response gene expression and resistance to oxidative stress.

Oxidative stress is associated with cardiovascular and neurodegenerative diseases, diabetes, cancer, psychiatric disorders and aging. In order to counteract, eliminate and/or adapt to the sources of stress, cells possess elaborate stress-response mechanisms, which also operate at the level of regulating transcription. Interestingly, it is becoming apparent that the metabolic state of the cell and certain metabolites can directly control the epigenetic information and gene expression. In the fission yeast Schizosaccharomyces pombe, the conserved Sty1 stress-activated protein kinase cascade is the main pathway responding to most types of stresses, and regulates the transcription of hundreds of genes via the Atf1 transcription factor. Here we report that fission yeast cells defective in fatty acid synthesis (cbf11, mga2 and ACC/cut6 mutants; FAS inhibition) show increased expression of a subset of stress-response genes. This altered gene expression depends on Sty1-Atf1, the Pap1 transcription factor, and the Gcn5 and Mst1 histone acetyltransferases, is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters, and results in increased cellular resistance to oxidative stress. We propose that changes in lipid metabolism can regulate the chromatin and transcription of specific stress-response genes, which in turn might help cells to maintain redox homeostasis.

Stress-induced cell depolarization through the MAP kinase-Cdc42 axis.

General stress responses, which sense environmental or endogenous signals, aim at promoting cell survival and fitness during adverse conditions. In eukaryotes, mitogen-activated protein (MAP) kinase-driven cascades trigger a shift in the cell's gene expression program as a cellular adaptation to stress. Here, we review another aspect of activated MAP kinase cascades reported in fission yeast: the transient inhibition of cell polarity in response to oxidative stress. The phosphorylation by a stress-activated MAP kinase of regulators of the GTPase cell division cycle 42 (Cdc42) causes a transient inhibition of polarized cell growth. The formation of growth sites depends on limiting and essential polarity components. We summarize here some processes in which inhibition of Cdc42 may be a general mechanism to regulate polarized growth also under physiological conditions.

A new negative feedback mechanism for MAPK pathway inactivation through Srk1 MAPKAP kinase.

The fission yeast mitogen-activated kinase (MAPK) Sty1 is essential for cell survival in response to different environmental insults. In unstimulated cells, Sty1 forms an inactive ternary cytoplasmatic complex with the MAPKK Wis1 and the MAPKAP kinase Srk1. Wis1 phosphorylates and activates Sty1, inducing the nuclear translocation of the complex. Once in the nucleus, Sty1 phosphorylates and activates Srk1, which in turns inhibits Cdc25 and cell cycle progression, before being degraded in a proteasome-dependent manner. In parallel, active nuclear Sty1 activates the transcription factor Atf1, which results in the expression of stress response genes including pyp2 (a MAPK phosphatase) and srk1. Despite its essentiality in response to stress, persistent activation of the MAPK pathway can be deleterious and induces cell death. Thus, timely pathway inactivation is essential to ensure an appropriate response and cell viability. Here, uncover a role for the MAPKAP kinase Srk1 as an essential component of a negative feedback loop regulating the Sty1 pathway through phosphorylation and inhibition of the Wis1 MAPKK. This feedback regulation by a downstream kinase in the pathway highlights an additional mechanism for fine-tuning of MAPK signaling. Thus, our results indicate that Srk1 not only facilitates the adaptation to stress conditions by preventing cell cycle progression, but also plays an instrumental role regulating the upstream kinases in the stress MAPK pathway.

A stress-blinded Atf1 can fully assemble heterochromatin in a RNAi-independent minimal mat locus but impairs directionality of mat2/3 switching.

