The Mechanism Of DNA Hydroxymethylation In Neural Tube Defects Induced By Folate Deficiency

Background:Neural tube defects(NTDs)are congenital birth defects of the central nervous system with a poor prognosis.Their etiology is complex,and the underlying mechanism remains unclear.Neural tube closure(NTC)is a highly coordinated process influenced by genetic and nutritional factors.Abnormalities at specific stages of the NTC process can result in the development of NTDs.Therefore,conducting in-depth research on the pathogenesis of NTDs is crucial for understanding their contributing factors,preventing and controlling their occurrence,and enhancing population well-being.Folate deficiency is the primary nutritional factor contributing to the development of NTDs.Disrupted metabolism of folate can lead to abnormal neural tube closure(NTC)and result in NTDs.The conversion of folate into tetrahydrofolate,acting as a methyl donor,plays a crucial role in the transfer of one-carbon units and the regulation of DNA methylation.DNA hydroxymethylation modification occurs through a cascade oxidation reaction mediated by the 10-11 translocation(TET)proteins,which convert 5mC to 5-hydroxymethylcytosine(5hmC),subsequently transforming into 5-formylcytosine(5fC)and 5-carboxylcytosine(5caC),thereby facilitating DNA hydroxymethylation modification.Previous studies have demonstrated significant variations in the distribution of 5hmC among different tissues in the human body,with the highest abundance observed in brain tissue.5hmC plays a crucial role in diverse regulatory processes within the central nervous system.Research has revealed that folate deficiency triggers an elevation in cellular redox reactions,resulting in abnormal DNA hydroxymethylation.Nevertheless,the precise regulatory and molecular mechanisms underlying DNA hydroxymethylation modification in the development of NTDs in the absence of folate remain unclear.Objective:This study aims to investigate the mechanism of DNA hydroxymethylation modification in the occurrence and development of NTDs in the presence of FA deficiency.Epigenetic methods will be employed to examine the impact of FA deficiency on the pathogenesis of NTDs,fostering novel insights into the field of NTD pathogenesis.Moreover,it will identify potential targets for preventing,treating,and screening drugs for NTDs,while also offering valuable references for similar birth and developmental abnormalities.Methods:1.The mouse embryonic stem cells(mESCs)model of folate deficiency will be established,and differentially expressed genes will be identified through RNA sequencing(RNA-seq).Functional changes resulting from folate deficiency will be predicted using gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.2.RT-qPCR was employed to assess changes in the transcription levels of DNA hydroxymethylases and DNMTs in the folate-deficient mESCs model.3.Hydroxymethylated DNA immunoprecipitation sequencing(hMeDIP-seq)and methylated DNA immunoprecipitation sequencing(MeDIP-seq)were performed.4.Chromatin immunoprecipitation sequencing(ChIP-seq)was utilized to assess the enrichment of histone 3 lysine 27 acetylation(H3K27ac)in the mESCs model of folate deficiency.5.The mESCs model will be established to simulate folate deficiency and folate supplementation.The transcription levels of NTDs-related genes and the changes in DNA hydroxymethylation modifications in these genes will be detected using RT-qPCR and hMeDIP-qPCR.6.The fetal mouse model of NTDs will be developed through low folate feeding combined with methotrexate(MTX)administration.Changes in DNA hydroxymethylation,DNA hydroxymethylase,and DNA methylation will be assessed using DNA dot blot and immunohistochemistry(IHC).Additionally,RT-qPCR will be employed to detect alterations in the transcription levels of NTDs.Result:1.RNA-seq analysis revealed substantial gene expression alterations in mESCs under folate deficiency,particularly in genes associated with oxygen metabolism,cell growth and development,and DNA and histone methylation pathways.Additionally,RT-qPCR demonstrated a significant increase in Tet1,Tet2,and Dnmt1 expression,while Tet3,Tdg,Dnmt3a,and Dnmt3b showed a significant decrease in response to folate deficiency.2.hMeDIP-seq and MeDIP-seq results demonstrated that folate deficiency led to a pronounced enrichment of 5hmC in mESCs,particularly within the Sonic hedgehog(Shh)signaling pathway and its related genes,including Gli2,Csnk1g3,Hhatl,and Smurf1.3.ChIP-seq analysis revealed that folate deficiency enhanced the enrichment of H3K27 ac in mESCs,particularly within the Shh pathway and its related genes,including Gli2,Csnk1g3,Hhatl,and Smurf1.4.The comprehensive analysis of hMeDIP-seq,MeDIP-seq,and ChIP-seq data demonstrated that folate deficiency promoted the enrichment of 5hmC and H3K27 ac in the promoter regions of Gli2,Csnk1g3,Hhatl,and Smurf1,with a co-localization of their enrichment regions.Notably,RT-qPCR and hMeDIP-qPCR revealed a significant increase in Gli2 and Hhatl expression under folate deficiency conditions.FA supplementation can rescue the downregulated levels of genes Gli2 and Hhatl in the Shh pathway caused by FA deficiency.5.In mouse model of methotrexate-induced neural tube defect under folate deficiency,the result of DNA dot blot showed upregulation of 5hmC level in the brain tissue of NTDs;IHC showed that TET1 and DNMT1 were significantly upregulated,and TET2,TET3 and DNMT3B was significantly downregulated;The result of RT-qPCR indicate that the expressions of Gli2 and Hhatl were significantly upregulated in the mouse brain tissue of NTDs.Conclusion:Folate deficiency induces the activation of the Shh pathway and downstream genes through the promotion of H3K27ac and DNA hydroxymethylation.Supplementation with folate can mitigate the alterations in the expression levels of Gli2 and Hhatl,as well as the enrichment levels of 5hmC,in the Shh pathway resulting from folate deficiency.