Regulation of the pleiotropic drug resistance transcription factors Pdr1 and Pdr3 in yeast

Aim: To understand how transcriptional factors Pdr1 and Pdr3, belonging to the pleiotropic drug resistance system, are activated, and regulated after introducing chemical toxins to the cell in the model organism Saccharomyces cerevisiae. Methods: Series of molecular methods were applied using different strains of S. cerevisiae over-expressing proteins of interest as a eukaryotic cell model. The chemical stress introduced to the cell is represented by menadione. Results were obtained performing protein detection and analysis. Additionally, the regulation of the DNA binding of the transcriptional activators after stimulation is quantified using chromatin immunoprecipitation, employing epitope-tagged factors and real-time qPCR. Results: Our results indicated higher expression levels of the Pdr1 transcriptional factor, compared to its homologous Pdr3 after treatment with menadione. The yeast-cell defence system was tested against various organic solvents to exclude the possibility of their presence potentially affecting the results. The results indicate that Pdr1 is most abundant after 30 minutes from the beginning of the treatment, compared with 240 minutes after the treatment when the function of the transcription factor is faded. It appears that Pdr1 binding to the PDR5 and SNQ2 promoters, which are both activated by Pdr1, peaks around the same time, or more precisely after 40 minutes from the start of the treatment. Conclusion: The tendency of Pdr1 reduction after its activation by menadione is detected. One possibility is that Pdr1, after recognizing the xenobiotic menadione, is removed by a degradation mechanism. Given the fact that Pdr1 directly binds the xenobiotic molecule, its destruction might help the cells to remove toxic levels of menadione. It is possible that overexpressing the part of Pdr1 which recognizes menadione alone was sufficient to detoxify and hence produce a tolerance towards menadione

Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast

[EN] Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense.We thank Lorena Latorre and Javier Forment for their help with the microarray experiments and data analysis. This work was funded only in the initial phase by a grant from Ministerio de Economía y Competitividad (BFU2011-23326). We thank the Fond for Open Access Publication from Consejo Superior de Investigaciones Científicas (CSIC) for supporting publication costs of this article.Vanacloig-Pedrós, E.; Proft, MH.; Pascual-Ahuir Giner, MD. (2016). Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast. Toxins. 8(10):1-20. https://doi.org/10.3390/toxins8100273S120810Bennett, J. W., & Klich, M. (2003). Mycotoxins. Clinical Microbiology Reviews, 16(3), 497-516. doi:10.1128/cmr.16.3.497-516.2003Marroquín-Cardona, A. G., Johnson, N. M., Phillips, T. D., & Hayes, A. W. (2014). Mycotoxins in a changing global environment – A review. Food and Chemical Toxicology, 69, 220-230. doi:10.1016/j.fct.2014.04.025Moretti, A., Susca, A., Mulé, G., Logrieco, A. F., & Proctor, R. H. (2013). Molecular biodiversity of mycotoxigenic fungi that threaten food safety. International Journal of Food Microbiology, 167(1), 57-66. doi:10.1016/j.ijfoodmicro.2013.06.033Wu, F., Groopman, J. D., & Pestka, J. J. (2014). Public Health Impacts of Foodborne Mycotoxins. Annual Review of Food Science and Technology, 5(1), 351-372. doi:10.1146/annurev-food-030713-092431Möbius, N., & Hertweck, C. (2009). Fungal phytotoxins as mediators of virulence. Current Opinion in Plant Biology, 12(4), 390-398. doi:10.1016/j.pbi.2009.06.004Doi, K., & Uetsuka, K. (2014). Mechanisms of Mycotoxin-induced Dermal Toxicity and Tumorigenesis Through Oxidative Stress-related Pathways. Journal of Toxicologic Pathology, 27(1), 1-10. doi:10.1293/tox.2013-0062Escrivá, L., Font, G., & Manyes, L. (2015). In vivo toxicity studies of fusarium mycotoxins in the last decade: A review. Food and Chemical Toxicology, 78, 185-206. doi:10.1016/j.fct.2015.02.005Vettorazzi, A., González-Peñas, E., & de Cerain, A. L. (2014). Ochratoxin A kinetics: A review of analytical methods and studies in rat model. Food and Chemical Toxicology, 72, 273-288. doi:10.1016/j.fct.2014.07.020Wang, Y., Wang, L., Liu, F., Wang, Q., Selvaraj, J., Xing, F., … Liu, Y. (2016). Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms. Toxins, 8(3), 83. doi:10.3390/toxins8030083Kőszegi, T., & Poór, M. (2016). Ochratoxin A: Molecular Interactions, Mechanisms of Toxicity and Prevention at the Molecular Level. Toxins, 8(4), 111. doi:10.3390/toxins8040111Faucet, V., Pfohl-Leszkowicz, A., Dai, J., Castegnaro, M., & Manderville, R. A. (2004). Evidence for Covalent DNA Adduction by Ochratoxin A following Chronic Exposure to Rat and Subacute Exposure to Pig. Chemical Research in Toxicology, 17(9), 1289-1296. doi:10.1021/tx049877sMantle, P. G., Faucet-Marquis, V., Manderville, R. A., Squillaci, B., & Pfohl-Leszkowicz, A. (2010). Structures of Covalent Adducts between DNA and Ochratoxin A: A New Factor in Debate about Genotoxicity and Human Risk Assessment. Chemical Research in Toxicology, 23(1), 89-98. doi:10.1021/tx900295aPfohl-Leszkowicz, A., & Manderville, R. A. (2011). An Update on Direct Genotoxicity as a Molecular Mechanism of Ochratoxin A Carcinogenicity. Chemical Research in Toxicology, 25(2), 252-262. doi:10.1021/tx200430fRahimtula, A. D., Béréziat, J.-C., Bussacchini-Griot, V., & Bartsch, H. (1988). Lipid peroxidation as a possible cause of ochratoxin a toxicity. Biochemical Pharmacology, 37(23), 4469-4477. doi:10.1016/0006-2952(88)90662-4Sorrenti, V., Di Giacomo, C., Acquaviva, R., Barbagallo, I., Bognanno, M., & Galvano, F. (2013). Toxicity of Ochratoxin A and Its Modulation by Antioxidants: A Review. Toxins, 5(10), 1742-1766. doi:10.3390/toxins5101742BRAGULAT, M., MARTINEZ, E., CASTELLA, G., & CABANES, F. (2008). Ochratoxin A and citrinin producing species of the genus Penicillium from feedstuffs. 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Biochemical Journal, 404(2), 317-326. doi:10.1042/bj20061875Kumar, M., Dwivedi, P., Sharma, A. K., Sankar, M., Patil, R. D., & Singh, N. D. (2012). Apoptosis and lipid peroxidation in ochratoxin A- and citrinin-induced nephrotoxicity in rabbits. Toxicology and Industrial Health, 30(1), 90-98. doi:10.1177/0748233712452598Kumar, R., Dwivedi, P. D., Dhawan, A., Das, M., & Ansari, K. M. (2011). Citrinin-Generated Reactive Oxygen Species Cause Cell Cycle Arrest Leading to Apoptosis via the Intrinsic Mitochondrial Pathway in Mouse Skin. Toxicological Sciences, 122(2), 557-566. doi:10.1093/toxsci/kfr143Máté, G., Gazdag, Z., Mike, N., Papp, G., Pócsi, I., & Pesti, M. (2014). Regulation of oxidative stress-induced cytotoxic processes of citrinin in the fission yeast Schizosaccharomyces pombe. Toxicon, 90, 155-166. doi:10.1016/j.toxicon.2014.08.005Pascual-Ahuir, A., Vanacloig-Pedros, E., & Proft, M. (2014). Toxicity Mechanisms of the Food Contaminant Citrinin: Application of a Quantitative Yeast Model. Nutrients, 6(5), 2077-2087. doi:10.3390/nu6052077Ribeiro, S. M. R., Chagas, G. M., Campello, A. P., & Kluppel, M. L. W. (1997). Mechanism of citrinin-induced dysfunction of mitochondria. V. Effect on the homeostasis of the reactive oxygen species. Cell Biochemistry and Function, 15(3), 203-209. doi:10.1002/(sici)1099-0844(199709)15:33.0.co;2-jSingh, N. D., Sharma, A. K., Dwivedi, P., Leishangthem, G. D., Rahman, S., Reddy, J., & Kumar, M. (2013). Effect of feeding graded doses of citrinin on apoptosis and oxidative stress in male Wistar rats through the F1generation. Toxicology and Industrial Health, 32(3), 385-397. doi:10.1177/0748233713500836Yu, F.-Y., Liao, Y.-C., Chang, C.-H., & Liu, B.-H. (2006). Citrinin induces apoptosis in HL-60 cells via activation of the mitochondrial pathway. Toxicology Letters, 161(2), 143-151. doi:10.1016/j.toxlet.2005.08.009Föllmann, W., Behm, C., & Degen, G. H. (2014). Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro. Archives of Toxicology, 88(5), 1097-1107. doi:10.1007/s00204-014-1216-8Klarić, M., Rašić, D., & Peraica, M. (2013). Deleterious Effects of Mycotoxin Combinations Involving Ochratoxin A. Toxins, 5(11), 1965-1987. doi:10.3390/toxins5111965Afshari, C. A., Hamadeh, H. K., & Bushel, P. R. (2010). The Evolution of Bioinformatics in Toxicology: Advancing Toxicogenomics. Toxicological Sciences, 120(Supplement 1), S225-S237. doi:10.1093/toxsci/kfq373Yasokawa, D., & Iwahashi, H. (2010). Toxicogenomics using yeast DNA microarrays. Journal of Bioscience and Bioengineering, 110(5), 511-522. doi:10.1016/j.jbiosc.2010.06.003Arbillaga, L., Azqueta, A., van Delft, J. H. M., & López de Cerain, A. (2007). In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A. 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Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray. BMC Genomics, 8(1), 95. doi:10.1186/1471-2164-8-95Toone, W. M., Morgan, B. A., & Jones, N. (2001). Redox control of AP-1-like factors in yeast and beyond. Oncogene, 20(19), 2336-2346. doi:10.1038/sj.onc.1204384Luo, Y., Wang, J., Liu, B., Wang, Z., Yuan, Y., & Yue, T. (2015). Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption. PLOS ONE, 10(8), e0136045. doi:10.1371/journal.pone.0136045Piotrowska, M., & Masek, A. (2015). Saccharomyces Cerevisiae Cell Wall Components as Tools for Ochratoxin A Decontamination. Toxins, 7(4), 1151-1162. doi:10.3390/toxins7041151Jungwirth, H., & Kuchler, K. (2005). Yeast ABC transporters - A tale of sex, stress, drugs and aging. FEBS Letters, 580(4), 1131-1138. doi:10.1016/j.febslet.2005.12.050Prasad, R., & Goffeau, A. (2012). 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Chipper: discovering transcription-factor targets from chromatin immunoprecipitation microarrays using variance stabilization

