叶酸缺乏影响胚胎干细胞H3K9巴豆酰化和神经发育相关基因表达

Folic acid deficiency affects H3K9 crotonylation and neurodevelopment-related gene expression in embryonic stem cells

目的采用染色质免疫共沉淀测序技术(chromatin immunoprecipitation sequencing,ChIP-seq)分析叶酸正常小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)与叶酸缺乏mESCs组蛋白H3K9巴豆酰化(H3K9cr)修饰的全基因图谱,以期探究叶酸缺乏条件下mESCs中H3K9cr全基因组状态变化以及受到调控的通路基因。方法mESCs分为叶酸正常组(FA=4 mg/L,FA4)和叶酸缺乏组(FA=0 mg/L,FA0),利用Western blot和免疫组织化学染色分别检测叶酸缺乏mESCs和神经管畸形(neural tube defects,NTDs)小鼠胚脑组织中H3K9cr蛋白表达水平;利用ChIP-seq技术对获得的特异性结合的DNA片段展开序列鉴定,对差异表达基因进行基因本体论(gene ontology,GO)以及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路分析并可视化,分析叶酸缺乏条件下mESCs全基因组并对受调控基因进行验证。结果叶酸缺乏mESCs和NTDs小鼠胚脑组织中整体H3K9cr水平降低(P<0.05),叶酸缺乏mESCs中Bdnf、Pax6、App等神经发育相关基因下调(P<0.05),FA4与FA0两组差异富集基因的KEGG通路富集于轴突引导、神经活性配体-受体相互作用、谷氨酸能突触等与神经系统相关的通路。结论胚胎干细胞中叶酸缺乏通过H3K9cr修饰参与调控神经发育相关基因,提示组蛋白巴豆酰化修饰可能参与叶酸缺乏引起的神经发育类疾病。

Objective To analyze the genome-wide profiles of H3K9 crotonylation(H3K9cr)in mouse embryonic stem cells(mESCs)in normal or deficient folic acid(FA)by chromatin immunoprecipitation sequencing(ChIP-seq)in order to observe the genome-wide changes in H3K9cr status and the regulated pathway genes under FA deficiency condition.Methods The mESCs were divided into normal FA group(4 mg/L,FA4)and FA deficient group(0 mg/L,FA0).Western blotting and immunohistochemical(IHC)assay were used to detect the protein level of H3K9cr in the FA deficient mESCs and the embryonic brain tissues of neural tube defects(NTDs)mice,respectively.ChIP-seq technology was carried out for sequence identification of the obtained specific binding DNA fragments.Differentially expressed genes(DEGs)were analyzed and visualized by gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathways,with the aim of analyzing the whole genome of mESCs with FA deficiency and validating the regulated genes.Results Overall H3K9cr level was significantly lower in the FA deficient mESCs and the embryonic brain tissues of NTDs mice than in the corresponding normal FA mESCs and mice(P<0.05).Neurodevelopmental-related genes such as Bdnf,Pax6,and App were down-regulated in the FA deficient mESCs(P<0.05).The KEGG pathway analysis indicated that the DEGs were concentrated in axon-guiding,neuroactive ligand-receptor interaction,glutaminergic synapses and other nervous system-related pathways.Conclusion FA deficiency in embryonic stem cells participates in the regulation of neurodevelopmental genes through modification of H3K9cr,suggesting that the modification of histone crotonylation may be involved in neurodevelopmental diseases induced by FA deficiency.