Pro- and Antioxidant Functions of the Peroxisome-Mitochondria Connection and Its Impact on Aging and Disease

[EN] Peroxisomes and mitochondria are the main intracellular sources for reactive oxygen species. At the same time, both organelles are critical for the maintenance of a healthy redox balance in the cell. Consequently, failure in the function of both organelles is causally linked to oxidative stress and accelerated aging. However, it has become clear that peroxisomes and mitochondria are much more intimately connected both physiologically and structurally. Both organelles share common fission components to dynamically respond to environmental cues, and the autophagic turnover of both peroxisomes and mitochondria is decisive for cellular homeostasis. Moreover, peroxisomes can physically associate with mitochondria via specific protein complexes. Therefore, the structural and functional connection of both organelles is a critical and dynamic feature in the regulation of oxidative metabolism, whose dynamic nature will be revealed in the future. In this review, we will focus on fundamental aspects of the peroxisome-mitochondria interplay derived from simple models such as yeast and move onto discussing the impact of an impaired peroxisomal and mitochondrial homeostasis on ROS production, aging, and disease in humans.Work from the authors’ laboratory was supported by grants from Ministerio de Economía, Industria y Competitividad (BFU2016-75792-R) and from Ministerio de Economía y Competitividad (BFU2011-23326).Pascual-Ahuir Giner, MD.; Manzanares-Estreder, S.; Proft, M. (2017). Pro- and Antioxidant Functions of the Peroxisome-Mitochondria Connection and Its Impact on Aging and Disease. Oxidative Medicine and Cellular Longevity. (9860841). https://doi.org/10.1155/2017/9860841S986084

Stress-Activated Degradation of Sphingolipids Regulates Mitochondrial Function and Cell Death in Yeast

[EN] Sphingolipids are regulators of mitochondria-mediated cell death in higher eukaryotes. Here, we investigate how changes in sphingolipid metabolism and downstream intermediates of sphingosine impinge on mitochondrial function. We found in yeast that within the sphingolipid degradation pathway, the production via Dpl1p and degradation via Hfd1p of hexadecenal are critical for mitochondrial function and cell death. Genetic interventions, which favor hexadecenal accumulation, diminish oxygen consumption rates and increase reactive oxygen species production and mitochondrial fragmentation and vice versa. The location of the hexadecenal-degrading enzyme Hfd1p in punctuate structures all along the mitochondrial network depends on a functional ERMES (endoplasmic reticulum-mitochondria encounter structure) complex, indicating that modulation of hexadecenal levels at specific ER-mitochondria contact sites might be an important trigger of cell death. This is further supported by the finding that externally added hexadecenal or the absence of Hfd1p enhances cell death caused by ectopic expression of the human Bax protein. Finally, the induction of the sphingolipid degradation pathway upon stress is controlled by the Hog1p MAP kinase. Therefore, the stress-regulated modulation of sphingolipid degradation might be a conserved way to induce cell death in eukaryotic organisms.The authors thank Eulalia de Nadal, William Prinz, Benoit Kornmann, Stephen Manon, Benedikt Westermann, and Frank Madeo for the kind gift of yeast strains and plasmids. The authors thank Alba Calatayud for her help with Bax expression experiments and Benito Alarcon for his help with the confocal microscopy. This work was supported by the grants from the Ministerio de Economia y Competitividad (BFU2011-23326 and BFU2016-75792-R).Manzanares-Estreder, S.; Pascual-Ahuir Giner, MD.; Proft, M. (2017). Stress-Activated Degradation of Sphingolipids Regulates Mitochondrial Function and Cell Death in Yeast. Oxidative Medicine and Cellular Longevity. (2708345):1-15. https://doi.org/10.1155/2017/2708345S115270834

Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell

[EN] The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. We will start by comparing recent in vivo procedures to capture gene expression in real time. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. 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The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription invivo and is partially redundant with the histone acetyltransferase, Gcn5. The EMBO Journal, 18(11), 3101-3106. doi:10.1093/emboj/18.11.3101Barbaric, S., Luckenbach, T., Schmid, A., Blaschke, D., Hörz, W., & Korber, P. (2007). Redundancy of Chromatin Remodeling Pathways for the Induction of the Yeast PHO5 Promoter in Vivo. Journal of Biological Chemistry, 282(38), 27610-27621. doi:10.1074/jbc.m700623200Proft, M., & Struhl, K. (2002). Hog1 Kinase Converts the Sko1-Cyc8-Tup1 Repressor Complex into an Activator that Recruits SAGA and SWI/SNF in Response to Osmotic Stress. Molecular Cell, 9(6), 1307-1317. doi:10.1016/s1097-2765(02)00557-9Lemieux, K., & Gaudreau, L. (2004). Targeting of Swi/Snf to the yeast GAL1 UASG requires the Mediator, TAFIIs, and RNA polymerase II. The EMBO Journal, 23(20), 4040-4050. doi:10.1038/sj.emboj.7600416Rienzo, A., Poveda-Huertes, D., Aydin, S., Buchler, N. E., Pascual-Ahuir, A., & Proft, M. (2015). Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-R

Divergence of alternative sugar preferences through modulation of the expression and activity of the Gal3 sensor in yeast

[EN] Optimized nutrient utilization is crucial for the progression of microorganisms in competing communities. Here we investigate how different budding yeast species and ecological isolates have established divergent preferences for two alternative sugar substrates: Glucose, which is fermented preferentially by yeast, and galactose, which is alternatively used upon induction of the relevant GAL metabolic genes. We quantified the dose-dependent induction of the GAL1 gene encoding the central galactokinase enzyme and found that a very large diversification exists between different yeast ecotypes and species. The sensitivity of GAL1 induction correlates with the growth performance of the respective yeasts with the alternative sugar. We further define some of the mechanisms, which have established different glucose/galactose consumption strategies in representative yeast strains by modulating the activity of the Gal3 inducer. (1) Optimal galactose consumers, such as Saccharomyces uvarum, contain a hyperactive GAL3 promoter, sustaining highly sensitive GAL1 expression, which is not further improved upon repetitive galactose encounters. (2) Desensitized galactose consumers, such as S. cerevisiae Y12, contain a less sensitive Gal3 sensor, causing a shift of the galactose response towards higher sugar concentrations even in galactose experienced cells. (3) Galactose insensitive sugar consumers, such as S. cerevisiae DBVPG6044, contain an interrupted GAL3 gene, causing extremely reluctant galactose consumption, which is, however, improved upon repeated galactose availability. In summary, different yeast strains and natural isolates have evolved galactose utilization strategies, which cover the whole range of possible sensitivities by modulating the expression and/or activity of the inducible galactose sensor Gal3.Ahmedabad University, Grant/Award Number: AU/SUG/SAS/DBLS/2019-20/01; DBT- Ramalinagaswami, Grant/Award Number: AU/SAS/DBT-RLS/20-21/04_KS_03.25; Ministerio de Ciencia e Innovacion, Grant/Award Number: PID2019-104214RB-I00Fita-Torró, J.; Swamy, KBS.; Pascual-Ahuir Giner, MD.; Proft, MH. (2023). Divergence of alternative sugar preferences through modulation of the expression and activity of the Gal3 sensor in yeast. Molecular Ecology (Online). 32(13):3557-3574. https://doi.org/10.1111/mec.1695435573574321

Live-cell assays reveal selectivity and sensitivity of the multidrug response in budding yeast

[EN] Pleiotropic drug resistance arises by the enhanced extrusion of bioactive molecules and is present in a wide range of organisms, ranging from fungi to human cells. A key feature of this adaptation is the sensitive detection of intracellular xenobiotics by transcriptional activators, activating expression of multiple drug exporters. Here, we investigated the selectivity and sensitivity of the budding yeast (Saccharomyces cerevisiae) multidrug response to better understand how differential drug recognition leads to specific activation of drug exporter genes and to drug resistance. Applying live-cell luciferase reporters, we demonstrate that the SNQ2, PDR5, PDR15, and YOR1 transporter genes respond to different mycotoxins, menadione, and hydrogen peroxide in a distinguishable manner and with characteristic amplitudes, dynamics, and sensitivities. These responses correlated with differential sensitivities of the respective transporter mutants to the specific xenobiotics. We further establish a binary vector system, enabling quantitative determination of xenobiotic-transcription factor (TF) interactions in real time. Applying this system we found that the TFs Pdr1, Pdr3, Yrr1, Stb5, and Pdr8 have largely different drug recognition patterns. We noted that Pdr1 is the most promiscuous activator, whereas Yrr1 and Stb5 are selective for ochratoxin A and hydrogen peroxide, respectively. We also show that Pdr1 is rapidly degraded after xenobiotic exposure, which leads to a desensitization of the Pdr1-specific response upon repeated activation. The findings of our work indicate that in the yeast multidrug system, several transcriptional activators with distinguishable selectivities trigger differential activation of the transporter genes.This work was supported by Ministerio de Economia y Competitividad Grant BFU2016-75792-R.Vanacloig-Pedrós, ME.; Lozano-Pérez, C.; Alarcon, B.; Pascual-Ahuir Giner, MD.; Proft, MH. (2019). Live-cell assays reveal selectivity and sensitivity of the multidrug response in budding yeast. Journal of Biological Chemistry. 294(35):12933-12946. https://doi.org/10.1074/jbc.RA119.009291S12933129462943

Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast

[EN] Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.This work was funded by a grant from Ministerio de Ciencia, Innovación y Universidades PID2019-104214RB-I00 to AP-A and MP. JG-H received a pre-doctoral fellowship from Generalitat Valenciana (ACIF/2021/171).Garrido-Huarte, JL.; Fita-Torró, J.; Viana, R.; Pascual-Ahuir Giner, MD.; Proft, MH. (2023). Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast. Frontiers in Microbiology. 14:1-15. https://doi.org/10.3389/fmicb.2023.11522491151

Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast

[EN] Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.This work was supported by the Ministerio de Economa y Competitividad (grant BFU2011-23326 to M.P.) and the Ministerio de Ciencia e Innovacion (predoctoral FPI grant to A.R.).Dolz Edo, L.; Rienzo, A.; Poveda Huertes, D.; Pascual-Ahuir Giner, MD.; Proft, MH. (2013). Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast. Molecular and Cellular Biology. 33(11):2228-2240. https://doi.org/10.1128/MCB.00240-13222822403311Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., … Brown, P. O. (2000). Genomic Expression Programs in the Response of Yeast Cells to Environmental Changes. Molecular Biology of the Cell, 11(12), 4241-4257. doi:10.1091/mbc.11.12.4241Ni, L., Bruce, C., Hart, C., Leigh-Bell, J., Gelperin, D., Umansky, L., … Snyder, M. (2009). Dynamic and complex transcription factor binding during an inducible response in yeast. Genes & Development, 23(11), 1351-1363. doi:10.1101/gad.1781909Posas, F., Chambers, J. R., Heyman, J. A., Hoeffler, J. P., de Nadal, E., & Ariño, J. (2000). The Transcriptional Response of Yeast to Saline Stress. Journal of Biological Chemistry, 275(23), 17249-17255. doi:10.1074/jbc.m910016199Rep, M., Krantz, M., Thevelein, J. M., & Hohmann, S. (2000). The Transcriptional Response ofSaccharomyces cerevisiaeto Osmotic Shock. Journal of Biological Chemistry, 275(12), 8290-8300. doi:10.1074/jbc.275.12.8290Yale, J., & Bohnert, H. J. (2001). Transcript Expression inSaccharomyces cerevisiaeat High Salinity. Journal of Biological Chemistry, 276(19), 15996-16007. doi:10.1074/jbc.m008209200Causton, H. C., Ren, B., Koh, S. S., Harbison, C. T., Kanin, E., Jennings, E. G., … Young, R. A. (2001). Remodeling of Yeast Genome Expression in Response to Environmental Changes. Molecular Biology of the Cell, 12(2), 323-337. doi:10.1091/mbc.12.2.323Martínez-Pastor, M. T., Marchler, G., Schüller, C., Marchler-Bauer, A., Ruis, H., & Estruch, F. (1996). The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). The EMBO Journal, 15(9), 2227-2235. doi:10.1002/j.1460-2075.1996.tb00576.xSchmitt, A. P., & McEntee, K. (1996). Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences, 93(12), 5777-5782. doi:10.1073/pnas.93.12.5777Beck, T., & Hall, M. N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature, 402(6762), 689-692. doi:10.1038/45287Gorner, W., Durchschlag, E., Martinez-Pastor, M. T., Estruch, F., Ammerer, G., Hamilton, B., … Schuller, C. (1998). Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes & Development, 12(4), 586-597. doi:10.1101/gad.12.4.586Saito, H., & Posas, F. (2012). Response to Hyperosmotic Stress. Genetics, 192(2), 289-318. doi:10.1534/genetics.112.140863De Nadal, E., Ammerer, G., & Posas, F. (2011). Controlling gene expression in response to stress. Nature Reviews Genetics, 12(12), 833-845. doi:10.1038/nrg3055Martínez-Montañés, F., Pascual-Ahuir, A., & Proft, M. (2010). Toward a Genomic View of the Gene Expression Program Regulated by Osmostress in Yeast. OMICS: A Journal of Integrative Biology, 14(6), 619-627. doi:10.1089/omi.2010.0046Alepuz, P. M. (2003). Osmostress-induced transcription by Hot1 depends on a Hog1-mediated recruitment of the RNA Pol II. The EMBO Journal, 22(10), 2433-2442. doi:10.1093/emboj/cdg243Nadal, E. d., Casadome, L., & Posas, F. (2003). Targeting the MEF2-Like Transcription Factor Smp1 by the Stress-Activated Hog1 Mitogen-Activated Protein Kinase. Molecular and Cellular Biology, 23(1), 229-237. doi:10.1128/mcb.23.1.229-237.2003Proft, M. (2001). Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress. The EMBO Journal, 20(5), 1123-1133. doi:10.1093/emboj/20.5.1123Proft, M., & Serrano, R. (1999). Repressors and Upstream Repressing Sequences of the Stress-RegulatedENA1Gene inSaccharomyces cerevisiae: bZIP Protein Sko1p Confers HOG-Dependent Osmotic Regulation. Molecular and Cellular Biology, 19(1), 537-546. doi:10.1128/mcb.19.1.537Rep, M., Reiser, V., Gartner, U., Thevelein, J. M., Hohmann, S., Ammerer, G., & Ruis, H. (1999). Osmotic Stress-Induced Gene Expression inSaccharomyces cerevisiaeRequires Msn1p and the Novel Nuclear Factor Hot1p. Molecular and Cellular Biology, 19(8), 5474-5485. doi:10.1128/mcb.19.8.5474Ruiz-Roig, C., Noriega, N., Duch, A., Posas, F., & de Nadal, E. (2012). The Hog1 SAPK controls the Rtg1/Rtg3 transcriptional complex activity by multiple regulatory mechanisms. Molecular Biology of the Cell, 23(21), 4286-4296. doi:10.1091/mbc.e12-04-0289Vendrell, A., Martínez-Pastor, M., González-Novo, A., Pascual-Ahuir, A., Sinclair, D. A., Proft, M., & Posas, F. (2011). Sir2 histone deacetylase prevents programmed cell death caused by sustained