Regulating Copper Ion Stress-induced Gene Expression In Yeast By MBF1 Protein

Multi-protein Bridging Factor 1（MBF1） was one of the auxiliary activating factors with conservative evolution that widely existed in eukaryotes. MBF1 regulated downstream gene transcription through combining transcription-activating factors with TATA binding proteins. Recent research demonstrated that MBF1 was of great importance during the response to biotic and abiotic stress. However, reported researches mainly focused on exploring the functions of MBF1 in animals and plants, and the functions of MBF1 in low class eukaryotes were rarely investigated.Heavy metal stress was one important abiotic stress and showed significant influence on organisms within all these threats, and the research of copper stress to organisms was drawing wide attention. Previous reports mostly focused on invesgating the influence mechanisms of copper ions on plants. Whereas, the mechanisms of cooper ion stress to micro-organism were of less attention. The affect of Copper stress to the expression of MBF1 protein in saccharomyces cerevisiae were also lack of investigation in the world wide.Herein, we investigated the transcriptomic sequence of Wild-type yeast Y258, mbf1 genetic defect-type yeast mbf1（-）, and mbf1（-） after copper ion stress treatment（6m M） through Illumina high-throughput sequencing. In comparison with differentially expressed genes wild-type yeast Y258 and mbf1（-）, 2060 genes with significantly differentially expressions were discovered（FDR≤0.001 AND |log2Ratio|≥1）. Compared with mbf（-）, the expression of 1568 genes were down-regulated and 492 genes were up-regulated in Y258, which indicated that the expressions of several genes were negatively-controlled and the expressions of most genes were actively-controlled by MBF1. By KEGG pathway analysis, BMF1-regulated genes mainly accumulated in oxidative phosphorylation pathway（Qvalue=0.028）, especially in cytochrome C oxidase in electron transport chain. This indicated the important role o f MBF1 protein in regulating ATP synthesis within cells, especially in the critical pathway that accepting electrons from the cytochrome C and reducing oxygen molecules into water. Moreover, MBF1-regulated genes also accumulated in the pathways of yeast meiosis, cell cycle regulation, homologous recombination, starch sugar metabolism, and histidine metabolism. This also dedicated that the great importance of MBF1 in transcriptional regulation.In comparison with differentially expressed gene mbf1（-） and copper ion stress-treated Cu²+（+）mbf1（-）, 1770 differentially expressed genes were found（FDR≤0.001 AND |log2Ratio|≥1）. The expressions of 750 genes were up-regulated and the expressions of 1020 genes were down-regulated under copeer ion stress treatment. The results showed that some copper stress-responding genes, including superoxide dismutase（SOD）, catalase（CAT）, heat shock protein（HSP）, and molecular chaperone, had been proven to be related to the metabolism of copper ions and had been reported in previous works. KEGG pathway analysis demonstrated that copper stress-responding genes mainly accumulated in peroxysome pathway（Qvalue=0.011）, and starch and sucrose metabolism pathway（Q value=0.00018）. The expressions of fatty acid metabolic genes in peroxysome pathway were up-regulated, the expressions of SOD, CAT enzymes were up-regulated, and the expressions of genes in starch and sucrose metabolism pathway were mainly up-regulated. The data indicated that the stress response was taken place under copper ion stress in Saccharomyces Cerevisiae. The yeast gained energy supply and simultaneously tried to restore the balance of free radicals through regulating the protein expression of superoxide dismutase（SOD） and catalase（CAT） to reduce cytotoxicity.Overlapping the two groups of differentially e xpressed genes above, 775 differentially expressed genes were obtained; indicating that these genes were both impacted by copper ion stress and mbf1 genes. Herein, the expressions of 120 genes were up-regulated, which mianly accumulated in oxidative phosphorylation pathway, transcriptional regulation, and DNA replication. This indicated that copper ions and MBF1 could widely affect the whole vital process. The expressions of 447 genes were both down-regulated under copper ion stress and up-regulated with MBF1 deficiency. And these d ifferentially expressed genes with larger multiples all enriched in the subunits of RNA ribosomes, which demonstrated that copper ion and MBF1 could regulate the cellular translation process, and further regulate the functions of expressed proteins.To identify the precision of transcriptome sequencing results, Real- Time PCR was applied to make relative quantitative inspection to differential expressed genes. 6 genes influenced by copper ion stress were selected from peroxisome pathway（YAR035W, YMR246 W, YLR174 W, YNL202 W, YJL210 W, and YOL147C）, 5 genes regulated by MBF1 were chosen from oxidative phosphorylation pathway（Q0045, Q0160, Q0055, Q0050, and Q0065）, and 7 genes both affected by copper ions and MBF1 were also selected from oxidative phosphorylation pathway（Q0070, Q0045, Q0160, Q0055, Q0050, Q0065, and Q0085） for relative quantitative validation. The results demonstrated the uniformity with transcriptome sequencing results, which indicated that transcriptome sequencing was a precise way for gene regulation research.Moreover, yeast expressed plasmid p EGH-rad24 and p EGH-mbf1 of yeast protein 14-3-3 BMH1 gene rad24 and MBF1 gene mbf1 were constructed. These two plasmids were utilized to transfer into Y258 to construct recombinant yeast which could both express BMH1 and BMF1. These works extablished material preparations for the research on to understand the interacting protein under MBF1 and BMH protein stress.