Spatiotemporal Control of Forkhead Binding to DNA Regulates the Meiotic Gene Expression Program.

Meiosis is a differentiated program of the cell cycle that is characterized by high levels of recombination followed by two nuclear divisions. In fission yeast, the genetic program during meiosis is regulated at multiple levels, including transcription, mRNA stabilization, and splicing. Mei4 is a forkhead transcription factor that controls the expression of mid-meiotic genes. Here, we describe that Fkh2, another forkhead transcription factor that is essential for mitotic cell-cycle progression, also plays a pivotal role in the control of meiosis. Fkh2 binding preexists in most Mei4-dependent genes, inhibiting their expression. During meiosis, Fkh2 is phosphorylated in a CDK/Cig2-dependent manner, decreasing its affinity for DNA, which creates a window of opportunity for Mei4 binding to its target genes. We propose that Fkh2 serves as a placeholder until the later appearance of Mei4 with a higher affinity for DNA that induces the expression of a subset of meiotic genes.

Transcription start site associated RNAs in bacteria.

Here, we report the genome-wide identification of small RNAs associated with transcription start sites (TSSs), termed tssRNAs, in Mycoplasma pneumoniae. tssRNAs were also found to be present in a different bacterial phyla, Escherichia coli. Similar to the recently identified promoter-associated tiny RNAs (tiRNAs) in eukaryotes, tssRNAs are associated with active promoters. Evidence suggests that these tssRNAs are distinct from previously described abortive transcription RNAs. ssRNAs have an average size of 45 bases and map exactly to the beginning of cognate full-length transcripts and to cryptic TSSs. Expression of bacterial tssRNAs requires factors other than the standard RNA polymerase holoenzyme. We have found that the RNA polymerase is halted at tssRNA positions in vivo, which may indicate that a pausing mechanism exists to prevent transcription in the absence of genes. These results suggest that small RNAs associated with TSSs could be a universal feature of bacterial transcription.

Strand-specific deep sequencing of the transcriptome.

Several studies support that antisense-mediated regulation may affect a large proportion of genes. Using the Illumina next-generation sequencing platform, we developed DSSS (direct strand specific sequencing), a strand-specific protocol for transcriptome sequencing. We tested DSSS with RNA from two samples, prokaryotic (Mycoplasma pneumoniae) as well as eukaryotic (Mus musculus), and obtained data containing strand-specific information, using single-read and paired-end sequencing. We validated our results by comparison with a strand-specific tiling array data set for strain M129 of the simple prokaryote M. pneumoniae, and by quantitative PCR (qPCR). The results of DSSS were very well supported by the results from tiling arrays and qPCR. Moreover, DSSS provided higher dynamic range and single-base resolution, thus enabling efficient antisense detection and the precise mapping of transcription start sites and untranslated regions. DSSS data for mouse confirmed strand specificity of the protocol and the general applicability of the approach to studying eukaryotic transcription. We propose DSSS as a simple and efficient strategy for strand-specific transcriptome sequencing and as a tool for genome annotation exploiting the increased read lengths that next-generation sequencing technology now is capable to deliver.

Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway.

Either calorie restriction, loss-of-function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient-dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 mitogen-activated protein (MAP) kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.

C-terminal truncation of the peroxiredoxin Tpx1 decreases its sensitivity for hydrogen peroxide without compromising its role in signal transduction.

Peroxiredoxins (Prxs) participate in hydrogen peroxide (H2O2) scavenging. Eukaryotic Prxs suffer H2O2-dependent inactivation, due to the oxidation of its catalytic cysteine to sulfinic acid, a modification which can be enzymatically reversed. This substrate-mediated reversible inactivation has been suggested to allow eukaryotic Prxs to act as floodgates, permitting high levels of H2O2 to trigger signal transduction. To test this hypothesis, we used the fission yeast Prx Tpx1, which acts as a H2O2 scavenger during aerobic metabolism and also participates in peroxide-induced signal transduction pathways. High concentrations of peroxide reversibly inactivate Tpx1.Here, we describe the characterization of a Tpx1 derivative, which lacks a carboxy-terminal extension present only in eukaryotic Prxs. This mutant protein is not inactivated by high doses of H2O2. Exclusive expression of this truncated version of Tpx1 is deleterious for aerobic growth, but H2O2-dependent signal transduction is not impaired in this strain. Instead, the ability of Tpx1.DeltaCTD to detect and detoxify peroxides is impaired. Our results indicate that inactivation of Tpx1 by excess peroxides is not required for H2O2 signaling towards the Sty1 pathway, as expected from the floodgate model, and that the carboxy-terminal extension of Tpx1 concomitantly improves H2O2 scavenging and increases susceptibility to inactivation.

The peroxiredoxin Tpx1 is essential as a H2O2 scavenger during aerobic growth in fission yeast.

Peroxiredoxins are known to interact with hydrogen peroxide (H(2)O(2)) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H(2)O(2) sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H(2)O(2). We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169.

Oxidative stress in Schizosaccharomyces pombe: different H2O2 levels, different response pathways.