activation of the Hog1 stress-activated protein kinase. EMBO reports, 12(10), 1062-1068. doi:10.1038/embor.2011.154De Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., & Posas, F. (2004). The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes. Nature, 427(6972), 370-374. doi:10.1038/nature02258Proft, M., & Struhl, K. (2002). Hog1 Kinase Converts the Sko1-Cyc8-Tup1 Repressor Complex into an Activator that Recruits SAGA and SWI/SNF in Response to Osmotic Stress. Molecular Cell, 9(6), 1307-1317. doi:10.1016/s1097-2765(02)00557-9Zapater, M., Sohrmann, M., Peter, M., Posas, F., & de Nadal, E. (2007). Selective Requirement for SAGA in Hog1-Mediated Gene Expression Depending on the Severity of the External Osmostress Conditions. Molecular and Cellular Biology, 27(11), 3900-3910. doi:10.1128/mcb.00089-07Capaldi, A. P., Kaplan, T., Liu, Y., Habib, N., Regev, A., Friedman, N., & O’Shea, E. K. (2008). Structure and function of a transcriptional network activated by the MAPK Hog1. Nature Genetics, 40(11), 1300-1306. doi:10.1038/ng.235Cook, K. E., & O’Shea, E. K. (2012). Hog1 Controls Global Reallocation of RNA Pol II upon Osmotic Shock in Saccharomyces cerevisiae. G3: Genes|Genomes|Genetics, 2(9), 1129-1136. doi:10.1534/g3.112.003251Proft, M., Gibbons, F. D., Copeland, M., Roth, F. P., & Struhl, K. (2005). Genomewide Identification of Sko1 Target Promoters Reveals a Regulatory Network That Operates in Response to Osmotic Stress inSaccharomyces cerevisiae. Eukaryotic Cell, 4(8), 1343-1352. doi:10.1128/ec.4.8.1343-1352.2005Vincent, A. C., & Struhl, K. (1992). ACR1, a yeast ATF/CREB repressor. Molecular and Cellular Biology, 12(12), 5394-5405. doi:10.1128/mcb.12.12.5394Wong, K. H., & Struhl, K. (2011). The Cyc8-Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein. Genes & Development, 25(23), 2525-2539. doi:10.1101/gad.179275.111Ikner, A., & Shiozaki, K. (2005). Yeast signaling pathways in the oxidative stress response. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 569(1-2), 13-27. doi:10.1016/j.mrfmmm.2004.09.006Temple, M. D., Perrone, G. G., & Dawes, I. W. (2005). Complex cellular responses to reactive oxygen species. Trends in Cell Biology, 15(6), 319-326. doi:10.1016/j.tcb.2005.04.003Toone, W. M., & Jones, N. (1999). AP-1 transcription factors in yeast. Current Opinion in Genetics & Development, 9(1), 55-61. doi:10.1016/s0959-437x(99)80008-2Brombacher, K., Fischer, B. B., Rüfenacht, K., & Eggen, R. I. L. (2006). The role of Yap1p and Skn7p-mediated oxidative stress response in the defence ofSaccharomyces cerevisiae against singlet oxygen. Yeast, 23(10), 741-750. doi:10.1002/yea.1392Lee, J., Godon, C., Lagniel, G., Spector, D., Garin, J., Labarre, J., & Toledano, M. B. (1999). Yap1 and Skn7 Control Two Specialized Oxidative Stress Response Regulons in Yeast. Journal of Biological Chemistry, 274(23), 16040-16046. doi:10.1074/jbc.274.23.16040Fernandes, L., Rodrigues-Pousada, C., & Struhl, K. (1997). Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions. Molecular and Cellular Biology, 17(12), 6982-6993. doi:10.1128/mcb.17.12.6982Gulshan, K., Rovinsky, S. A., Coleman, S. T., & Moye-Rowley, W. S. (2005). Oxidant-specific Folding of Yap1p Regulates Both Transcriptional Activation and Nuclear Localization. Journal of Biological Chemistry, 280(49), 40524-40533. doi:10.1074/jbc.m504716200Kuge, S. (1997). Regulation of yAP-1 nuclear localization in response to oxidative stress. The EMBO Journal, 16(7), 1710-1720. doi:10.1093/emboj/16.7.1710Delaunay, A., Isnard, A.-D., & Toledano, M. B. (2000). H2O2 sensing through oxidation of the Yap1 transcription factor. The EMBO Journal, 19(19), 5157-5166. doi:10.1093/emboj/19.19.5157Kuge, S., Arita, M., Murayama, A., Maeta, K., Izawa, S., Inoue, Y., & Nomoto, A. (2001). Regulation of the Yeast Yap1p Nuclear Export Signal Is Mediated by Redox Signal-Induced Reversible Disulfide Bond Formation. Molecular and Cellular Biology, 21(18), 6139-6150. doi:10.1128/mcb.21.18.6139-6150.2001Koziol, S., Zagulski, M., Bilinski, T., & Bartosz, G. (2005). Antioxidants protect the yeastSaccharomyces cerevisiaeagainst hypertonic stress. 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Science, 285(5429), 901-906. doi:10.1126/science.285.5429.901Galiazzo, F., & Labbe-Bois, R. (1993). Regulation of Cu,Zn- and Mn-superoxide dismutase transcription in Saccharomyces cerevisiae. FEBS Letters, 315(2), 197-200. doi:10.1016/0014-5793(93)81162-sGaray-Arroyo, A., & Covarrubias, A. A. (1999). Three genes whose expression is induced by stress inSaccharomyces cerevisiae. Yeast, 15(10A), 879-892. doi:10.1002/(sici)1097-0061(199907)15:10a3.0.co;2-qKwon, M. (2003). Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA) - General Subjects, 1623(1), 1-5. doi:10.1016/s0304-4165(03)00151-xPascual-Ahuir, A., Posas, F., Serrano, R., & Proft, M. (2001). Multiple Levels of Control Regulate the Yeast cAMP-response Element-binding Protein Repressor Sko1p in Response to Stress. Journal of Biological Chemistry, 276(40), 37373-37378. doi:10.1074/jbc.m105755200Schüller, C., Brewster, J. L., Alexander, M. 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Activator and repressor functions of the Mot3 transcription factor in the osmostress response of Saccharomyces cerevisiae

