Transcriptional Profiling of Saccharomyces cerevisiae Upon Exposure to Saxitoxin
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae Schizosaccharomyces pombe
PROVIDER: GSE16652 | GEO | 2009/06/18
SECONDARY ACCESSION(S): PRJNA116241
REPOSITORIES: GEO
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Project description:Saxitoxin is a potent neurotoxin produced by several species of dinoflagellates and cyanobacteria. The molecular target of saxitoxin in higher eukaryotes is the voltage-gated sodium channel; however, its target in lower eukaryotic organisms remains unknown. The goal of this study was to obtain the transcriptional fingerprint of the model lower eukaryote Saccharomyces cerevisiae upon exposure to saxitoxin to identify potential genes suitable for biomarker development. Microarray analyses identified multiple genes associated with copper and iron homeostasis and sulfur metabolism as significantly differentially expressed upon exposure to saxitoxin; these results were verified with quantitative reverse-transcriptase PCR (qRT-PCR). Additionally, the qRT-PCR assays were used to generate expression profiles in a subset of the differentially regulated genes across multiple exposure times and concentrations, the results of which demonstrated that overall, genes tended to respond in a consistent manner to the toxin. In general, the genes encoding the metallothioneins CUP1 and CRS5 were induced following exposure to saxitoxin, while those encoding the ferric/cupric reductase FRE1 and the copper uptake transporter CTR1 were repressed. The gene encoding the multicopper ferroxidase FET3, part of the high-affinity iron uptake system, was also induced in all treatments, along with the STR3 gene, which codes for the cystathionine beta-lyase found in the methionine biosynthetic pathway. Experiment Overall Design: Cultures of S. cerevisiae S288C were grown to OD600=1.0 in YPD (1% yeast extract, 2% peptone, 2% dextrose). Four hundred microliters from individual 10 mL overnight cultures were transferred to sterile tubes and saxitoxin added to a final concentration of 16 µM. Sample were mixed gently and incubated at 30° C for 45 min. Controls consisted of deionized water. Three biological replicates were performed under these conditions for microarray analysis. Cells were harvested via sequential centrifugation and placed immediately in liquid nitrogen.Total RNA was extracted from frozen cell pellets using a protocol that combined the hot acid phenol method with the RNEasy kit. Total RNA was processed by the Affymetrix Core Facility at the University of Tennessee (Knoxville, TN) following the protocols of the Affymetrix GeneChip Expression Analysis Technical Manual. MAS5.0 was used to provide present/absent calls; genes absent in all six samples were then removed from the data set. Intensity values for all transcripts were obtained from the scanned image files of the chips. The intensity values of the six arrays were background-corrected and normalized with the GC-RMA algorithm.
2009-07-01 | E-GEOD-16652 | biostudies-arrayexpress
Project description:In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates Cu-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiae genes, including CTR1, CTR3, FRE1, FRE7, YJL217w and YFR055w. Two of the last three newly identified Mac1 target genes have no known function, the third, YFR055w, is homologous to cystathionine gamma-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1up1, are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells due to constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1 and CRS5, and two genes, FET3 and FTR1, in the iron uptake system. Cu-induced FET3 and FTR1 expression arises from an indirect Cu effect on cellular Fe pools. This study is described in more detail in Gross C et al.(2000) J Biol Chem 275:32310-6 Keywords: other
2002-02-12 | GSE26 | GEO
Project description:Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications. yap1yap8 mutant cells independently transformed with pRS416 and YcpLac111, YcpLac111-YAP1, or pRS416-YAP8 were grown in triplicates in SC-URA-LEU containing 2mM of AsV until exponential growth phase, and RNA was extracted, labeled, and hybridized to Affymetrix Yeast Genome S98 arrays.
2011-11-04 | E-GEOD-33427 | biostudies-arrayexpress
Project description:Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications.
