Study On The Function Of Saccharomyces Cerevisiae Elp3 Gene In Yap1 Oxidative Stress Pathway

The transcription elongation complex Elongator has six subunits,of which Elp3 is the catalytic subunit.Elp3 can acetylate histones and other non-histone proteins,but more and more studies have shown that Elp3 also has the function of t RNA acetyltransferase.Studies from different organisms have shown that Elp3 plays a role in a variety of biological processes such as stress response,development and neuronal degeneration.Preliminary research in our laboratory found that yeast strains lacking Elp3 are highly sensitive to hydrogen peroxide,and the lack of Elp3 under oxidative stress affects the accumulation of transcription factor Yap1.Yap1 enters the nucleus to regulate the expression of antioxidant genes is the key to the oxidative stress response mechanism.When the cell encounters oxidative stress,Yap1 enters the nucleus with the help of Gpx3,Trx2,and Ybp1 to regulate the expression of antioxidant-related proteins together with the transcription factor Skn7.In order to further study the function of Elp3 in the Yap1pathway,we carried out the following work:(1)Constructed Gpx3,Trx2,Ybp1 co-deleted and overexpressed elp3?strains,and passed hydrogen peroxide sensitivity experiments,ROS,MDA and Yap1nuclear accumulation Detect and explore the function of Elp3 in Gpx3,Trx2,Ybp1 assisting Yap1 to enter the nucleus;(2)Transfer the Yap-GFP fusion expression plasmid into each mutant strain,and detect the co-deletion pair of Elp3 and Yap1 nuclear entry auxiliary protein by fluorescence microscopy.The influence of Yap1 nuclear accumulation;(3)Construct Elp3 bait expression vector,and detect whether it interacts with Yap1 pathway related proteins such as Gpx3,Trx2,YBP,Yap1,Skn7,etc.;(4)Construct myc-Elp3 fusion Expression vector,through Ch IP-PCR to determine whether Elp3 is involved in the transcription extension of Yap1 pathway related genes.The above experimental results show that under oxidative stress,the co-deletion of Gpx3,Trx2,and Ybp1 can significantly increase the H₂O₂sensitivity of the elp3?strain,increase ROS,MDA,and significantly reduce the nuclear accumulation of Yap1;in the wild case of Elp3,three Overexpression can significantly increase the tolerance of wild-type H₂O₂,and significantly reduce ROS and MDA.However,the transfer of the same Gpx3,Trx2,Ybp1 overexpression plasmids into the elp3?strain cannot improve its hydrogen peroxide sensitive phenotype.The levels of MDA and ROS are similar to those of the elp3?strain.It shows that Elp3 is necessary for Gpx3,Trx2,Ybp1 to assist Yap1 into the nucleus.The transcriptional activity detection of Yap1 and Yap1-Skn7 found that the deletion of Trx2 will not reduce the transcriptional activity of Yap1,but it can reduce the transcriptional activity of Yap1-Skn7,which may be related to the dual regulatory function of Trx2 in the Yap1 pathway;Gpx3 and Ybp1 lack Yap1 The transcriptional activities of Yap1-Skn7 and Yap1-Skn7 were significantly reduced;however,the transcriptional activities of Yap1 and Yap1-Skn7 were significantly increased after Elp3 deletion.It is speculated that after Elp3 deletion,the high levels of ROS and MDA in the cell trigger other unknown pathways in the redox system to activate Yap1 to counter the cell Excessive peroxide in the cell damages cells.Although the two-hybrid experiment did not find a direct interaction between Elp3 and Gpx3,Trx2,YBP,Yap1,and Skn7;the Ch IP-PCR experiment provided that Elp3 participates in the regulation of oxidative stress in Saccharomyces cerevisiae by promoting the transcription extension of Yap1pathway-related genes.evidence.