Molecular Mechanism Of Nickel Tolerance Of The Histone Point Mutant H4I26A In Saccharomyces Cerevisiae

Nickel is a heavy metal that is widely distributed in the environment.Environmental pollution caused by nickel may be derived from excessive mining and industrial waste.Exposure to high concentrations of nickel can induce a variety of human diseases,such as allergy,cardiovascular and kidney diseases,pulmonary fibrosis,lung cancer and nasal cancer.Nickel can cause cytotoxicity such as DNA damage,protein damage and lipid peroxidation by substituting essential metal in metallothionein,binding with catalytic residues of non-metallic enzymes or indirectly causing oxidative stress.Cells can regulate gene expression to protect against Nickel stress and detoxify it by improving antioxidant capacity or activating metabolic pathways.H4I26A,a histone point mutation strain of Saccharomyces cerevisiae,whose isoleucine at point 26 of histone H4 mutates to alanine was screened by our previous work.It's resistance to nickel,but the mechanism is not clear.In this study,adopted H4I26A as the research object,oxidative damage,antioxidant system and apoptosis of Saccharomyces cerevisiae cells under different concentrations of NiCl₂stress were observed.The transcriptome of Saccharomyces cerevisiae under 5 mMNiCl₂stress was analyzed by RNA-seq.The content of trehalose and the expression of TSL1 and TPS1,Trehalose synthesis genes,in yeast cell was determined.The nucleosome occupancy in the upstream of TSL1 and TPS1 genes transcription start sites was observed by MNase-q PCR.The main results were summarized as following:1.With 5 mMNiCl2 stress,the growth of BY4741,wild-type BY4741 was seriously inhibited,while H4I26A,the mutant strain,grew well;Ni2+is distributed in both cytoplasm and nucleus;when the concentration of NiCl₂ stress increased,the Ni²⁺content of H4I26A had a significant increase,and the content was significantly higher than BY4741.2.Treated with different NiCl₂ concentration,the intracellular ROS and MDA contents of wild-type BY4741 were significantly higher than that of mutant H4I26A;the intracellular SOD activity of wild-type BY4741 and mutant H4I26A enhanced with the increase of NiCl₂stress concentration,and that of mutant H4I26A was significantly lower than control group.With NiCl₂stress,the intracellular GSH content of wild-type BY4741 and mutant H4I26A decreased significantly;however,the change of intracellular GSH content of mutant H4I26A was not significant with the increase of NiCl₂ stress concentration.3.With the increase of NiCl₂ stress concentration,in wild-type BY4741,the mitochondrial membrane potential decreased gradually while the concentration of Ca²⁺increased gradually,and its apoptosis was significantly higher than mutant H4I26A;the influence of Ni²⁺on the cell cycle of H4I26A was not significant.4.Transcriptome sequencing analysis of Saccharomyces cerevisiae treated with 5mMNiCl₂ showed that compared with the control group,in the wild-type BY4741,955 genes were up-regulated and 722 genes were down-regulated;in the mutant strain H4I26A,949genes were up-regulated.687 genes were down-regulated.The GO function analysis of differentially expressed genes under nickel stress revealed that in the two strains,the differentially expressed genes were all related to biological processes such as metabolic processes,redox processes,ion transport,and cell wall formation;enrichment analysis of the KEGG pathway found that nickel The stress mainly affects the energy metabolism,lipid metabolism,steroid biosynthesis,oxidative phosphorylation,trehalose synthesis metabolism and other related pathways of Saccharomyces cerevisiae.Under nickel stress,GO function enrichment analysis of up-regulated genes in wild-type BY4741 and down-regulated genes in mutant strains revealed that they are related to energy metabolism,while down-regulated genes in wild-type BY4741 and up-regulated genes in mutant strains are mainly related to ion transport.5.Without NiCl₂ stress,the trehalose content in wild-type BY4741 cells was significantly higher than mutant H4I26A;with NiCl₂ stress,the trehalose content in mutant H4I26A cells was significantly increased;with 5 mMNiCl₂ stress,the expression of TSL1and TPS1,trehalose synthesis genes of mutant H4I26A significantly,was significantly raised,while the expression of wild-type BY4741 was significantly descended;meanwhile,with 5mMNiCl₂ stress,the nucleosome occupancy rate in the upstream of TSL1 and TPS1 genes transcription initiation sites in mutant H4I26A was decreased.In conclusion,the mutant H4I26A suffered little oxidative damage and possessed strong antioxidant capacity with NiCl₂ stress;NiCl₂ stress mainly affected the pathways such as energy metabolism,lipid metabolism,steroid biosynthesis,oxidative phosphorylation and trehalose anabolism of Saccharomyces cerevisiae.Trehalose is an important protective agent in cells.Therefore,with NiCl₂ stress,the nucleosome occupancy rate in the upstream of TSL1 and TPS1 genes transcription initiation sites in mutant H4I26A decreased and the expression level was significantly increased.The increase of trehalose content can protect the strain,which may be one of the reasons for the Nickel tolerance of H4I26A.This study provides a theoretical basis for the understand of Saccharomyces cerevisiae tolerance mechanism under heavy metal stress.