PGC7 Regulates DNMT1/DNMT3a Function By Maintaining ERK Pathway Activity

PGC7(also known as Stella or Dppa3)was first identified as a highly expressed gene in primordial germ cells(PGCs),or a maternal effector protein involved in imprinting maintenance for the development of early embryo.DNA methylation is an important epigenetic modification,and the activity of DNA methyltransferases(DNMTs)is crucial to it.The MEK/ERK signaling pathway responds to various extracellular signals and converts them into a variety of specific biological responses,and also participates in the regulation of DNMT1/DNMT3 a activity.RACK1 is a scaffold protein,which interacts with different cellular proteins.It is a key mediator in various pathways,and contributes to many types of cell functions.Some reports have revealed the mechanism of imprinting maintenance from the perspective of the antagonistic relationship between PGC7 and TET3 or DNMT3 a protein,but whether PGC7 has a post-translational regulatory mechanism regulating DNMT1/DNMT3 a function remains to be studied.To this end,the research revealed the regulatory mechanism of ERK phosphorylation by PGC7,and further researched the effect of PGC7-mediated ERK phosphorylation on DNMT1/DNMT3 a function and the effect of ERK activity on the overall DNA methylation level and the effect of phosphorylation of DNMT1S717 by ERK on the overall DNA methylation level by co-immunoprecipitation(CO-IP),western blot,immunostaining,and methylation staining and other methods.The results are listed below:1.It was found that knockdown of PGC7 in cells could down-regulate ERK phosphorylation,but there was no interaction between PGC7 and ERK.Co-Immunoprecipitation and western blot further reveled that RACK1 could interact with PGC7,MEK1,ERK1,and ERK2 respectively,and RACK1 could weaken the interaction between MEK1 and ERK1,and PGC7 could weaken the interaction between RACK1 and ERK1/2.The results showed that PGC7 could preferentially combine with RACK1 and indirectly promote the combination of MEK and ERK.Furthermore,knock-down of RACK1 could promote ERK phosphorylation.Therefore,one of the mechanisms by which PGC7 regulates ERK phosphorylation is through RACK1.2.ERK could regulate the localization and stability of DNMT1/DNMT3 a protein.Immunostaining results showed that U0126,an inhibitor of MEK,could promote the nuclear localization of DNMT1 and the cytoplasmic localization of DNMT3 a.It was further verified by western blot results of nucleus and cytoplasm separation after PD treatment,which also indicated that DNMT1 entry into the nucleus caused by PD treatment was not mediated by UHRF1.It was also found that inhibition of ERK activity could improve the stability of DNMT3 a protein after treatment with SCH,a specific inhibitor of ERK,and CHX,an inhibitor of eukaryotic protein synthesis.3.There were interactions and co-localization of cytoplasm and nucleus between ERK1 and DNMT1,ERK1 and DNMT3 a.In this study,GPS3.0 software predicted that there were multiple ERK modification sites on DNMT1/DNMT3 a.Combined with the identified phosphorylation modification sites in the phosphorylation mass spectrometry data,several phosphorylation modification sites with high scores were selected.Eukaryotic expression vectors of p3×Flag-DNMT1S15 A,DNMT1S717A,DNMT1S958 A and DNMT1S1421 A were constructed respectively.Eukaryotic expression vectors of p3×Flag-DNMT3 a S102A,DNMT3 a S251A,DNMT3 a T257A and DNMT3 a S251T257AA were constructed respectively.Immunostaining results showed that the intracellular localization of DNMT1S717 A protein changed into the nucleus,which was consistent with the inhibition of ERK activity.The results suggested that ERK can phosphorylate DNMT1S717 and influence its intracellular localization.However,the intracellular location of the site-mutated DNMT3 a protein did not change significantly.4.It was also found that knock-down of PGC7 could increase the nuclear import of DNMT1 and the nuclear export of DNMT3 a.The results suggested that PGC7 can regulate the intracellular localization of DNMT1/DNMT3 a by down-regulating ERK phosphorylation.Methylation staining showed that U0126-treatment significantly increased the overall DNA methylation level.DNMT1S717 A improved the overall DNA methylation level.The results showed that phosphorylation of DNMT1S717 by ERK would affect the overall DNA methylation level.To conclude,the research revealed that PGC7 could maintain ERK phosphorylation,and revealed its maintenance mechanism from the perspective of protein interactions.It was also found that PGC7-mediated ERK phosphorylation would affect DNMT1/DNMT3 a function,and the inhibition of ERK activity would increase the overall DNA methylation level and phosphorylation of DNMT1S717 by ERK would affect the overall DNA methylation level.It was confirmed that PGC7 could regulate DNMT1/DNMT3 a function by maintaining ERK pathway activity.The function and mechanism of PGC7 in imprinting maintenance were further expanded.