Screening For Enzymes Of Carbamylation Of Histone H3K23 From Genome-wide Deletion Library Of Saccharomyces Cerevisiae

Objective:Enzymes of carbamylation of Histone H3K23 were screened from genome-wide deletion library of Saccharomyces cerevisiae,and candidate genes were verified to further study the function and molecular regulation mechanism of lysine carbamylation and provide new ideas for related diseases.Methods:1.Immunoblotting assay was performed to examine the specificity antibody against histone H3K23 carbamylation.2.Compared the feasibility of yeast lysozyme and acid-washed glass beads in terms of experimental consumables,time-consuming and immunoblotting assay.3.The first screening of enzymes of carbamylation of histone H3K23 from genome-wide deletion library of saccharomyces cerevisiae was carried out by immunoblotting assay.4.The KEGG function annotations of 245 carbamylation-modifying genes were analyzed by bioinformatics.5.The second screening of 245 carbamylation-modifying genes was carried out by immunoblotting assay.6.Immunoblotting assay was performed to detect the effect of different amino acid conditions on the carbamylation of histone H3K23.7.Immunoblotting assay was performed to detect the effect of histone deacetylase inhibitors on carbamylation of histone H3K23 of Saccharomyces cerevisiae.8.Immunoblotting assay was performed to detect the effect of protein synthesis inhibitors on carbamylation of histone H3K23 of Saccharomyces cerevisiae.9.Immunoblotting assay was performed to explore the degradation pathway of histone H3K23 carbamylase of Saccharomyces cerevisiae.10.Molecular cloning was performed to construct candidate gene expression vector of enzymes of carbamylation of histone H3K23 and yeast transformation method was used to construct the corresponding over-expressed Saccharomyces cerevisiae strain.11.Candidate genes were overexpressed in Saccharomyces cerevisiae.Immunoblotting assay was performed to detect the carbamylation level of Histone H3K23 of Saccharomyces cerevisiae.Results:1.Site-specific antibody against H3K23 carbamylation can specifically recognize K23 modification of histone H3 in Saccharomyces cerevisiae2.Considering the cost and time-consuming of the experiment,pickling glass beads is more suitable for screening enzymes of Enzymes of carbamylation of Histone H3K23 in the whole genome of Saccharomyces cerevisiae.3.Through the first round of screening,36 genes with enhanced carbamoylation level and 209 genes with decreased carbamoylation level were obtained.4.KEGG enrichment analysis of 245 carbamylation-modifying genes affecting histone H3K23 showed significant enrichment of phagocytosis,RNA degradation,RNA detection and oxidative phosphorylation.5.Through the second round of screening,49 genes affecting histone H3K23 carbamylation modification were obtained.6.Immunoblotting assay showed that amino acid deficient medium inhibited the growth of Saccharomyces cerevisiae and reduced the carbamylation of histone H3K23.7.Immunoblotting assay showed that histone deacetylase inhibitors did not cross-regulate histone H3K23 carbamylation in Saccharomyces cerevisiae.8.Immunoblotting assay showed that histone H3K23 carbamylase of Saccharomyces cerevisiae did not degrade through ubiquitination.9.Immunoblotting assay showed that histone H3K23 carbamylase of Saccharomyces cerevisiae did not degrade through autophagy.10.The expression vectors of LSM7,MRP10,ADA2,SPT6,SPT16,SPT5,SPT10,TRM1 and LSM1 were successfully constructed by molecular cloning.The wild-type yeast cells(BY4742)were transformed into 0.5 ug plasmids of each group for 1 hour,then screened on CSH-URA agar medium for 2 days.The target yeast strains were obtained by screening monoclonal and monoclonal expansion culture and identification.11.Immunoblotting assay showed that the carbamylation level of histone H3K23 of Saccharomyces cerevisiae overexpressing LSM7,LSM1,ADA2 and SPT6 decreased,the carbamylation level of histone H3K23 of Saccharomyces cerevisiae overexpressing SPT16 increased,while the carbamylation level of histone H3K23 of Saccharomyces cerevisiae overexpressing SPT5,SPT10,MRP 10 and TRM1 had no effects.Conclusion:Under the condition of deficient amino acid culture,the growth of Saccharomyces cerevisiae was inhibited,and the level of histone H3K23 carbamylation was decreased.Saccharomyces cerevisiae histone H3K23 carbamylase did not degrade by ubiquitination and autophagy.LSM7,MRP10,ADA2,SPT6 and SPT16 may be involved in the regulation of histone H3K23 carbamylation in Saccharomyces cerevisiae.