Molecular Mechanism And Function Of DNA Oxygenase TET1 In Regulating Oligodendrocyte Differentiation

Background:In the Central Nervous System（CNS）,the myelin sheath structure ensures the rapid transmission of nerve impulses,insulation between axons,and energy supply to neurons.However,as the myelin-forming cells in CNS,understanding oligodendrocyte（OLs）differentiation and myelinization is still insufficient.5-hydroxymethylcytosine（5hm C）has the highest abundance in the CNS,which is closely related to the development and neural function of the nervous system.Ten-eleven translocation 1（TET1）is the key enzyme catalyzing the formation of 5hm C and interacting with transcription factors to regulate gene expression.Currently,there is a lack of sufficient research on the mechanism of TET1 regulating oligodendrocytes.In the previous study,our research group found that the expression level of DNA oxygenase TET1 and the abundance of 5hm C exhibits dynamic changes during the differentiation and maturation of OLs^[1].TET1 is highly expressed in the oligodendrocyte progenitor cells（OPCs）stage and participates in the differentiation of OLs.According to my master’s degree results,we preliminary showed that lack of TET1 causes oligodendrocyte cell cycle arrest and intrinsic dysdifferentiation in OL,ultimately hypomyelination in the mouse brain^[2].Therefore,studying the mechanism of TET1regulating oligodendrocytes is of great significance for nervous system development and myelin-related diseases.However,the cellular and molecular mechanisms related to TET1regulation of oligodendrocyte differentiation and how it affects neuronal function remain unclear.This study will focus on these issues for in-depth exploration.Methods:The study was divided into two parts.In the first part,we identified that TET1 regulates the expression of relevant target genes in two ways,the oxygenase pathway catalyzing 5hm C modification and the non-oxygenase pathway combining with transcription factors.In order to clarify the molecular mechanisms by which TET1 influences oligodendrocyte differentiation and myelination.1)Analysis of Tet1 epigenetic modification function by high-throughput sequencing:Firstly,we detected the differential gene expression in the OPCs of Tet1 conditional knock out（Tet1c KO）mice through transcriptome sequencing（RNA seq）.Gene Ontology（GO）and gene set enrichment analysis（GSEA）were performed to determine the primary functions of the differential genes in TET1 deficient OPCs.Second,through transcriptome-associated hydroxymethylation sequencing（5hm C seq）analysis,we investigated the correlation between the modification sites of TET1-5hm C and target gene expression.Finally,CUT&RUN-seq was used to clarify the binding sites and transcriptional regulation of TET1in the genome.2)The effect of TET1 deletion on the expression levels of calcium ion transport genes in OPC cells were verified by GSEA analysis and q RT PCR.Calcium activity was measured in two groups of OPC cells using a calcium indicator.Cellular immunofluorescence staining was performed to determine the expression of TET1-5hm C target gene inositol-1,4,5-trisphosphate receptor 2（ITPR2）,an endoplasmic reticulum calcium channel,during oligodendrocyte differentiation.For in vitro experiments,interfering with Itpr2 expression using si RNA,OPC differentiation was visualized using a combination of Western blot,q RT-PCR,and cellular immunofluorescence.Itpr2 conditional knock out mice in oligodendrocytes were constructed in vivo to observe oligodendrogenesis and myelin developmental changes by immunofluorescence and scanning electron microscopy.3)Proteins interacting with TET1 were collected by Co-immunoprecipitation（Co-IP）experiments followed by mass spectrometry（MS）to identify the protein type.Combined analysis of RNA seq results and TET1 bound proteins in OPC and OL states in relation to their expression amounts.Finally,TET1 and OLIG2 were overexpressed by plasmid transfection in Hela cell lines,and the interaction between TET1 and transcription factors in OPCs and OLs was verified by CO-IP combined with Western blot experiments.The second part is a preliminary study of the impact of white matter deficiency on neurons caused by TET1 deletion.1)Behavioral testing:Tet1 c KO mice were examined for changes in cognition and sensory gating function through novel object recognition（NOR）and pre-pulse inhibition（PPI）experiments.2)Neuronal morphological changes:GSEA and q RT-PCR experiments validate OPC neurotrophic factor related genes expression changes in Tet1 c KO mice.Alterations in the extracellular matrix and the morphology and number of different types of neurons in the prefrontal cortex（PFC）of Tet1 c KO mice were observed by immunofluorescence staining,combined with Imaris graphic analysis.3)Parvalbumin（PV）neurons functional assays:Investigation of altered calcium activity in single PV neurons at PFC sites in isolated brain slices by