Regulation Of The Bdf1p Transcription Factor In The Salt Stress Response Of Saccharomyces Cerevisiae

Cellular organisms have developed several autonomous mechanisms to adjust themselves to new conditions,because they are constantly challenged by changing environmental conditions,.Under stress conditions,the cells undergoes a series of changes to protect the external system from the detrimental effects of stress,which were referred to as the environmental stress responses(ESR).The studies of yeast stress responses have provided a powerful tool for investigation of the genes function and elucidation of the nature of important life activities.In addition,research on stress response has important role for the improvement of the stress resistance of plant corps and industrial microbial,and has a large impact on medical issues.Saccharomyces cerevisiae was the first eukaryotic organism whose whole genome was completely sequenced(http://genome-www.stanford.edu/Saccharomyces).The rapid growth,ease of genetic manipulation,and a well-defined genetic system of the yeast Saccharomyces cerevisiae make it ideal for fundamental and applied studies in eukaryotic species.Salt-stress adaptation mechanisms are being intensively studied in model organisms Saccharomyces cerevisiae.Under high salt conditions,the yeast cells are mainly challenged by ion and hyperosmotic stress.Several mechanisms involved in yeast salt stress response have been identified.For instance,under NaCl stress,the high osmolarity glycerol mitogen activated protein kinase signaling transduction pathway (HOG-MAPK)is activated,which mediates the production and accumulation of the osmolyte glycerol.Calcineurin functions through the Crz 1 p/Ten 1 p transcription factor to induce several salt stress-responsive genes expression,such as the ENA1 gene which encodes a plasma membrane P-type Na~+-ATPase ion extrusion pump. Additionally,the TRK1/2p transport system increased affinity for K~+ and prevent entry of excess Na~+,to increase NaCl tolerance.Yeast mutants with impaired K~+ transport system showed increased sensitivity to salt stress due to accumulate of more Na~+ than wild type strain.The S.cerevisiae BDF1 gene,which encodes a bromodomain-containing transcription factor,was isolated in a large-scale screen for salt-sensitivity mutants following transposon mutugenesis.Bromodomain transcription factor 1 protein(Bdf1p)contains two bromodomains and an ET(extra-terminal)domain.It is a member of the BET protein family.In yeast, Bdf1p was not essential for cell viability.Bromodomains are evolutionarily conserved and act as acetryl-lysine binding domains that process the molecular information conveyed by lysine acetylation modification.To understanding the biological functions ofbromdomain-containing proteins in cellular processes were currently at a fledgling stage.By using gene knockout and drugs block techniques,the relationship between BDF1 and several signaling transduction pathways/genes have been known in salt tolerance,was investigated.These included HOG-MAPK pathway,Calcineurin pathway,ENA1 and TRK1 genes.Genetic analysis indicated that the function of Bdf1p in salt tolerance is independent of the HOG-MAPK pathway or the calcineurin pathway,K~+ transportation system and is not meditated by Enalp,the major determinant of Na~+ extrusion system.The yeast genome-scale cDNA microarray platform provides an effective research tool to investigated the global changes in gene expression under a given salt stress condition.To gain further insight into the role of Bdf1p in salt tolerance on a global scale,DNA microarray analysis was conducted.The global gene expression changes in the wild-type and a bdf1△mutant responding to a salt stress treatment(0.6 mol/L NaCl,45 min)were measured,respectively.Total yeast RNA was isolated after treated by0.6 mol/L NaCl for45 min,and the cDNA were labeled with Cy5-dCTP and Cy3-dCTP,respectively.Then the Cy5/cy3-1abeled cDNA were hybridized on the yeast genome-wide DNA microarrays containing 5935 ORFs.Obtained images were analyzed with a GenePix Pro 4.0.A space and intensity-dependent normalization based on the LOWESS program was employed.We determined the levels of gene expression based on the statistical method student's t-test in combination with a 2-fold cutoff Genes whose ratio changed＞2-fold with p＜0.05 were determined to be have significantly different gene expression.To eliminate the dye-bias artifact,the dye-swap experiment was performed for two different RNA populations.And two biologically independent experiments of each of the samples(wild-type,wild-type salt treated,bdf1△,bdf1△salt treated)were analyzed.Only genes having consistently altered expression in two independent experiments were selected for further analysis,and the final fold changes were derived from the average of two independent experiments.Based on this experiment design,totally 217 differentially expressed genes were identified in salt-treated yeast cells(wild