The MAP kinase Sty1 phosphorylates and activates the transcription factor Atf1 in response to several stress conditions, which then shifts from a transcriptional repressor to an activator. Atf1 also participates in heterochromatin assembly at the mat locus, in combination with the RNA interference (RNAi) machinery. Here, we study the role of signal-dependent phosphorylation of Atf1 in heterochromatin establishment at mat, using different Atf1 phospho mutants. Although a hypo-phosphorylation Atf1 mutant, Atf1.10M, mediates heterochromatin assembly, the phosphomimic Atf1.10D is unable to maintain silencing. In a minimal mat locus, lacking the RNAi-recruiting cis elements and displaying intermediate silencing, Atf1.10M restores full heterochromatin and silencing. However, evolution experiments with this stress-blinded Atf1.10M show that it is unable to facilitate switching between the donor site mat3 and mat1. We propose that the unphosphorylated, inactive Atf1 contributes to proper heterochromatin assembly by recruiting repressive complexes, but its stress-dependent phosphorylation is required for recombination/switching to occur.

Native RNA sequencing in fission yeast reveals frequent alternative splicing isoforms.

The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies higher than 20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make about 80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects.

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.

TOR and MAP kinase pathways synergistically regulate autophagy in response to nutrient depletion in fission yeast.

General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired.Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.

SWI/SNF and RSC remodeler complexes bind to MBF-dependent genes.

In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism that involves the co-repressors Yox1 and Nrm1 that are loaded onto MBF at the end of S phase, while positive transactivation is achieved through the constitutive binding of the co-activator Rep2. Here we show that Rep2 is required to fully recruit the chromatin remodelers SWI/SNF and RSC to MBF-regulated promoters. On the contrary, Nrm1 and Yox1, when bound to the MBF complex, block the approximation of these chromatin remodelers to MBF-regulated promoters. We propose that SWI/SNF and RSC are recruited to MBF-regulated genes, and RSC together with SAGA complex are important to regulate the G1-to-S transcriptional wave. Mutants of these remodeler complexes are highly sensitive when cells are exposed to insults that challenge DNA synthesis.

Evolution of the Early Spliceosomal Complex-From Constitutive to Regulated Splicing.

Pre-mRNA splicing is a major process in the regulated expression of genes in eukaryotes, and alternative splicing is used to generate different proteins from the same coding gene. Splicing is a catalytic process that removes introns and ligates exons to create the RNA sequence that codifies the final protein. While this is achieved in an autocatalytic process in ancestral group II introns in prokaryotes, the spliceosome has evolved during eukaryogenesis to assist in this process and to finally provide the opportunity for intron-specific splicing. In the early stage of splicing, the RNA 5' and 3' splice sites must be brought within proximity to correctly assemble the active spliceosome and perform the excision and ligation reactions. The assembly of this first complex, termed E-complex, is currently the least understood process. We focused in this review on the formation of the E-complex and compared its composition and function in three different organisms. We highlight the common ancestral mechanisms in S. cerevisiae, S. pombe, and mammals and conclude with a unifying model for intron definition in constitutive and regulated co-transcriptional splicing.

Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42.

Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1-kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation.

Cross talk between the upstream exon-intron junction and Prp2 facilitates splicing of non-consensus introns.

Splicing of mRNA precursors is essential in the regulation of gene expression. U2AF65 recognizes the poly-pyrimidine tract and helps in the recognition of the branch point. Inactivation of fission yeast U2AF65 (Prp2) blocks splicing of most, but not all, pre-mRNAs, for reasons that are not understood. Here, we have determined genome-wide the splicing efficiency of fission yeast cells as they progress into synchronous meiosis in the presence or absence of functional Prp2. Our data indicate that in addition to the splicing elements at the 3' end of any intron, the nucleotides immediately upstream the intron will determine whether Prp2 is required or dispensable for splicing. By changing those nucleotides in any given intron, we regulate its Prp2 dependency. Our results suggest a model in which Prp2 is required for the coordinated recognition of both intronic ends, placing Prp2 as a key regulatory element in the determination of the exon-intron boundaries.

The Mitochondria-to-Cytosol H2O2 Gradient Is Caused by Peroxiredoxin-Dependent Cytosolic Scavenging.

Fluorescent protein-based reporters used to measure intracellular H2O2 were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular H2O2, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible H2O2 in the cytosol from mitochondrial origin unless the main H2O2 scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and ∆tpx1 cells. We conclude that there is a bidirectional flux of H2O2 across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.

