Interaction Between PGC7 And YY1 Targets Regulation Of PRC2-Mediated Global H3K27me3 Levels

PGC7,also known as Dppa3 or Stella,is an inherently disordered unfolded protein of 150 amino acids in mice.It mainly participates in the regulation of epigenetic modification and the expression of imprinted genes through interaction with other proteins.YY1 is a complex transcription factor with both transcriptional activation and transcriptional repression functions.YY1 can mediate transcriptional inhibition by recruiting the PRC2 complex to the chromosome.YY1 can promote m TOR-mediated phosphorylation of AKT,PRC2 complex It mainly mediates the methylation of histone H3 at position 27,and AKT can regulate the trimethylation of histone H3 at position 27 by phosphorylating EZH2 at position 21 of serine.In this study,through the protein profile data of PGC7 in F9 cells,it was found that YY1 may interact with PGC7.In this study,Western blot,Co-IP,immunofluorescence staining,Chromatin immunoprecipitation(Ch IP),real-time quantitative PCR(Q-PCR)and other technologies have explored the functions of PGC7 and YY1 in F9 cells.The results are as follows:1.In this study,the p CMV-MYC-YY1 expression vector was constructed,and the interaction between mouse YY1 protein and PGC7 protein was verified by protein immunoprecipitation(Co-IP);and Flag-YY1 and HA-PGC7 were used in F9 mice In teratoma cell lines,the co-localization of YY1 protein and PGC7 protein was detected;on the basis of interaction,expression vectors of truncated bodies of three major domains of mouse YY1 protein were constructed,and protein immunoprecipitation(Co-IP)It was determined that the binding position of the PGC7 protein and the YY1 protein was located in the transcription repression domain of the YY1 protein.2.In this study,it was found that overexpression of PGC7 in mouse F9 cells will cause the total amount of H3K27me3 in the cells to decrease,and interference with PGC7 will cause the total amount of H3K27me3 in the cells to increase;overexpression of YY1 in mouse F9 cells will cause the total amount of H3K27me3 in the cells As the amount increases,interference with YY1 will cause the total amount of H3K27me3 in the cell to decrease.3.In order to explain the possible mechanism of PGC7 regulating H3K27me3,the enrichment of PGC7 protein for H3 wild type and H3K27 R mutant was compared by immunoprecipitation to determine that PGC7 can bind to H3K27;and to verify that PGC7 protein can bind to EZH2 of PRC2 complex,The interaction of RBBP4 and EED verifies that YY1 can directly interact with EZH2,EED,SUZ12 and RBBP4;through the experiment of PGC7,it is verified that PGC7 has no effect on the interaction of EZH2 and SUZ12,EZH2 and EED,and SUZ12 and EED.PGC7 cannot directly affect the structure of the PRC2 complex;PGC7 was added to the experiment,and it was found that PGC7 can competitively inhibit the interaction between YY1 and EZH2,SUZ12,and RBBP4,that is,the presence of PGC7 can competitively inhibit the combination of YY1 and PRC2 complex.4.To study the role of PGC7 in the two processes of YY1 promoting AKT phosphorylation and AKT-mediated EZH2 phosphorylation.This study verified the direct interaction between AKT protein and EZH2 protein through protein immunoprecipitation;through the addition of PGC7,it was found that the addition of PGC7 can promote the direct interaction between AKT and EZH2;in F9 cells,the inhibitor MK2206 inhibits AKT Activity and overexpression verified that AKT can regulate the content of H3K27me3,and verified that the presence of AKT in F9 cells can increase the content of phosphorylated EZH2(p EZH2-S21),and found that overexpression of PGC7 can cause the content to increase,that is,PGC7 can Inhibit the enzyme activity of EZH2.5.Through protein immunoprecipitation,the direct interaction between the YY1 protein and AKT protein was verified,and the co-localization of AKT and YY1 in F9 cells was verified by immunofluorescence;through the PGC7 addition experiment,it was found that the addition of PGC7 can promote AKT Interaction with YY1;overexpression of YY1 verified that it can significantly promote AKT activation,but PGC7 cannot promote AKT activation.In summary,this study determined that PGC7 can interact with YY1,and PGC7 and YY1 can regulate the content of H3K27me3 in F9 cells.It was found that PGC7 can interact with PRC2,but cannot destroy the structure of the PRC2 complex,YY1 Can directly interact with PRC2 to recruit PRC2,PGC7 can competitively inhibit the interaction between YY1 and PRC2,PGC7 can promote the interaction between AKT and EZH2 to regulate p EZH2-S21,PGC7 can promote the interaction between AKT and YY1,but cannot promote YY1 Activation of AKT.