The Regulation Mechanism And Function Of Ubiquitination On Transcription Factor Cbf1 In Yeast

As a critical transcription factor in yeast cells,Cbf1participates in and regulates the transcriptional activation of genes belong to various pathways,including respiration,lipid synthesis and methionine synthesis processes.In previous researches in our lab,we found that only took charge of DNA pulldown methods alone to enrich Cbf1 transcriptional complex could identified Cbf1 with high-degree ubiquitination,relative to the immunoprecipitation strategy to enrich all the cellular Cbf1.These results impiled that only a subset of Cbf1could be modified with ubiquitin chains,abd the ubiquitination modification on K271 sites might involved in transcriptional activation regulation.Therefore,We aimed at exporling the regulation mechanism of ubiquitination modifications on Cbf1 K271 site in some of thebiological processes in Saccharomyces cerevisiae and verified its biological function.Methods:1.ConstructionofJMP024-cbf1?,JMP024-3HA-Cbf1and JMP024-3HA-Cbf1K271 strains1.1 Construction of JMP024-cbf1?strainThe gene sequence of the Cbf1 protein was obtained from the SGD database,and synthesized a pair of primers which containing the upstream and downstream homologous sequences of Cbf1 gene and the upstream and downstream ORF sequence of kanamycin,respectively.Then,took the kanamycin resistance gene as a template,amplified the knockout element by PCR amplification technology.The knockout element was transformed into JMP024 competent cells,screened on YPD-G418 plates,thepositive clones were screened by colony PCR.1.2 Construction of JMP024-3HA-Cbf1 strainFirst of all,the upstream and downstream primers for amplifying the 3HA fragment and the Cbf1 fragment were synthesized.The 3HA fragment and the Cbf1 fragment were amplified respectively.3HA-Cbf1 fragment could obtain by a fusion PCR.The 3HA-Cbf1 fragment was linked onto the pMD18-T vector and sequenced.The 3HA-Cbf1 fragment was subjected to abundance amplification and transformed into JMP024-cbf1?cells.The transformants were selected on SD-Met plates,and then gained the positive clones by colony PCR.1.3 Construction of JMP024-3HA-Cbf1K271R strainThe Cbf1K271R site mutation primers were designed and synthesized,and the 3HA-Cbf1-T plasmid was used as a template for PCR amplification.After completion of the reaction,Added Dpn I restriction endonuclease to eliminate template plasmid DNA,the cells were transformed,and the plasmid was extracted and sequenced.The 3HA-Cbf1K271R fragment was amplified by using the correct plasmid as a template,transformed into JMP024-cbf1?competent cell.Screened on SD-Met plate,and the positive clones were screened by colony PCR.2.Comparison the Cbf1 expression level and growth status between Cbf1site mutat strain and wild-type strain2.1 Detection of Cbf1 expressionCultured Cbf1 site mutation and wild-type strains in YPD medium,total cellular proteins were extracted under denaturing conditions and protein concentrations were determined.Whether there is a difference in Cbf1 content between Cbf1 site mutant strain and wild-type strain was determined by Weastern Blot and SILAC quantitative proteomics.2.2 Growth status characterizationCbf1 site mutant and wild-type strains were cultured.Transferred to fresh SC and SD+Met liquid medium at the same starting 0D_600,and the OD₆₀₀00 was measured every 2 h.Two parallel biological replicates were set for each strain and the standard deviation was calculated.Took 1 OD of the cells in the exponential growth phase into a centrifuge tube,and series diluted to four concentrations.Point on SC and SD+Met plates,and took photos for every 12 h.3.Role of the ubiquitin chain modificated on Cbf1K271 site in the methionine synthesis pathway3.1 SILAC quantitative proteomicsCbf1 site mutation and wild-type strains were cultured using the SILAC forward and reverse labeling strategy,and biological replicates were set.The total cell protein was extracted under the denaturing condition after mixing the light and heavy yeast cells.After trypsin digestion,mass spectrometry samples were prepared by StageTip rapid separation method,and the data were analyzed by MaxQuant?version 1.5.6.0?software.3.2 Real Timen-Polymerase Chain ReactionIncubated Cbf1 site mutant and wild-type strains in SD+Met medium,and the cells