[EN] Mot3 and Rox1 are transcriptional repressors of hypoxic genes. Both factors recently have been found to be involved in the adaptive response to hyperosmotic stress, with an important function in the adjustment of ergosterol biosynthesis. Here, we determine the gene expression profile of a mot3 rox1 double mutant under acute osmostress at the genomic scale in order to identify the target genes affected by both transcription factors upon stress. Unexpectedly, we find a specific subgroup of osmostress-inducible genes to be under positive control of Mot3. These Mot3-activated stress genes also depend on the general stress activators Msn2 and Msn4. We confirm that both Mot3 and Msn4 bind directly to some promoter regions of this gene group. Further-more, osmostress-induced binding of the Msn2 and Msn4 factors to these target promoters is severely affected by the loss of Mot3 function. The genes repressed by Mot3 and Rox1 preferentially encode proteins of the cell wall and plasma membrane. Cell conjugation was the most significantly enriched biological process which was negatively regulated by both factors and by osmotic stress. The mating response was repressed by salt stress dependent on Mot3 and Rox1 function. Taking our findings together, the Mot3 transcriptional regulator has unanticipated diverse functions in the cellular adjustment to osmotic stress, including transcriptional activation and modulation of mating efficiency.This work was supported by grants BFU2008-00271 and BFU2011-23326 from the Ministry of Science and Innovation (Madrid, Spain). F. Martinez-Montanes was supported by an FPI predoctoral fellowship from the Ministry of Science and Innovation (Madrid, Spain).Martínez Montañés, FV.; Rienzo, A.; Poveda Huertes, D.; Pascual-Ahuir Giner, MD.; Proft, MH. (2013). Activator and repressor functions of the Mot3 transcription factor in the osmostress response of Saccharomyces cerevisiae. Eukaryotic Cell. 12(5):636-647. https://doi.org/10.1128/EC.00037-13S63664712