The Hsp40 Mas5 Connects Protein Quality Control and the General Stress Response through the Thermo-sensitive Pyp1.

Upon heat shock, the fission yeast Hsp40 chaperone Mas5 drives temperature-sensitive proteins toward protein aggregate centers (PACs) to avoid their degradation until lower temperatures favor their refolding. We show here that cells lacking Mas5 are resistant to oxidative stress. Components of the general stress pathways, the MAP kinase Sty1 and the transcription factor Atf1, are suppressors of this phenotype. Strain Δmas5 expresses higher levels of Sty1- and Atf1-dependent stress genes than wild-type cells. Pyp1, the main tyrosine phosphatase maintaining Sty1 inactive in the absence of stress, is a temperature-sensitive protein that aggregates upon temperature up-shifts in a Mas5-dependent manner. In strain Δmas5, Pyp1 is sent to proteasomal degradation even in the absence of stress. We propose that Pyp1 is a thermo-sensitive phosphatase, which during heat stress coalescences into PACs in a Mas5-dependent manner, to promote full activation of the anti-stress Sty1-Atf1 cascade.

Phosphorylation of the Transcription Factor Atf1 at Multiple Sites by the MAP Kinase Sty1 Controls Homologous Recombination and Transcription.

Transcription factors are often the downstream effectors of signaling cascades. In fission yeast, the transcription factor Atf1 is phosphorylated by the MAP kinase Sty1 under several environmental stressors to promote transcription initiation of stress genes. However, Sty1 and Atf1 have also been involved in other cellular processes such as homologous recombination at hotspots, ste11 gene expression during mating and meiosis, or regulation of fbp1 gene transcription under glucose starvation conditions. Using different phospho-mutants of Atf1, we have investigated the role of Atf1 phosphorylation by Sty1 in those biological processes. An Atf1 mutant lacking the canonical MAP kinase phosphorylation sites cannot activate fbp1 transcription when glucose is depleted, but it is still able to induce recombination at ade6.M26 and to induce ste11 after nitrogen depletion; in these last cases, Sty1 is still required, suggesting that additional non-canonical sites are activating the transcription factor. In all cases, an Atf1 phosphomimetic mutant bypasses the requirement of the Sty1 kinase in these diverse biological processes, highlighting the essential role of the DNA binding factor Atf1 on chromatin remodeling and cell adaptation to nutritional changes. We propose that post-translational modifications of Atf1 by Sty1, either at canonical or non-canonical sites, are sufficient to activate some of the functions of Atf1, those involving chromatin remodeling and transcription initiation. However, in the case of fbp1 where Atf1 acts synergistically with other transcription factors, elimination of the canonical sites is sufficient to hamper some of the interactions required in this complex scenario and to impair transcription initiation.

Chaperone-Facilitated Aggregation of Thermo-Sensitive Proteins Shields Them from Degradation during Heat Stress.

Cells have developed protein quality-control strategies to manage the accumulation of misfolded substrates during heat stress. Using a soluble reporter of misfolding in fission yeast, Rho1.C17R-GFP, we demonstrate that upon mild heat shock, the reporter collapses in protein aggregate centers (PACs). They contain and/or require several chaperones, such as Hsp104, Hsp16, and the Hsp40/70 couple Mas5/Ssa2. Stress granules do not assemble at mild temperatures and, therefore, are not required for PAC formation; on the contrary, PACs may serve as nucleation centers for the assembly of stress granules. In contrast to the general belief, the dominant fate of these PACs is not degradation, and the aggregated reporter can be disassembled by chaperones and recovers native structure and activity. Using mass spectrometry, we show that thermo-unstable endogenous proteins form PACs as well. In conclusion, formation of PACs during heat shock is a chaperone-mediated adaptation strategy.

Identification of ubiquitin-proteasome system components affecting the degradation of the transcription factor Pap1.

Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H2O2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade.