Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology

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Background In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative

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  BioMed   Central Page 1 of 18 (page number not for citation purposes) BMC Genomics Open Access Research article Comparison of gene expression signatures of diamide, H 2 O 2 and menadione exposed  Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology IstvánPócsi* 1 , MártonMiskei 1 , ZsoltKarányi 2 , TamásEmri 1 , PatriciaAyoubi 3 , TündePusztahelyi 1 , GyörgyBalla 4  and RolfAPrade 5  Address: 1 Department of Microbiology and Biotechnology, Faculty of Science, University of Debrecen, P.O.Box 63, H-4010 Debrecen, Hungary, 2 Department of Medicine, Faculty of Medicine, University of Debrecen, P.O. Box 19, H-4012 Debrecen, Hungary, 3 Department of Biochemistry and Molecular Biology, Oklahoma State University, 348E Noble Research Center, Stillwater, OK 74078, USA, 4 Department of Neonatology, Faculty of Medicine, University of Debrecen, P.O.Box 37; H-4012 Debrecen, Hungary and 5 Department of Microbiology and Molecular Genetics, Oklahoma State University, 307 LSE, Stillwater, OK 74078, USA Email: IstvánPócsi*-istvanpocsi@yahoo.com; MártonMiskei-miskeim@freemail.hu; ZsoltKarányi-karanyi@internal.med.unideb.hu;  TamásEmri-emri@freemail.hu; PatriciaAyoubi-ayoubi@okstate.edu; TündePusztahelyi-pusztahelyi@yahoo.com; GyörgyBalla-balla@jaguar.dote.hu; RolfAPrade-prade@okstate.edu* Corresponding author Abstract Background: In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diversecellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology inoxidative stress-exposed  Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O 22- ),superoxide (O 2•- ) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses. Results: Genome-wide transcriptional changes triggered by diamide, H 2 O 2 and menadione in  A. nidulans vegetativetissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress inducedby diamide and H 2 O 2 were pulse-like, with recovery after 1 h exposure time while no recovery was observed withmenadione. The distribution of stress-responsive gene probes among major physiological functional categories wasapproximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O 22- , O 2•- concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groupsresponsive to diamide, H 2 O 2 and menadione treatments and gene groups influenced by GSH/GSSG, O 22- and O 2•- wereonly partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redoxstate influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfAtranscription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins,defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms.Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsivetranscription factors, and some elements of sexual development and sporulation was ROS responsive. Conclusion: The existence of separate O 22- , O 2•- and GSH/GSSG responsive gene groups in a eukaryotic genome hasbeen demonstrated. Oxidant-triggered, genome-wide transcriptional changes should be analyzed considering changes inoxidative stress-responsive physiological conditions and not correlating them directly to the chemistry andconcentrations of the oxidative stress-inducing agent. Published: 20 December 2005 BMC Genomics  2005, 6 :182doi:10.1186/1471-2164-6-182Received: 23 September 2005Accepted: 20 December 2005This article is available from: http://www.biomedcentral.com/1471-2164/6/182© 2005 Pócsi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  BMC Genomics  2005, 6 :182http://www.biomedcentral.com/1471-2164/6/182Page 4 of 18 (page number not for citation purposes) tions is an excellent model to perform transcriptome anal- yses using EST-based DNA microarrays [8-10]. Physiological parameters indicative of oxidative stress canalso be measured with high reproducibility in  A. nidulans [11,12].In this study, we assay physiologically well-defined oxida-tive stress-inducing systems to describe the global tran-scriptional changes observable in  A. nidulans  vegetativehyphae exposed to diamide, H 2 O 2 and menadione. Inaddition, we demonstrate large gene groups responsive tochanges in intracellular peroxide (O 22- ) and superoxide(O 2•- ) levels or glutathione/glutathione disulfide (GSH/GSSG) redox imbalance. Results Optimization of oxidative stress inducing conditions For diamide, H 2 O 2 and menadione treatments, 1.8, 75and 0.8 mM were selected, respectively, which were wellbelow the " dosis lethalis minima " (DLM) determined in liq-uid  A. nidulans cultures (Additional file 1:Supplement1for physiological changes).Diamide at 1.8 mM decreased GSH/GSSG value without effecting intracellular O 22- or O 2•- levels (Figure 1, Addi-tional file 1:Supplement1 for physiological changes).H 2 O 2 and menadione increased intracellular O 22- and O 2•- levels, respectively, but also disturbed GSH/GSSG redox balance at all concentrations tested. In addition, menadi-one added at all tested concentrations facilitated intracel-lular accumulation of O 22- (Figure 1, Additional file1:Supplement1 for physiological changes). Increases inintracellular O 22- levels induced by 75 mM H 2 O 2 or 0.8mM menadione and decreases in GSH/GSSG ratios whendiamide, H 2 O 2 or menadione was present were compara-ble to each other, respectively (Figure 1). The stressobserved in diamide and H 2 O 2 -exposed cultures waspulse-like with a maximum intensity under 1 h exposuretime and followed with full recovery (3–9 h). Meanwhile,all stress-related physiological parameters tested changedsteadily up to 9 h exposure time in menadione-treated cul-tures indicating accumulation of stress with no recovery.Short time (1 h) exposures to 1.8 mM diamide increasedthe specific glutathione S -transferase (GST) and catalaseactivities while superoxide dismutase (SOD) activity went up only after extended treatments (6 h; Additional file1:Supplement1 for physiological changes). The specific SOD, GST and catalase activities all responded to short 0.8mM menadione treatments (1 h) while 75 mM H 2 O 2 trig-gered elevations only in catalase activity (Additional file1:Supplement1 for physiological changes). After extended(6 h) incubation with H 2 O 2 , the increase in SOD and GST activities was significant at P < 5 % and P < 1 %, respec-tively (Additional file 1:Supplement1 for physiologicalchanges).Similar to previous observations [6,7], 1.8 mM diamide, 75 mM H 2 O 2 and 0.8 mM menadione did not influencecell survival rates significantly even after extended (9 h)treatment periods (data not shown).Northern blot mRNA accumulation analysis of selectedgenes expressed under various kinds of oxidative stress areshown in Figure 2. M Northern = log  2 (optical density  stress-exposed *optical density  control-1 ) values were calculated for each exposure time and each stress-inducing agent con-centration tested. Among the selected genes were:  gstA (glutathione S -transferase) clearly up-regulated by diamide and menadione (Figures 2 A and 2B);  sodA (Cu,Zn-superoxide dismutase), which responded solely tomenadione treatments (Figure 2 A). In addition,  sodA  wasdown-regulated in control cultures after 6 h, whichresulted in fluctuations in M Northern = f(t) functions when Correlation between the transcriptional changes recorded for selected genes ( aoxA ,  gstA , sodA and sconC  ) in parallel DNA microarray and Northern blot hybridizations Figure 3 Correlation between the transcriptional changes recorded for selected genes ( aoxA ,  gstA , sodA and sconC  ) in parallel DNA microarray and Northern blot hybridizations. In DNA microarray experiments, gene expressions were read on spots representing the following contigs: aoxA (ORF ID: AN2099.2): OSU contig ID: contig2000Sep131300_2956);  gstA (ORF ID: AN4905.2): OSU contig ID: contig2000Sep131300_1307; sodA (ORF ID: AN0241.2): OSU contig ID: contig2000Sep131300_575, sconC (ORF ID: AN2302.2): contig2000Sep131300_757. All M'-M Northern data pairs are from 1.8 mM diamide, 75 mM H 2 O 2 and 0.8 mM menadione treatments (1–9 h). y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    l   o   g     2     (    R    *    G   -    1     )     D    N    A   c    h    i   p     (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    M   v   a    l   u   e   s    (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    l   o   g     2     (    R    *    G   -    1     )     D    N    A   c    h    i   p     (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M Northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    M   v   a    l   u   e   s    (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    l   o   g     2     (    R    *    G   -    1     )     D    N    A   c    h    i   p     (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    M   v   a    l   u   e   s    (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    l   o   g     2     (    R    *    G   -    1     )     D    N    A   c    h    i   p     (    M    ’    )   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M   y = 0.7632x + 0.1416R = 0.6918 -3.0-2.0-1.0 0.0 1.02.03.04.05.0-4.0 -2.0 0.0 2.0 4.0 6.0(M Northern )Log 2 (optical density stress-exposed *optical density control-1 )     L    O    E    S    S   n   o   r   m   a    l    i   z   e    d    M   v   a    l   u   e   s    (    M    ’    )  BMC Genomics  2005, 6 :182http://www.biomedcentral.com/1471-2164/6/182Page 5 of 18 (page number not for citation purposes) mycelia were exposed to diamide or H 2 O 2 (Figures 2 A and2C). Expression of a Ca 2+ -calmodulin-dependent serine-threonine-protein kinase homologue of Schizosaccharomy-ces pombe (ORF ID: AN4483.2) fluctuated in both stress-exposed and control cultures annulling each other whenM Northern = f(t) were calculated after 3 h (Figures 2 A and2D). Transcription of  sconC (a sulfur metabolic transcrip-tional regulator) and aoxA (mitochondrial alternative oxi-dase) was not effected by oxidative stress with M Northern  values around or between +1 and -1 (data not shown).Gene activation or repression was dose-dependent whendiamide and menadione concentrations were elevatedfrom 1.0 to 1.8 mM and from 0.35 to 0.8 mM, respectively (Figure 2). When the concentration of H 2 O 2  was elevatedfrom 75 to 150 mM the mRNA pools were degraded at 1and 3 h exposure times while there was apparently noreduction in rRNA levels at any exposure time tested (Fig-ure 2). Evaluation of cDNA microarray gene expression data  The log  2 -ratios of primary DNA microarray readings for the gene probes (M values) were normalized by LOESSintensity-dependent block-by-block normalization (M' values). The validity of the normalized DNA microarray readings was estimated by correlating DNA microarray M'and the appropriate M Northern  values calculated for North-ern blots (Figures 2 and 3). The correlation between gene expression data determined for the same set of randomly selected genes was R  ≈ 0.7 (Figure 3). Time-dependence of gene expression profiles from cDNA microarrays showed that  gstA  was up-regulated by mena-dione and diamide while  sodA  was only induced by mena-dione (Figures 2E and 2F). The transcription of  sodA  wasnot fluctuating in the presence of H 2 O 2 and diamide, andall M' values were within [+1,-1] thresholds (Figure 2F). The expression of Ca 2+ -calmodulin-dependent serine-threonine-protein kinase was clearly fluctuating in micro-array experiments with a maximum down-regulation at 1h incubation and with a maximum induction of 3 h incu-bation under diamide and H 2 O 2 -treatments (Figure 2G). Time-course of gene expression ratios, which was affectedby stress-inducing agents more than two-fold, showed amaximum intensity response by 1 h exposure time (Figure4). The expression ratio decreased sharply after 1 h expo-sure to diamide, decreased slightly and more slowly withH 2 O 2 and remained high up to 9 h in the presence of menadione with a transient decrease at 3–6 h and a sec-ond peak at 9 h (Figure 4). With diamide and menadione,maximum 16.9–17.4 % of the gene probes responded with at least two-fold change in expression to stress, whilethis ratio was lower (maximum 12 %) for H 2 O 2 . It is note- worthy that 4–6 % of the M' values were below or abovethe [+2,-2] thresholds, i.e . higher than 4-fold changerecorded, when the ratio of affected gene probes was max-imal for the agents (Figure 4).In total, 2499 gene probes (available in Additional file2:Supplement2 for the list of oxidative stress responsivegene probes) were affected by at least one of the stress-inducing compounds tested. Prior to gene distributionanalysis, recovery phase diamide (3–6 h) and H 2 O 2 (3–9h) data were disregarded, and only gene probes with at least 60 % of the M' values available for each stress-induc-ing agent (diamide: 15 min – 1 h, H 2 O 2 : 15 min – 1 h,menadione: 30 min – 9 h) were processed further result-ing in1502 gene probes in total (Figure 5, Additional file2:Supplement2 for the list of oxidative stress responsivegene probes). Half (52.7 %) of the selected gene probesresponded to more than one agent, 35.1 % were influ-enced by the agents equally – up- or down-regulated, while 17.6 % of them were effected differentially. Thenumber of gene probes affected by stress increased in theorder of H 2 O 2 (42.0 %), diamide (49.2 %) and menadi-one (81.6 %). Similar tendencies were observed for geneprobes affected only by one agent, H 2 O 2 (6.3 %), diamide(8.4 %) and menadione (32.6 %). There were large sets of transcripts within the genome that were concomitantly responsive to several agents, e.g. diamide and H 2 O 2 andmenadione (20.2 %); H 2 O 2 and menadione (11.9 %);diamide and H 2 O 2  (3.7 %), and diamide and menadione(17.0 %) (Figure 5, Additional file 2:Supplement2 for the list of oxidative stress responsive gene probes). After filter-ing out data where the agents caused more than two-foldbut opposite changes in transcription the ratios of diamide-H 2 O 2 -menadione, H 2 O 2 -menadione, diamide-H 2 O 2 and diamide-menadione responsive gene probes went down to 13.0, 7.7, 2.6 and 11.8 %, respectively (Fig-ure 5).In order to perform gene enrichment calculations, thegroup of stress-responsive gene probes (1502; Additionalfile 2:Supplement2 for the list of oxidative stress respon-sive gene probes) was supplemented with a set of geneprobes not influenced by oxidative stress but obeying the60 % data availability filtering criteria (484 in total). Thecombined set of gene probes (1986 in total; Figure 6 A, Additional file 3:Supplement3 for the list of gene probesconsidered in significant enrichment calculations) wasused for functional data sorting and statistical analysis.Stress-responsive gene probes were recorded in highest numbers in categories "Transport, cytoskeleton, cell wall","Carbon metabolism" and "RNA splicing and translation,protein maturation". The ratio of "Function-unknown"genes was around 50 % independently of stress-inducing agent (Figures 6B–D). The distribution of stress-respon-sive gene-probes among functional categories was approx-imately the same for each stress-inducing agent (Figures
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