two-photon imaging.Calcium signaling changes in PV neurons during activity in mice were studied using brain stereotaxic injections of adeno-associated virus（AAV）combined with recording by fiber-photometry.PFC excitatory and inhibitory synapse number alterations were observed by immunofluorescence staining.Results:The first part1).RNA SEQ results showed that knock out of TET1 resulted in significant downregulation of the expression levels of several functionally relevant genes,including myelination,oligodendrocyte differentiation,cell cycle,calcium ion transport,and extracellular matrix,etc.The 5hm C seq results indicated that the overall 5hm C modification level in OPC cells was significantly decreased after Tet1 knock out.The results of the differential combined analysis of h Me DIP seq and RNA seq showed that the genes with reduced levels of both transcription and 5hm C modification mainly included cell division,myelination,calcium ion transport and other related functional sets.TET1 CUT&RUN seq results showed that TET1 binds at the promoter regions of the above genes and is associated with transcriptional activation.2)GSEA and q RT PCR results showed that several calcium ion transport related genes,including Itpr2,Cacna1a,and Cacna1c were significantly downregulated.OPC cell calcium imaging showed that Tet1 c KO OPCs exhibited diminished function of voltage-gated calcium channels and endoplasmic reticulum calcium release channels,and the amplitude of cellular calcium oscillations was significantly reduced.Itpr2,as a post mitotic OPC marker,was expressed at the highest level in the stage of oligodendrocytes differentiated 1 day in vitro（OL1DIV）.Interfering with Itpr2 expression affects OPC differentiation,and activation of OPC calcium channels by agonists rescues Tet1 c KO OPC cell differentiation defects.Knock out of Itpr2 in oligodendrocytes resulted in OLs differentiation defects at the developmental stage in mice,with reduced expression of myelin proteins and hypomyelination.3)The results of Mass spectrometry showed that the types of TET1 binding proteins in OPC and OL stages were different.The combined analysis of IP-MS and RNA seq showed that the binding of TET1 to these proteins was independent of its own expression level.GO analysis showed the protein combined by TET1 in OPC and OL was mainly related to protein stability,molecular metabolism,Dicarboxylic acid metabolic mitochondrial process and cell redox homeostasis.The KEGG database（Kyoto encyclopedia of genes and genes,KEGG）analysis of biological functions and signaling pathways showed that TET1interacting proteins were mainly related to membrane trafficking,myelin sheath,carbon metabolism,and axon guidance.Western blot validated that TET1 could bind to the transcription factor OLIG2 in OPC and OL,while TET1 binds to HDAC1 only in OL.The second part:1)NOR experiments showed that Tet1 c KO mice had a reduced proportion of time spent in novel object exploration and impaired cognitive function.Results from PPI experiments showed that Tet1 c KO mice had significantly reduced suppression efficiency of 70-,76-,and 82-d B prepulse stimulation,respectively.2)Genes involved in neuronal synapse development,such as Tnr,Gdnf,Ank3,Reelin were down regulated in OPC cells from Tet1 c KO mice.The brightness of PV neuron staining at PFC was reduced in Tet1 c KO mice,and the number of PV high expressing cells was reduced.There was no difference in the number of remaining interneurons or pyramidal neurons.3)Diminished calcium ion activity in PFC PV neurons of Tet1 c KO mice.The number of inhibitory synapses onto excitatory neurons and excitatory synapses onto PV neurons is reduced in PFC.Conclusion:In summary,we investigated the mechanism of TET1 regulation in oligodendrocytes.First,TET1 promotes the expression of this gene by mediating the cell cycle,oligodendrocyte differentiation,and other modifications of 5hm C on genes associated with oligodendrocyte development.Meanwhile,TET1 binding at the promoter region correlates with the expression activation.Second,we found the important role of calcium ion activity for OPC differentiation,and Itpr2,a novel target gene of TET1-5hm C,was involved in oligodendrocyte differentiation and myelination.Subsequently,we analyzed TET1 binding to different protein regulatory networks in OPCs and OLs,and TET binding to oligodendrocyte related transcription factors.Furthermore,the present study observed the effects of TET1 deletion on PFC neuron development and local excitatory/inhibitory synapse ratios.The above findings suggest that abnormal oligodendrocyte function in Tet1 c KO mice leads to diminished PV neuron inhibitory function and disrupted PFC local excitation/inhibition balance,which in turn produces schizophrenia-like behavioral manifestations.This study provides a theoretical basis for investigating the important role of oligodendrocytes in the pathogenesis and treatment of schizophrenia.