type and bdf1△mutant),in which 144 total genes(119 up-regulated genes and down-regulated 25 genes)displayed the same altered tendency in the wild type and bdf1△mutant after the salt treatment.Functional classification of the 144 differential expressed genes were base on the MIPS Functional Catalogue Database(http://mips.gif.de/proj/funcatDB/search_main_fra me.html).The above genes are associated with functions,such as glycerol metabolism,trehalose metabilism,ion transport,heat shock protein,unclassified protein,and so on.Interestingly,the expression level of ENA1 had a similar increase in the wild type and bdf1△mutant(5.25- and 6.32- fold,respectively),suggesting that the function of ENA1 in Na~+ tolerance is independent of BDF1.Additionally,The glycerol-3-phosphate dehydrogenase gene(GDP1),which is a key enzyme of glycerol metabolism,was highly induced in the wild type and bdf1△mutant(35.79- and 35.52- fold, respectively)after the salt treatment.The determination of intracellular content of the yeast cells showed that there was a similar level of glycerol in wild type and bdf1△mutant after the salt treatment,which is consistent with the data from the microarray analysis.Change in expressions of two randomly selected genes(MEF1 and YNL208w)were further confirmed by real-time quantitative RT-PCR.And the results suggested good agreement between the microarray and real-time quantitative PCR analysis. In yeast,BDF2 gene is a homolog of the BDF1 gene.Construction of TRK1 deletion mutants was performed by long flanking homology(LFH)-PCR and the kanMX4 deletion marker was used.In spot dilution growth assays,we have not observed a salt sensitive phenotype of the bdf2△mutant compared to the wild-type at various NaCl concentrations,which suggests that the BDF2 gene is not essential for salt resistance. In addition,the microarray results showed that,in response to the salt stress,the expression of BDF2 gene was down regulated in bdf1△mutant but not in the wild type strain.This result indicated that there is some genetic interaction between BDF1 and BDF2 gene,and the roles of Bdf1p and Bdf2p in salt tolerance remains to be determined.It has been known that the mitochondrial functions are important for energy metabolism and have an intimate relation with cell death.The microarray results showed that 7 genes associated with mitochondrial function were downregulated in the bdf1△strain but not in the wild type strain after the salt treatment(0.6M NaCl for 45min).These included four mitochondrial ribosomal genes(MRPL3,MRP4,MRPL7, MRPL11),Cytochrome c Oxidase encoding gene CYC1,a gene encoding a mitochondrial elongation factor MEF1and a gene encoding a mitochondrial valyl-tRNA synthetase VAS1.This result suggested a possible role for Bdf1p in the control of mitochondrial functions under salt stress conditions.Mitochondrial membrane potential(△Ψm)and reactive oxygen species(ROS) production are useful indicators of mitochondrial function.To measure△Ψ, Mitotracker red CMRos was used,which CMRos(Molecular Probes),which stains mitochondria in a△Ψm-dependent fashion.The MitoTracker specifically stains mitochondria when the△Ψm is high,but stains in a diffused pattern when△ΨM is lost.After treated with 0.6 M NaCl for 45 min,the wild type cells displayed a very nice,linear MitoTracker staining,but about 20%of bdf1△cells showed a diffused staining,indicating that absence of Bdf1p caused△Ψloss under salt stress conditions. To monitor the ROS production of yeast cells,we used dihydrorhodamine 123(DHR), which can be oxidized by the intracellular ROS to become fluorescent chromophore rhodamine.After treated with 0.6 M NaCl for 45 min,wild-type cells showed a very dim,red fluorescence after incubation with DHR,whereas bdf1△cells had intense red fluorescence staining with DHR after treatment with NaCl.Relative fluorescence intensities were further quantified on a fluorescent microplate reader.About 1.5-fold increase in fluorescence intensities was observed in the bdf1△mutant compare with the wild-type strain.Superoxide dismutase(SOD)is the key enzyme providing cells protection from the ROS toxicity.After the salt treatment,the SOD activity increased significantly in bdf1△,and was 38 unit higher than in wild type cells.This result indicated that the SOD activity responded normally in bdf1△mutant and the accumulation of ROS in bdf1△cells was not due to the inhibition of Superoxide dismutase activity in the mutant.Apoptosis is an intrinsic cell death process.Mitochondrial dysfunction has been described as a key event in triggering yeast apoptosis.DAPI staining and examination with transmission electron microscopy revealed that the salt-stress treated bdf1△cells displayed randomly distributed nuclear fragments,extensive chromatin condensation and margination along the nuclear envelope,which is a typical marker of apoptosis. This result indicated that absence of Bdf1p caused mitochondria-dependent yeast apoptosis under salt stress conditions.