Effect Of O-GlcNAc Modification On The Nuclear Localization Of TET3 Protein

The O-Glc NAc(O-linked N-acetylglucosamine,O-GlcNAc)modification is a carbohydrate post-translational modification on hydroxyl groups of serine and/or threonine residues of cytosolic and nuclear proteins. The O-GlcNAcylation has been shown to participate in diverse biological processes via regulating diverse protein functions,including protein subcelluar localization, activity and stability. O-GlcNAcylation is regulated by O-GlcNAc transferase(OGT) and O-GlcNAase(OGA).The OGT transfers the GlcNAc moiety to substrate proteins,while OGA hydrolyzes O-GlcNAc from proteins.TET3,one of Ten Eleven translocation(TET) enzymes,is a 2-oxoglutarate- and Fe(II)-dependent dioxygenases.The TET3 that is able to selectively conduct active and rapid demethylation to male genome after fertilization plays crucial roles in early embryonic development and reprogramming.In recent study,it reported that human OGT was not only a TET3-interacting protein but also regulated TET3 protein subcellular localization and enzymatic activity.The results suggested that OGT may affect the DNA demethylation of early embryos though regulating TET3 protein subcellular localization and function,which then influenced early embryonic development and reprogramming. Here we systematically explored the mechanism of the regulation of TET3 protein by OGT in mouse,the sequences of TET3 and OGT genes from mouse,human,bovine goat and porcine were compared,and the expression of TET3 gene and the genes related to O-GlcNAc modification in bovine IVF and SCNT embryos were detected.The main research contents and results are as follows:1.Firstly,mouse TET3 and OGT genes were cloned into vectors to overexpress the two proteins. Immunofluorescent staining revealed that TET3 protein exhibited a predominant nuclear staining pattern in NIH3T3 cells when expressed alone.However, TET3 protein exhibited a predominant cytoplasmic localization when it was co-expressed with OGT in NIH3T3 cells.This results demonstrated that OGT regulated TET3 protein subcelluar localization.2.To explore if OGT regulated TET3 protein subcellular localization in a manner depending on OGT’s enzymatic activity. We treated the NIH3T3 cells transfected with TET3 and OGT vectors with OGT inhibitor Alloxan and observed that the treatment disabled the regulation of OGT to TET3 protein.Then we treated the NIH3T3 cells transfected with TET3 vector with PUGNAc, a specific OGA inhibitor and observed that the PUGNAc treatment increase TET3 protein cytoplasmic localization; and we also discovered that the ability of TET3 protein nuclear localization was strengthened when the NIH3T3 cells transfected with TET3 vector were clutured in a lower concentration of glucose.The upper results testified that OGT regulated TET3 protein subcellular localization by influencing its O-GlcNAcylation.3.To further explain how OGT regulated TET3 subcelluar localization,we constructed TET3 vector with no nuclear localization signal(NLS) at first.Analogous to the subcellular localization of TET3 protein modified by OGT, the TET3 deletion mutant protein was predominant cytoplasmic localization.The result suggested that the O-GlcNAc modification of TET3 protein may regulate TET3 protein nuclear localization though interfering the NLS function of TET3 protein.4.The TET3 genes of mouse,human,bovine and goat were compared in a manner relying on bioinformatics.The result showed that the TET3 gene among those spieces had a high homology.The mouse TET3 protein also had enzymatic activity when ectopically expressed in bovine fibroblast.qRT-PCR assay was performed to study TET3 gene expression pattern in early embryos by IVF and SCNT.The results showed that the TET3 gene mainly expressed before 8-cell stage embryos during early embryo development, and hardly expressed in morula and blastocyst;the OGT gene also had a high expressions in early embryos.The level of 5mC in development of IVF and SCNT embryos were detected. The result showed that IVF and SCNT embryos both underwent DNA demethylation,which implyed that TET3 protein played a vital role in DNA demethylation of early embryos.The upper results suggested that the regulation of TET3 protein subcelluar localization by OGT was able to be applied to the research of improving the incompleted demethylation of SCNT embryos.In summary,this study revealed that OGT was able to regulate TET3 protein subcellular localization in a manner altering the O-GlcNAc modification level on TET3 protein in mouse; TET3 and OGT genes in mouse,human,bovine,goat and porcine had a high homology; TET3 and OGT gene both had a high expression in early embryos.All these results imply that the regulation of TET3 protein subcelluar localization by OGT is able to be applied to the research of improving the incompleted demethylation of SCNT embryos and provide new insight and theoretical basises for the research of improving the effeciency of bovie SCNT reprogramming.