were transforred into SD-Met medium and collected at 0 min,15 min,30 min,and 45 min,respectively,and frozen in liquid nitrogen.RNA was extracted by grinding,and cDNA was synthesized using a reverse transcription kit.Designed primers oftarget genefor RT-PCR amplification.4.Screening the specific substrates which regulated by the ubiquitination on Cbf1 protein K271 siteThe protein quantitative information was obtained by SILAC quantitative proteomics,and the cutoff standard was set to screen differential proteins.Analyed differential proteins'function,and selected some of them for transcription level verification by RT-PCR.Growth status were measured by spot plate phenotypic experiment and growth curve testing.5.Ubiquitination modification in Cbf1K271 siteThe biotin modified upstream and downstream primers were synthesized,and the Met4-2367-T plasmid was used as a template for PCR amplification.the recovered DNA fragment was mixed with Dynabeads?M-280 Streptavidin beads,then the beads were Mixed with whole cell lysates of Cbf1 site mutant strain and wild-type strain,to enrich the transcriptional complex related proteins which had interaction with that DNA motif.The purified proteins were digeasted and subjected to MS identification.Results:1.ConstructionofJMP024-cbf1?,JMP024-3HA-Cbf1and JMP024-3HA-Cbf1K271R strainsWe confirmed that the strip size from each strain was in line with expectations by colony PCR and agarose gel electrophoresis,and the strains were successfully constructed.2.Detection of Cbf1 expression and growth status in site mutation and wild-type strainWestern Blot result showed that there was no significant difference in Cbf1protein content between Cbf1 site mutation and wild-type strain at TCL level.The SILAC experimental results showed that the ratio of Cbf1 protein in the site mutation to the wild-type strain was within the range of 0.8¹.3,and the difference was not statistically significant,that is,there was no significant difference in the expression level of Cbf1 protein bwteen the site mutation and wild-type strains.Growth curve mapping and dot plate phenotypic experiments showed that the growth of Cbf1 site mutation and wild-type strain was consistent.3.Role of the ubiquitin chain on Cbf1 K271 site in the methionine synthesis pathwayThe SILAC quantitative results showed that the expression of the methionine synthesis pathway related proteins was not significantly different between the Cbf1 site mutation and wild-type strain except Met22.RT-PCR result showed that there was almost no difference in the transcriptional level of Met22 in Cbf1site mutation and wild-type strains.When strains were stimulated by methionine deficiency,proteins in the downstream of the pathway have no significant difference in the transcriptional activation between site mutation and the wild-type strains.The results of the spot plate phenotype showed that the Cbf1site mutation strain was not sensitive to various methionine concentration.4.Screening and validation proteins which specificly regulated by ubiquitination on Cbf1 K271 siteAccording to the selection criteria,there were 32 differential proteins in SC medium between wild-type strains and K271R site mutant strains,which were mainly involved in physiological processes such as cell stress,glucose metabolism and biosynthesis.RT-PCR results showed that the transcriptional levels of GPP2,GRX1 and TDH1 were significantly down-regulated in Cbf1site mutation strain.The growth phenotypic experiment showed that the growth of Cbf1 wild-type strain was significantly blocked on different concentrations of AgNO₃ solid plate.5.Role of ubiquitin chains on Cbf1 K271 site performedThe result of DNA Pulldown purification showed that the Cbf1 protein on the transcription complex in the wide-type strain was more abundant than Cbf1site mutant strain.Conclusions:1.The ubiquitination at the Cbf1 K271 site did not affect its own stability,suggesting the ubiquitin chain at the Cbf1K271 site did not mediate protein degradation.2.The ubiquitination of the Cbf1K271 site was not involved in transcriptional activation of genes associated with the methionine synthesis pathway.3.The ubiquitination at Cbf1 K271 site may affect the transcriptional activation of the protein by affecting the binding ability of Cbf1 to the promoter regions of various protein coding genes.