Identification Of A Novel Non-catalytic Role Of TET Proteins Negtively Regulating Neuronal Differentiation

DNA methylation plays important roles in cell differentiation,proliferation,and development.DNA methylation dynamics can effect brain function and lead to neurological disorders.Ten-eleven translocation(TET)family enzymes are Fe(II)and ?-ketoglutarate(?-KG)dependent dioxygenases,which can convert 5-methylcytosine(5mC)to 5-hydroxymethylcytosine(5hmC),and further 5-formylcytosine(5fC)and 5-carboxylcytosine(5caC)in DNA.TET proteins have been shown to facilitate active DNA demethylation.Furthermore,5hmC mediated epigenetic modification is also significant in neurodevelopment and diseases.TET proteins are discovered to be essential for various biological processes such as embryonic development,immune regulation,stem cell differentiation,and cancer formation.TET proteins play important roles in neural functions as well.It has been found that TET proteins can be involved in the regulation of various aspects in neurogenesis and memory formation.However,their exact roles in early neuronal differentiation are still poorly understood.In this study,the roles of TET proteins during neuronal differentiation were investigated using Neuro2 a cells as a model,which might also bring a differentiation therapy for neuroblastoma in the future.Firstly,the expression of TET proteins were not correlated with 5hmC level in Neuro2 a cells,implying that catalytic activities of TET proteins might be suppressed in Neuro2 a cells.Immunofluorescence staining was performed to visualize the subcellular localization of endogenous TET proteins.It could be clearly observed that all three TET proteins expressed at detectable levels and localized to the nuclei.However,dot blotting indicated that 5hmC level was low in Neuro2 a cells.Secondly,TET proteins negatively regulated neuronal differentiation of Neuro2 a cells.After the knockdown of TET proteins using RNAi,neuronal differentiation of Neuro2 a cells were promoted.Whereas,overexpression of TET proteins inhibited neuronal differentiation of VPA-induced Neuro2 a cells,and it also confirmed our conclusion above.Subsequently,we found overexpression of CXXC-deleted and CD inactive mutants of TET1 could also inhibit VPA-induced neuronal differentiation of Neuro2 a cells,indicating that TET1 affected neuronal differentiation of Neuro2 a cells in an enzymatic activity-independent manner.Lastly,TET1 protein acted through srGAP3 expression independent of its catalytic domain.In our previous study,srGAP3 was reported to reduce VPA-induced neuronal differentiation of Neuro2 a cells.Knockdown of TET1 could downregulate srGAP3 expression,while overexpression of TET1 could upregulate srGAP3 expression.Furthermore,TET1 might negatively regulate neuronal differentiation of VPA-induced Neuro2 a cells via modulating the expression of srGAP3.In summary,non-catalytic TET proteins could negatively regulate early stage of neuronal differentiation through srGAP3.The results present here might facilitate better understanding of the role of TET proteins in neuronal differentiation,and provide a possible therapy target for neuroblastoma.