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.This work was supported by grants from Ministerio de Economia y Competitividad (BFU2011-23326), Generalitat de Valencia (ACOMP2011/031), and the NIH Director's New Innovator Award (DP2 OD008654-01). Alessandro Rienzo was a recipient of a predoctoral FPI grant from Ministerio de Economia y Competitividad.Rienzo, A.; Poveda Huertes, D.; Aydin, S.; Buchler, NE.; Pascual-Ahuir Giner, MD.; Proft, MH. (2015). Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast. 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Dose dependent gene expression is dynamically modulated by the history, physiology and age of yeast cells

[EN] Cells respond to external stimuli with transient gene expression changes in order to adapt to environmental alterations. However, the dose response profile of gene induction upon a given stress depends on many intrinsic and extrinsic factors. Here we show that the accurate quantification of dose dependent gene expression by live cell luciferase reporters reveals fundamental insights into stress signaling. We make the following discoveries applying this non-invasive reporter technology. (1) Signal transduction sensitivities can be compared and we apply this here to salt, oxidative and xenobiotic stress responsive transcription factors. (2) Stress signaling depends on where and how the damage is generated within the cell. Specifically we show that two ROS-generating agents, menadione and hydrogen peroxide, differ in their dependence on mitochondrial respiration. (3) Stress signaling is conditioned by the cells history. We demonstrate here that positive memory or an acquired resistance towards oxidative stress is induced dependent on the nature of the previous stress experience. (4) The metabolic state of the cell impinges on the sensitivity of stress signaling. This is shown here for the shift towards higher stress doses of the response profile for yeast cells moved from complex to synthetic medium. (5) The age of the cell conditions its transcriptional response capacity, which is demonstrated by the changes of the dose response to oxidative stress during both replicative and chronological aging. We conclude that capturing dose dependent gene expression in real time will be of invaluable help to understand stress signaling and its dynamic modulation.The authors thank Daniel E. Gottschling (Fred Hutchinson Cancer Research Center, Seattle, US) for the kind gift of MEP yeast strain UCC4925. This work was supported by the Ministerio de Economia y Competitividad (grant number BFU2016-75792-R).Pascual-Ahuir Giner, MD.; González-Cantó, E.; Juyoux, P.; Pable, J.; Poveda-Huertes, D.; Saiz-Balbastre Sandra; Squeo, S.... (2019). Dose dependent gene expression is dynamically modulated by the history, physiology and age of yeast cells. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1862(4):457-471. https://doi.org/10.1016/j.bbagrm.2019.02.009S4574711862