2011-11-04 | GSE33427 | GEO
Project description:The present work describes the effects on iron homeostasis when copper transport was deregulated in Arabidopsis thaliana by overexpressing copper transporters (COPTOE). A genome-wide analysis conducted on COPT1OE plants, highlighted that iron homeostasis gene expression was affected, both under copper deficiency and excess. Among the inhibited genes were those encoding the iron uptake machinery and their transcriptional regulators. Subsequently, COPTOE seedlings contained less iron and were more sensitive than controls to iron deficiency. These results emphasized the importance of appropriate spatiotemporal copper uptake for iron homeostasis under copper deficiency. The deregulation of copper-I uptake difficulted the transcriptional activation of the subgroup Ib of basic helix-loop-helix (bHLH-Ib) factors. Oppositely, copper excess inhibited the expression of the master regulator FIT but activate bHLH-Ib expression in COPTOE plants, in both cases leading to the lack of an adequate iron uptake response. As copper increased in the media, iron-III was accumulated in roots, accounting for a defective iron mobilization to the aerial parts, and this effect was exacerbated in COPTOE plants. Thus, iron-III overloading in roots inhibited local iron deficiency responses, aimed to metal uptake from soil, leading to a general lower iron content in the COPTOE seedlings. The understanding of the role of copper uptake in iron metabolism could be applied for increasing crops resistance to iron deficiency
2020-02-04 | GSE143857 | GEO
Project description:Copper tolerance and sulfite tolerance are two well-studied phenotypic traits of Saccharomyces cerevisiae. The genetic bases of these traits are derived from allelic expansion at the CUP1 locus and reciprocal translocation at the SSU1 locus, respectively. Previous work identified a negative association between sulfite and copper tolerance in S. cerevisiae wine yeasts. Here we probe the relationship between sulfite and copper tolerance and show that an increase in CUP1 copy number does not impart copper tolerance in all S. cerevisiae wine yeast. Bulk-segregant QTL analysis was used to identify variance at SSU1 as a causative factor in copper sensitivity, which was verified by reciprocal hemizygosity analysis in a strain carrying 20 copies of CUP1. Transcriptional and proteomic analysis demonstrated that SSU1 over-expression did not suppress CUP1 transcription or constrain protein production but suggested that SSU1 overexpression induced sulfur limitation during exposure to copper. Finally, an SSU1 over-expressing strain exhibited increased sensitivity to moderately elevated copper concentrations in sulfur-limited medium, demonstrating that SSU1 over-expression burdens the sulfate assimilation pathway. Over-expression of MET 3/14/16, genes upstream of H2S production in the sulfate assimilation pathway increased the production of SO2 and H2S but did not improve copper sensitivity in an SSU1 overexpressing background. We conclude that copper and sulfite tolerance are conditional traits in S. cerevisiae and provide evidence of the metabolic basis for their mutual exclusivity. These findings suggest an evolutionary basis for the extreme amplification of CUP1 observed in some yeasts.
2023-03-22 | PXD037997 | Pride
Project description:Extrachromosomal circular DNA (eccDNA) facilitates adaptive evolution by allowing rapid and extensive gene copy number variation, and is implicated in the pathology of cancer and ageing. Here, we demonstrate that yeast aged under environmental copper accumulate high levels of eccDNA containing the copper resistance gene CUP1. Transcription of CUP1 causes CUP1 eccDNA accumulation, which occurs in the absence of phenotypic selection. We have developed a sensitive and quantitative eccDNA sequencing pipeline that reveals CUP1 eccDNA accumulation on copper exposure to be exquisitely site specific, with no other detectable changes across the eccDNA complement. eccDNA forms de novo from the CUP1 locus through processing of DNA double-strand breaks (DSBs) by Sae2 / Mre11 and Mus81, and genome-wide analyses show that other protein coding eccDNA species in aged yeast share a similar biogenesis pathway. Although abundant we find that CUP1 eccDNA does not replicate efficiently, and high copy numbers in aged cells arise through frequent formation events combined with asymmetric DNA segregation. The transcriptional stimulation of CUP1 eccDNA formation shows that age-linked genetic change varies with transcription pattern, resulting in gene copy number profiles tailored by environment.
2019-10-18 | GSE135542 | GEO
Project description:The effect of the heavy metal copper on the expression of a wide spectrum of genes was analyzed by using a DNA microarray. Gene expression profile of baker’s yeast Saccharomyces cerevisiae grown in a media containing sub-lethal concentration of cupric sulfate was compared with those of yeast grown in a normal media. Among about 6200 ORFs of yeast, 143 ORFs were induced more than two-fold to resist against copper toxicity after exposure to copper. As was expected, FRE1, FRE7, and CTR1, genes controlled by Mac1p, were strongly down-regulated. Copper metallothionein CUP1-1 and CUP1-2 were induced more than 20-fold. Some genes related to sulfur metabolism and oxidative stress response were also up-regulated. This DNA microarray technology indicated that plasma membrane, cell wall, protein, and DNA, were molecular targets of copper toxicity. Keywords: stress response
2007-11-01 | GSE9231 | GEO
Project description:Iron and copper are important environmental nutrients for plant growth. However, the molecular mechanisms of both iron and copper signaling that integrate the two pathways remain poorly understood. The Arabidopsis thaliana high affinity copper transporter COPT5, is a tonoplast localized permease involved in copper remobilization. Here, a global expression microarray analysis of the copt5 mutant points out the induction of iron deficiency responses, including NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 4 (NRAMP4), a tonoplast-localized iron transporter. The copper requirement in iron perception and uptake from the media becomes more evident in the double nramp3nramp4 mutant, unable to remobilize iron from vacuoles, that is highly sensitive to copper deficiency. Furthermore, COPT5 expression is altered under iron deficiency and the copt5 mutant is sensitive to iron deficiency and is unable to perceive iron in the media under copper deficiency. Noteworthy, iron deficiency post-transcriptionally restraints the copper-dependent superoxide dismutase protein levels and the subsequent activity. As a consequence of its increased iron deficiency responses, the copt5 mutant present lower levels of both copper- and iron-dependent superoxide dismutase activities. Moreover, the copt5 mutant mobilizes faster its iron storage pools and presents higher levels of iron in cotyledons and seeds. These results underline the importance of internal metal pools in the understanding of copper and iron deficiency responses and their crosstalk that are critical for governing proper plant development in response to combined metal scarcities in soils.
2017-05-30 | GSE91044 | GEO
Project description:Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast. FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed. Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner. Fit1p was localized exclusively to the cell wall by indirect immunofluorescence. Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin. FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake. Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources. Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains. Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space.
2005-09-27 | E-SMDB-1593 | biostudies-arrayexpress