The Function And Mechanism Of Cops2 And Cops5 In Mouse Embryonic Stem Cells

Embryonic stem cells(ESCs)have unlimited self-renewal ability and the potential to differentiate into all cell types in the body under appropriate culture conditions in vitro.These characteristics make ESCs have a broad application potential in regenerative medicine.The rapid proliferation ability of ESCs can ensure a sufficient number of cells for clinical treatment,and maintaining the stability of the genome is essential for the safety of embryonic stem cells in clinical applications.Therefore,studying the unique regulatory mechanism of cell cycle in embryonic stem cells to maintain rapid proliferation,and how embryonic stem cells maintain their genome stability while proliferating rapidly,has important scientific significance and will also promote the clinical application of embryonic stem cells.The rapid proliferation of embryonic stem cells depends on the short G1 and G2 phases.Previous studies have shown that the pluripotency factor Oct4 inhibits mitotic entry by inhibiting the activation of Cdk1,thus promoting the maintenance of genome stability.However,it is not clear how embryonic stem cells enter the mitotic stage in the presence of Oct4.We have previously reported that knockdown of Cops2 causes cell cycle arrest in the G2/M phase of mouse embryonic stem cells.Therefore,we further investigated how Cops2 regulates the cell cycle of mouse embryonic stem cells.In this study,through immunoprecipitation combined with mass spectrometry,we found that Cops2 interactswith Oct4 and Cdk1.It was further demonstrated that Cops2 could only enhance Cdk1/Cyclin B activity in the presence of Oct4.Consistently,in He La cells,Cops2 promoted the cell cycle transition from G2 phase to M phase only in the presence of Oct4.Mechanistically,Cops2 attenuates the interaction between Oct4 and Cdk1 by sequestering Oct4 and forming Cops2/Cdk1 complex,thus blocking the inhibitory effect of Oct4 on Cdk1 activation[1].It was reported that Cops2 deficiency leads to mouse embryo death at E3.5,which is consistent with our previous findings on the role of Cops2 in embryonic stem cell self-renewal.In contrast,Cops5 and Cops8 KO leads to E7.5-E8.5 lethality.Therefore,we hypothesized that Cops5 and Cops8 may play a role in the differentiation of mouse embryonic stem cells.In this study,we set out to investigate how Cops5 and Cops8 regulate ESC differentiation.We attempted to knock out Cops5 and Cops8 in mouse ESCs.But no Cops5 KO ESC clones were identified out of 127 clones,while three Cops8 KO ESC lines were established out of 70 clones.This suggested that Cops5 is essential for the self-renewal of mouse embryonic stem cells.We then constructed an inducible Cops5 KO ESC line.Cops5 KO leads to decreased expression of the pluripotency marker Nanog,proliferation defect,G2/M cell cycle arrest,apoptosis of ESC and severe DNA damage.Further analysis revealed dual roles of Cops5 in maintaining genomic stability of ESCs.On one hand,Cops5 suppresses the autophagic degradation of Mtch2 to direct cellular metabolism toward glycolysis and minimize reactive oxygen species(ROS)production,thereby reducing endogenous DNA damage.On the other hand,Cops5 is required for high DNA damage repair(DDR)activities in ESCs.Without Cops5,elevated ROS and reduced DDR activities lead to DNA damage accumulation in ESCs.Subsequently,p53 is activated to trigger G2/M arrest and apoptosis[2].Altogether,our studies revealedthe important role of Cops2 in G2/M transition,and elucidated that Cops5 maintains genomic stability by regulating cell metabolism and the DDR pathway.Our study lay the foundation for the application of ESCs.