Characterization Of The Nuclear Localization Signal Of The Mouse TET3 Protein And Preliminary Study On Its Exporting Mechanism

DNA demethylation is associated with gene activation and is mediated by a family of ten-eleven translocation (TET) dioxygenase. The TET3 protein is a 1668-amino-acid DNA demethylase. Its catalytic function is fullfiled in the nucleus. But its translation and modification is happened in the cytoplasm. TET3 protein is a big protein so that it can not be imported into nucleus. The one or more NLS is embedded in the TET3. In this study, we predicted that TET3 possess five NLS. In the following study, we confer whether these NLS is functional or not.We used a series of green fluorescent protein-tagged constructs to observe the nucleic localization of fusion proteins. In this way we define the poper location of NLS embedded between 1310 and 1668. In the mutation study we confered the NLS (KKRK) is functional, which is conserved in TET3 in all species listed in NCBI.We added the KKRK or the fragment containing KKRK to the EGFP-GH or EGFP-GST. Then these cytoplamic protein into the nucleus. When TET3 fragment only deleting KKRK was added to EGFP-GH, the fusion protein was in the cytoplam. So possessing the KKRK is the necessary and sufficient condition of TET3 translocation into nucleus.From previous study, we know that protein containing KKRK is mediate by importin-aand importin-β. This indicates that TET3 may also be mediated by importin-αå'Œ importin-βIn the study we found that some TET3 fragment containing KKRK is in the cytoplasmid. With others’ study about LBM inhibiting TET3 translocation, we proposed that O-GlcNAc of TET3 promote TET3 translocation. But whether the modification effect on its NES or binding to other protein translocation to cytoplasm, it needs more study. The translocation into cytoplasm of TET3 on high carbonhydrate condition indicate that nutrition may effect on globe level of 5hmC.