The Mechanism And Function Of The Dephosphorylation Of EZH2 Mediated By Protein Phosphatase PP1

EZH2(Enhancer of zeste homolog 2),as the catalytic subunit of Polycomb Repressive Complex2(PRC2),catalyzes the trimethylation of lysine 27 on histone H3 methylation and then silences the transcription of PRC2 target genes.Therefore,EZH2 plays an important role in proliferation,differentiation and tumorigenesis.Serine 21 of EZH2 can be phosphorylated by AKT protein kinase,and the phosphorylated protein reduces the binding affinity to histone H3,thereby attenuating the activity of histone methyltransferase of EZH2,and leading to the transcription derepression of PRC2 target genes.Epithelial-mesenchymal transition(EMT)is one of the most important pathological events of lens fibrosis disease,and this study aims t o elucidate the molecular mechanism of EZH2 S21de-phosphorylation and its regulation of gene expression during the EMT of lens epithelial cells.We began with the establishment of the in vitro EMT cell model,in which the immortal rabbit lens epithelial cell line was treated with recombinant TGF? ligand to induced mesenchymal morphology transition(EMT).It was found that the specific inhibitor of AKT2 could significantly attenuate TGF?-induced up-regulation of p-EZH2 S21,down-regulation of H3K27me3 and activation of EMT marker genes during the EMT process of NN1003 A cells.These results suggested that EZH2 S21 phosphorylation plays an important role in the EMT of lens epithelial cells.To define the protein phosphatase catalyzing the dephosphorylation of EZH2 S21,Okadaic acid(OA),the general inhibitor of serine protein phosphatase PP1 and PP2 A was used to treat NN1003 A cells.Western blot results showed that OA treatment induced dosage dependent increase in p-EZH2 phosphorylation at S21,indicating that PP1 is one of the important phosphatases dephosphorylating of EZH2 S21.CoImmunoprecipitation and immunofluorescence co-localization experiments further confirmed the interaction between EZH2 and the PP1 regulatory subunit,MYPT1.To investigate whether MYPT1 regulates EMT of lens epithelial cells via controlling EZH2 S21 phosphorylation,CRISPR-CAS9 technology was used to silence MYPT1 gene in human embryonic lens epithelial cell line(FHL124)and a stable cell line of FHL124 MYPT1-KO was established.Compared with Mock KO cells,FHL124 MYPT1-KO cells showed further significantly enhanced activation of EMT marker gene.On the contrary,overexpression of the S21 A mutant imitating constant dephosphorylation,but no the wild type,could significantly attenuate the EMT progression.In summary,our results revealed :(1)AKT-EZH2-H3K27me3 signaling pathway controls the EMT process of lens epithelial cells;(2)Protein phosphatase PP1-MYPT1 mediates dephosphorylation of EZH2.(3)MYPT1 gene deletion can accelerate the EMT process of human lens epithelial cell line.(4)EZH2 S21 dephosphorylation controls the activation of EMT genes in lens epithelial cells.Our study for the first time have identified that PP1-MYPT1 mediates the dephosphorylation of EZH2 S21,and elucidated that the phosphorylation of EZH2 S21 regulates the activation of EMT marker gene in lens epithelial cells.These results provide more information in our understanding of the mechanism mediating lens fibrosis.