Study On The Function Of Elp3 In Oxidative Stress Of S.cerevisiae

Elp3 is the catalyticsubunit ofthe Elongator complex which has histone acetyltransferase?HAT?activity and could acetylates histone H3 and H4.Earlier work in our labbased on the H₂O₂ sensitivity experiment showed that theElp3 deletion andH3K14/H4K8 mutation in the HAT target site of Elp3increasethe sensitivity of elp3?yeast H₂O₂,suggesting that Elp3 may affect the H₂O₂ tolerance of yeast by acetylating histones to promote the transcription of antioxidant genes.To further validate this hypothesis,we measured ROS,MDA,and CAT,SOD,and GPX mRNA levels of wild and mutant histone elp3?transformed with full-length and conserved domains deteted Elp3 using WT and elp3?as controls.The results showed that under the H₂O₂ stress,the contents of ROS and MDA in each strain were significantly increased.The transfer of yELP3 gene could decrease the levels of ROS and MDA in elp3?cells.The H3K14/H4K8 mutation further increased the levels of ROS and MDA in elp3?strain and reduced the compensation effect of yELP3.The results of qRT-PCR showed that the expression levels of CAT,SOD and GPX in elp3?strain were significantly lower than those in WT under oxidative stress,and increased by yELP3 transformed.The H3K14/H4K8 mutation further down-regulated the expression level of the antioxidant gene of the elp3?strain,and also significantly reduced the compensation effect of yELP3.The above results indicate that Elp3 can participate in the oxidative stress response of Saccharomyces cerevisiae through regulating transcription of antioxidant genes by acetylated histones.Moreover,deletion of the HAT or SAM domain in Elp3 reduced the compensation effect of ELP3 on the elp3?strain,The ROS?MDA and antioxidant gene levels are similar to those of the elp3?strain.indicating that the two coserved domain are important for the function of Elp3.It was known that Elp3 can also modify tRNA and control the translation efficiency ofprotein.Therefore,it is likely that Elp3 also affects the oxidative stress of yeastthrough its tRNA modification function.In order to validate this hypothesis,sensitivity detection and antioxidant index determination were conducted under H₂O₂ stress using WT?elp3?and elp3?strains in which tRNA_Lysys UUU and tRNA_GlnUUG overexpressed independently or simultaneously were used as materials.The results showed that simultaneous overexpressionof tRNA_LysUUUand tRNA_GlnUUG could significantly improve the H₂O₂ tolerance of elp3?strain and reduce the ROS and MDA levels.It was suggested thatElp3 may affect the translation efficiency of antioxidant enzymes through tRNA modification,thus affecting the cell H₂O₂ tolerance.Therefore,we determined the expression of antioxidant proteins by Western Blot,theresults showed that the total protein content,SOD,GPX3 and CAT protein expressions of tRNA_LysUUU/tRNA_Glnln UUG/elp3?strain were between elp3?and wild strain underH₂O₂ stress.These results indicated that the overexpression of modified tRNA could significantly improve antioxidant protein expression of ROS,MDA and H₂O₂ tolerance of elp3?strain cells under hydrogen peroxide stress.It is revealed that Elp3could participate in the oxidative stress reaction of s.cerevisiae by affectingthe translation of antioxidant proteins through tRNA modification.There is a certain relationship between oxidative stress and heat shock response.Oxidative stressrelated indexes of WT,elp3?train and the tRNA_LysUUU and tRNA_GlnUUG overexpressed lonely or simultaneously strains were determined.High temperature and H₂O₂ synergistic treatment further reduced the hydrogen peroxide tolerance of elp3?strain,and the modified tRNA overexpression significantly compensated the hydrogen peroxide and high temperature sensitive phenotype of elp3?strain.Moreover,underthe stress of H₂O₂,in the loss of Elp3 led to significantly down-regulated expressions of Hsp75 andHsp105.These results indicated that Elp3 is involved in the oxidative stress of saccharomyces cerevisiae byregulating the expression of oxidative stressrelated proteins and heat shock proteins.