The Mechanisms Of Histone Methylation Modification In Retinoic Acid-induced Neural Tube Defects

Objectives:Neural tube defects（NTDs）are common birth defects,which affected the quality of population seriously,but the etiology is complex,and the pathogenesis is still not clear.It is of great significance to investigate the mechanisms of NTDs for reducing the birth defects and improving the quality of population.The purposes of this study are:1 To understand the transcriptome profile of RA-induced NTDs mouse embryo,and select the important differentially expressed genes（DEGs）related NTDs.2 To study the expression pattern of Hox genes in NTDs,and investigate regulation of the histone methylation modification on Hox genes,which would provide new mechanisms of the NTDs induced by environmental factors from the epigenetic perspective.In addition,explore the function and mechanisms of Nr2e1 in the RA-induced NTDs occurance preliminary.Methods:1 Transcriptome profile of NTDs mouse embryos was detected by RNA sequencing（RNA-seq）technology.1.1 We established the NTDs mouse model by gavage of 28 mg/kg retinoic acid（RA）on pregnant mouse at E7.5 d,the control pregnant mice were treated with the same dose of sesame oil.The brain vesicle of mouse embryos were collected at E8.5d,E9.5d and E10.5d respectively,and sequenced by using Illumina HiSeqTM 2000 sequencing platform.1.2 The quantity of gene expression was calculated by RPKM method,and DEGs were selected according to the FDR≤ 0.001 and Log2Ratio≥ 1,and performed with GO and KEGG pathway enrichment analysis.1.3 The important DEGs were selected by taking intersection of DEGs among E8.5d,E9.5d and E10.5d,and validated by RT-PCR or RT-qPCR.2 The RNA-seq showed Hox genes expression was up-regualted,which the function and mechanism played on NTDs were investigated deeply in our study.2.1 The mRNA level of Hox genes in the RA-induced NTDs mouse,mouse embryonic stem cells（ESCs）and neural stem cells（NSCs）were detectd by RT-qPCR.2.2 The H3K27me3 modification level in the RA-induced NTDs mouse,mouse embryonic stem cells（ESCs）and neural stem cells（NSCs）were detectd by Western blot.2.3 The mRNA level of methylation transferase enzyme Ezh2 and demethylation Kdm6a/Kdm6 b in the RA-induced NTDs mouse,mouse embryonic stem cells（ESCs）and neural stem cells（NSCs）were detectd by RT-qPCR.2.4 The enrichment of H3K27me3 on Hox gene promoter region was detectd by ChIP-qPCR in RA induced ESCs.2.5 HOX genes expression in the human NTDs samples were detected by Nanostring,and H3K27me3 modification level was detected by Western blot.3 The RNA-seq data showed Nr2e1 expression was in highest abundance and largest difference ratio,which function and mechanism were investigated preliminary.3.1 The whole embryo in situ hybridization and RT-PCR technology were used to detect the spatio-temporal expression patterns of Nr2e1 gene in RA-induced NTDs mouse embryo.3.2 The proliferation and differentiation of two kinds of neural stem cells（NSCs）isolated from control and NTDs mouse embryo were compared by colony formation,CCK-8,flow cytometry and immunofluorescence method.And Nr2e1 gene expression in the two kinds of NSCs was detected by RT-PCR and Western blot method.3.3 NSCs were tansfected with LV3-Nr2e1 shRNA,which proliferation and differentiation were detected by colony formation,CCK-8 and immunofluorescence method.Results:1 The transcriptome profile analysis of NTDs mouse embryo.1.1 There were 587 DEGs in Brc8₅-vs-Bra8₅,including 296 up-regulated genes and 291 down-regulated genes,2256 DEGs in Brc9₅-vs-Bra9₅,including 1782 up-regulated genes and 474 down-regulated genes,and 2337 DEGs in Brc10₅-vs-Bra10₅,including 1464 up-regulated genes and 873 down-regulated genes.1.2 Through GO enrichment of functional analysis,it was found that the DEGs were mainly enriched in some cellular components such as axon,cells junction,cell membrane and extracellular matrix,molecular function such as binding,channel and transport capacity,and biological processes such as development process,differentiation and migration.However,GO terms were slightly different in the different developmental stage.1.3 Through KEGG pathway enrichment analysis,it was found that the DEGs were mainly involved in the axon guidance,basal cell carcinoma,cell adhesion molecules,cholinergic synapses,Hedgehog signaling pathways and Glycolysis/Gluameogenesis.1.4 196 DEGs were screened by the intersection method,which were mainly divided into 2 categories according to the gene expression pattern,the up-regulated genes and down-regulated genes.The up-regulated were mainly involved inregionalization,cell differentiation and anterior/posterior pattern specification,and down-regulated genes were mainly involved in CNS development,brain development,neuron differentiation,generation of neurons and neurogenesis.1.5 26 DEGs were screened by fold change≥ 20 meantime at E9.5d and E10.5d,and 25 genes were validated.2 The abnormal expression of Hox genes in NTDs occurance,regulated by H3K27me3 modification.2.1 10 Hox genes selected in the RNA-seq data were up-regualated in RA-induced NTDs mouse embryo,and had the same change trend in RA-induced Sv/129 ESCs and C57 BL / 6 NSCs.2.2 H3K27me3 level was decreased in RA-induced of ESCs,NSCs and NTDs mouse,which was regulated by the decrease of Ezh2 expression and increase of Kdm6 b expression.2.3 Ch IP-qPCR results showed that the combination of H3K27me3 on the promoters of 10 Hox genes was weakened in RA-induced ESCs,which suggested that the decrease of H3K27me3 could cause the increase of downstream Hox genes expression.2.4 10 HOX genes expression were all up-regulated in the human anencephaly samples,and H3K27me3 modification levels were declined,among which HOXC4 and HOXD1 had obvious negative correlation with H3K27me3.3 The function and mechanism of Nr2e1 in the NTDs occurance.3.1 The whole embryo in situ hybridization showed Nr2e1 was mainly expressed in the embryo forebrain,increased with the normal development,but not increased in RA-induced NTDs embryo.RT-PCR results showed Nr2e1 expression in NTDs embryo was significantly lower than that of the normal embryo.3.2 Colony formation and CCK-8 results showed the proliferation of NSCs from normal embryo was significantly higher than that of NSCs from NTDs embryo.FCM results showed that the NTDs NSCs were arrested in the G1 phase,and can’t enter S phase.Immunofluorescence results showed that the differentiation ability into astrocytes but neurons of NTDs NSCs were lower than normal NSCs,which suggested the NTDs NSCs was impaired.In addition,Nr2e1 expression in NTDs NSCs was significantly lower than normal NSCs.3.3 The inhibition of Nr2e1 expression significantly reduced the proliferation of NSCs,and make NSCs to differentiate into less neurons but more astrocytes,which suggested the NSCs differentiation was dysregulated.Conclusions:1 Transcriptome profile of NTDs mouse embryo changed dramatically,and 25 genes were selected as NTDs related genes,including Hox and Nr2e1 genes.2 The regulation of H3K27me3 on the Hox genes plays an important role in the occurrence of anencephaly,which would provide a new epigenetic mechanism of NTDs.3 Nr2e1 gene may be involved in the RA-induced NTDs through regulating the proliferation and differentiation of NSCs.