Chromatin immunoprecipitation combined with microarray technology (Chip(2)) allows genome-wide determination of protein-DNA binding sites. The current standard method for analyzing Chip(2 )data requires additional control experiments that are subject to systematic error. We developed methods to assess significance using variance stabilization, learning error-model parameters without external control experiments. The method was validated experimentally, shows greater sensitivity than the current standard method, and incorporates false-discovery rate analysis. The corresponding software ('Chipper') is freely available. The method described here should help reveal an organism's transcription-regulatory 'wiring diagram'

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

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

Relativistic effects on the Fukui function

The extent of relativistic effects on the Fukui function, which describes local reactivity trends within conceptual density functional theory (DFT), and frontier orbital densities has been analysed on the basis of three benchmark molecules containing the heavy elements: Au, Pb, and Bi. Various approximate relativistic approaches have been tested and compared with the four-component fully relativistic reference. Scalar relativistic effects, as described by the scalar zeroth-order regular approximation methodology and effective core potential calculations, already provide a large part of the relativistic corrections. Inclusion of spin-orbit coupling effects improves the results, especially for the heavy p-block compounds. We thus expect that future conceptual DFT-based reactivity studies on heavy-element molecules can rely on one of the approximate relativistic methodologie

PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae

Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression

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. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.This work was funded by Ministerio de Ciencia, Innovacion y Universidades, grant number BFU2016-75792-R.Pascual-Ahuir Giner, MD.; Fita-Torró, J.; Proft, MH. (2020). Capturing and Understanding the Dynamics and Heterogeneity of Gene Expression in the Living Cell. International Journal of Molecular Sciences. 21(21):1-19. https://doi.org/10.3390/ijms21218278S1192121Murray, J. I., Whitfield, M. L., Trinklein, N. D., Myers, R. M., Brown, P. O., & Botstein, D. (2004). Diverse and Specific Gene Expression Responses to Stresses in Cultured Human Cells. Molecular Biology of the Cell, 15(5), 2361-2374. doi:10.1091/mbc.e03-11-0799Gasch, 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. 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MutationTaster2021

Here we present an update to MutationTaster, our DNA variant effect prediction tool. The new version uses a different prediction model and attains higher accuracy than its predecessor, especially for rare benign variants. In addition, we have integrated many sources of data that only became available after the last release (such as gnomAD and ExAC pLI scores) and changed the splice site prediction model. To more easily assess the relevance of detected known disease mutations to the clinical phenotype of the patient, MutationTaster now provides information on the diseases they cause. Further changes represent a major overhaul of the interfaces to increase user-friendliness whilst many changes under the hood have been designed to accelerate the processing of uploaded VCF files. We also offer an API for the rapid automated query of smaller numbers of variants from within other software. MutationTaster2021 integrates our disease mutation search engine, MutationDistiller, to prioritise variants from VCF files using the patient's clinical phenotype. The novel version is available at https://www.genecascade.org/MutationTaster2021/. This website is free and open to all users and there is no login requirement

RegEl corpus: identifying DNA regulatory elements in the scientific literature

High-throughput technologies led to the generation of a wealth of data on regulatory DNA elements in the human genome. However, results from disease-driven studies are primarily shared in textual form as scientific articles. Information extraction (IE) algorithms allow this information to be (semi-)automatically accessed. Their development, however, is dependent on the availability of annotated corpora. Therefore, we introduce RegEl (Regulatory Elements), the first freely available corpus annotated with regulatory DNA elements comprising 305 PubMed abstracts for a total of 2690 sentences. We focus on enhancers, promoters and transcription factor binding sites. Three annotators worked in two stages, achieving an overall 0.73 F1 inter-annotator agreement and 0.46 for regulatory elements. Depending on the entity type, IE baselines reach F1-scores of 0.48–0.91 for entity detection and 0.71–0.88 for entity normalization. Next, we apply our entity detection models to the entire PubMed collection and extract co-occurrences of genes or diseases with regulatory elements. This generates large collections of regulatory elements associated with 137 870 unique genes and 7420 diseases, which we make openly available.Database URL: https://zenodo.org/record/6418451#.YqcLHvexVqgPeer Reviewe