Phosphorylation Regulation Of PGC7 Interaction Protein STAT1 Role In Pluripotency Of F9 Cells

PGC7 is a maternal effector that is specifically expressed in mammalian germ cells,oocytes,preimplantation embryos,and pluripotent cells.The expression status of PGC7 is related to the pluripotency level of mouse ES cells.The high expression level of PGC7 also helps to maintain the hypomethylation level of embryonic stem cells and inner cell mass.These results indicate that PGC7 plays a important role in maintaining cell pluripotency.Nanog consists of 305 amino acids with a conserved homeotic domain that is specifically expressed in pluripotent cells such as preimplantation embryos and ES cells,and decreases in expression as cell pluripotency decreases,indicating that Nanog There is a dose effect on the maintenance of pluripotency,and the level of expression is closely related to the degree of cell differentiation and pluripotency.STAT family proteins include STAT1-4,STAT5 a,STAT5b,and STAT6,and studies have shown that STAT family proteins contribute to the regulation of embryonic stem cell pluripotency.The LIF/gp130/STAT3 signaling pathway is the earliest mouse pluripotency maintenance pathway in mouse ES cells.LIF activates STAT3 to regulate target gene expression and inhibit ES cell differentiation,thereby maintaining the pluripotent state of mouse ES cells.Our previous study found a novel pathway independent of LIF-JAK-STAT3,in which Vc activates the JAK2/STAT2 signaling pathway,whereas activated STAT2 binds to the proximal region of the Nanog promoter and promotes Nanog in ESC Expression,thereby exerting its function of regulating the pluripotency of embryonic cells.STAT1 is involved in the regulation of gene expression in a variety of biological processes,such as embryonic development,programmed cell death,innate immunity,adaptive immunity,and regulation of cell proliferation in organisms.There is no report that STAT1 is involved in the regulation of pluripotency of embryonic stem cells.In the early stage,our group identified the PGC7 interaction protein network by mass spectrometry and found that STAT1 is an interaction candidate protein of PGC7.This experiment focused on the interaction between STAT1 and PGC7 to study its mechanism of action in stem cell pluripotency maintenance.The main research contents and results are as follows:(1)Analysis of PGC7-STAT1 interaction and the effect of PGC7 on STAT1 subcellular localization.Firstly,this study analyzed the domain composition of STAT1,and constructed a truncated recombinant eukaryotic expression vector for its five domains,and used protein immunoprecipitation to detect the binding of PGC7 to STAT1.The results showed that the deletion of any domain of STAT1 did not affect the interaction between STAT1 and PGC7,that is,PGC7 and STAT1 had extensive interactions.Subsequently,the subcellular localization of PGC7 and STAT1 was observed by immunofluorescence.It was found that both PGC7 and STAT1 nuclear were distributed,but when PGC7 and STAT1 were co-expressed,STAT1 was nucleated,in other words PGC7 could significantly promote the nuclear localization of STAT1.(2)PGC7 regulates the activity of STAT1.Firstly,the effect of PGC7 on the interaction between STAT1 and its phosphatase PTPN6 a was detected by Co-iP.The results showed that PGC7 could inhibit the interaction between STAT1 and its phosphatase.Subsequently,the effect of PGC7 on the phosphorylation level of STAT1 was detected by WB.The results showed that PGC7 can increase the phosphorylation level of STAT1.Combined with the above results,PGC7 can enhance the hydrolysis of its phosphate group by inhibiting the phosphatase of STAT1.Phosphorylation level of STAT1.Finally,the effect of PGC7 on STAT1 transcriptional activity was examined by a dual luciferase reporter assay,which indicated that PGC7 can activate the transcriptional activity of STAT1.In summary,PGC7 antagonizes the hydrolysis of its phosphate group by antagonizing the specific phosphatase of STAT1,thereby increasing the phosphorylation level of STAT1,and finally promoting its transport into the nucleus,which activates the transcriptional activity of STAT1.(3)The interaction of PGC7 and STAT1 on pluripotency.Firstly,the promoter activity of Nanog was detected by a dual luciferase reporter assay.The results showed that both PGC7 and STAT1 can enhance the promoter activity of Nanog,and the interaction between the two can further enhance the activity of Nanog promoter.Subsequently,real-time fluorescence quantitative detection of Nanog and OCT4 transcription levels showed that the interaction of PGC7 with STAT1 can significantly increase the expression levels of Nanog and OCT4 genes.Finally,the overall pluripotency of F9 cells was detected by AP staining.The results showed that the interaction between PGC7 and STAT1 could antagonize the decrease of pluripotency induced by RA,significantly increase the pluripotency of F9 cells,and participate in the maintenance of pluripotency of embryonic stem cells.