Schizosaccharomyces pombe triggers different signalling pathways depending on the severity of the oxidative stress exerted, the main ones being the Pap1 and the Sty1 pathways. The Pap1 transcription factor is more sensitive to hydrogen peroxide (H(2)O(2)) than the MAP kinase Sty1 pathway, and is designed to induce adaptation, rather than survival, responses. The peroxiredoxin Tpx1 acts as a H(2)O(2) sensor and the upstream activator of the Pap1 pathway. Therefore, sensitivity to H(2)O(2) depends on this thioredoxin peroxidase. In order to achieve maximal activation of the MAP kinase pathway, the concentration of H(2)O(2) needs to be at least fivefold higher than that to fully activate Pap1. Tpx1 is a H(2)O(2) scavenger, thus its peroxidase activity is essential for aerobic growth. As described for other eukaryotic peroxiredoxins, high doses of H(2)O(2) temporarily inactivate Tpx1 and delay Pap1 activation, whereas the Sty1 pathway remains fully functional under these conditions. As part of the Sty1-dependent transcriptional response, the expression of Srx1 is induced and this reductase re-activates the over-oxidised Tpx1. Therefore, the antioxidant pathways of the fission yeast are perfectly designed so that the transcriptional programs triggered by the different signalling pathways never overlap.

The glycolytic metabolite methylglyoxal activates Pap1 and Sty1 stress responses in Schizosaccharomyces pombe.

Methylglyoxal, a toxic metabolite synthesized in vivo during glycolysis, inhibits cell growth. One of the mechanisms protecting eukaryotic cells against its toxicity is the glyoxalase system, composed of glyoxalase I and II (glo1 and glo2), which converts methylglyoxal into d-lactic acid in the presence of glutathione. Here we have shown that the two principal oxidative stress response pathways of Schizosaccharomyces pombe, Sty1 and Pap1, are involved in the response to methylglyoxal toxicity. The mitogen-activated protein kinase Sty1 is phosphorylated and accumulates in the nucleus following methylglyoxal treatment. Moreover, glo2 expression is induced by methylglyoxal and environmental stresses in a Sty1-dependent manner. The transcription factor Pap1 also accumulates in the nucleus, activating the expression of its target genes following methylglyoxal treatment. Our studies showed that the C-terminal cysteine-rich domain of Pap1 is sufficient for methylglyoxal sensing. Furthermore, the redox status of Pap1 is not changed by methylglyoxal. We propose that methylglyoxal treatment triggers Pap1 and Sty1 nuclear accumulation, and we describe the molecular basis of such activation mechanisms. In addition, we discuss the potential physiological significance of these responses to a natural toxic metabolite.

A cysteine-sulfinic acid in peroxiredoxin regulates H2O2-sensing by the antioxidant Pap1 pathway.

The Schizosaccharomyces pombe transcription factor Pap1 regulates antioxidant-gene transcription in response to H2O2. Pap1 activation occurs only at low, but not elevated, H2O2 concentrations that instead strongly trigger the mitogen-activated protein kinase Sty1 pathway. Here, we identify the peroxiredoxin Tpx1 as the upstream activator of Pap1. We show that, at low H2O2 concentrations, this oxidant scavenger can transfer a redox signal to Pap1, whereas higher concentrations of the oxidant inhibit the Tpx1-Pap1 redox relay through the temporal inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid. This cysteine modification can be reversed by the sulfiredoxin Srx1, its expression in response to high doses of H2O2 strictly depending on active Sty1. Thus, Tpx1 oxidation to the cysteine-sulfinic acid and its reversion by Srx1 constitutes a previously uncharacterized redox switch in H2O2 signaling, restricting Pap1 activation within a narrow range of H2O2 concentrations.

Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration.

The transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide-induced responses in Schizosaccharomyces pombe. Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations. The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant. Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide-induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor. We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1-regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide. In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant. We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation-activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1. Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway.

Schizosaccharomyces pombe cells lacking the Ran-binding protein Hba1 show a multidrug resistance phenotype due to constitutive nuclear accumulation of Pap1.

In Schizosaccharomyces pombe, the transcription factor Pap1, and the mitogen-activated protein kinase Sty1 are excluded from the nucleus in a Crm1-dependent manner under non-stressed conditions. Upon oxidant treatment, both Sty1 and Pap1 concentrate into the nucleus, due to an enhanced import or an impaired export. Hba1, a protein that when overexpressed confers brefeldin A resistance, contains a Ran binding domain. The purpose of this project was to understand at the molecular level the role of Hba1 in the S. pombe oxidative stress response. Fluorescent and confocal microscopy studies demonstrate that Hba1 is located at the nucleoplasm and not at the nuclear envelope. We also demonstrate that either multiple copies or deletion of the hba1 gene induces nuclear accumulation of Pap1 and Sty1. We propose that Hba1 assists Crm1 to export some nuclear export signal-containing proteins. Pap1 nuclear accumulation is sufficient for constitutive activation of its specific antioxidant response. On the contrary, constitutive nuclear localization of Sty1 in the Deltahba1 strain does not trigger the Sty1-specific, Atf1-dependent antioxidant response in the absence of stress. We conclude that the increased multidrug resistance of strains lacking or overexpressing Hba1 is due to the accumulation of Pap1 in the nucleus under non-stressed conditions.