The Epigenetic Characteristics Of Neural Tube Defects Susceptibility Genes Caused By Folate Deficiency-induced DNA Double-strand Breaks And Screening Of New Susceptibility Genes

Objective:By using the high-throughput sequencing of epigenomics sequencing and transcriptomics sequencing technology,the chromatin epigenetic characteristics of neural tube defects(Neural tube defects,NTDs)susceptibility genes caused by the folate deficiency induced DNA double-strand break(DNA double-strand break,DSB)were analyzed and investigated.Then the above-mentioned epigenetic characteristics of NTDs susceptibility genes were screened for new NTDs susceptibility genes,in the hope of providing new theoretical support and research basis for the occurrence of NTDs triggered by folate deficiency.Methods:1.After being treated for 24hours with the folate antagonist methotrexate(MTX)mouse embryonic stem cells(mESCs)were collected for ChIP-seq with H3K4me1,H3K4me3?H3K27me3?H3K27ac and CTCF,in the wake of which the chromatin states were characterized by ChroHMM software;2.On the foundation of our previous research,the hypergeometric algorithm was utilized to calculate the distribution characteristics of DSB and DSB-induced changes of the epigenetic modification in genome;3.After interacting with MTX for 24h,the mESCs were collected to performRNA-seq,screening and analyzing the transcriptional changes of NTDs susceptibility genes;4.Following the treatment of mESCs by MTX for 24h,they were collected to conduct ATAC-seq sequencing in an effort to unmask the connection between gene expression and open chromatin localization;5.By adopting bioinformatics methods,the MTX-induced epigenetic chromatin characteristics of the DSB region of NTDs susceptible genes were analyzed and calculated,screening new NTDs susceptibility genes according to the results;6.The expression of aforementioned new susceptibility genes in low folate human NTDs fetal brain tissues were verified by Nanostring.Results:1.MTX-induced DSB in mESCs accumulate at the promoter and enhancer regions of genome.2.Co-occurrence of DSB and enriched epigenetic modification at active promoters leads to gene dysregulation.3.MTX-induced DSB in mESCs in the gene active enhancer region can regulate the activity of gene promoters.4.MTX-induced DSB in mESCs changed the transcription of NTDs susceptibility genes through CTCF topoisomeric region.5.The screened new NTDs susceptibility genes have been verified in the neural tissues of low folate human NTDs samples.Conclusion:1.Under folate deficiency,DSB make active promoters fragile,leading to the the dysregulation of downstream NTDs genes.2.Under folate deficiency,distal DSB occur at distal enhancers,breaking the interactions between gene promoters and enhancers,and affecting the transcriptional activity of NTD genes.3.PABPC4?MED13L?TPGS1?FAM208A?SF3B5?BMYC?FAM109A are new NTDs susceptibility genes.