活化转录因子4参与甲基化修饰与巨噬细胞炎症反应的关联研究

Association between activating transcription factor 4 and methylation modification in macrophage inflammatory response

目的探究活化转录因子4(activating transcription factor 4,ATF4)参与甲基化修饰与脂多糖(lipopolysaccharide,LPS)诱导巨噬细胞炎症反应的潜在关联。方法对比分析应用LPS(LPS组)和PBS(对照组)处理小鼠单核巨噬细胞白血病细胞系(即RAW264.7细胞系)8 h后染色质免疫共沉淀测序(ChIP-seq)和RNA高通量测序(RNA-seq)转录组分析表达谱的变化。采用京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)、基因本体(Gene Ontology,GO)分析,寻找与甲基化修饰相关通路,以及ATF4调控的甲基化修饰核心基因。应用MethPrimer平台在线预测ATF4参与巨噬细胞炎症反应甲基化修饰的关键靶基因的胞嘧啶-磷酸-鸟嘌呤(CpG)岛的分布情况。应用蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络分析,获取ATF4与这些关键蛋白质之间的调控关系。结果LPS组ATF4蛋白质和mRNA的相对表达量均分别显著高于对照组(t=7.507、4.771,P值均<0.001)。对LPS组开展ATF4的ChIP-seq和RNA-seq分析实验,并经KEGG和GO通路富集分析发现,ATF4参与巨噬细胞炎症反应的甲基化修饰相关基因为40个,其功能分别聚焦于组蛋白甲基化、组蛋白去甲基化、蛋白质甲基化、mRNA甲基化通路。进一步结合文献检索结果,根据上述40个基因是否存在CRE位点调控,筛选并获得ATF4调控甲基化修饰相关的7个潜在靶基因,即Brca 1、Btg 1、Carm 1、Ezh 2、Kdm 3 a、Kdm 4 b和Prmt 6。应用MethPrimer平台在线预测7个基因翻译起始位点的CpG岛,发现Prmt 6、Ezh 2、Carm 1分别有1个CpG岛,Kdm 3 a、Brca 1、Kdm 4 b分别有2个CpG岛,Btg 1有3个CpG岛;提示ATF4潜在靶基因受甲基化修饰影响。PPI网络分析结果显示,Brca 1、Btg 1、Carm 1、Ezh 2、Kdm 3 a、Kdm 4 b、Prmt 6之间分别存在交互作用。进一步通过Sangerbox 3.0在线分析网站的GO分析发现,Brca 1、Btg 1、Carm 1、Ezh 2、Kdm 3 a、Kdm 4 b、Prmt 6的功能主要聚焦于组蛋白甲基化、组蛋白修饰、染色质的共价修饰等。结论ATF4可能是巨噬细胞炎症反应过程中参与甲基化修饰的新型调节因子。

Objective To explore the association between activating transcription factor 4(ATF4)and methylation modification in lipopolysaccharide(LPS)induced inflammatory response of macrophage.Methods The expression profiles of ATF4 ChIP-Seq and RNA-seq transcriptomes of RAW264.7 cells treated with LPS or PBS for 8 h were analyzed and compared.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)pathway enrichment analysis were performed to find the key signaling pathways.Through co-expression analysis,the potential target genes with methylation modification of ATF4 were screened.The distribution of CpG islands of key target genes of ATF4 involved in methylation modification was online predicted on the MethPrimer platform.The relationship between ATF4 and these key proteins involved in inflammatory response were obtained by protein-protein interaction(PPI)network analysis.Results The relative expression levels of ATF4 protein and mRNA in the LPS group were significantly higher than those in the PBS group(t=7.507,4.771,both P<0.001).Totally 40 core genes were involved in the methylation modification and their functions were focused on histone methylation,histone demethylation,protein methylation and mRNA methylation pathways.Moreover,based on the conserved cAMP response element(CRE)site in the regulatory region of genes,Brca1,Btg1,Carm1,Ezh2,Kdm3a,Kdm4b,and Prmt 6 were screened as potential targets of ATF4 involved in methylation regulation.It was found that there was one CpG island in the Prmt6,Ezh2,and Carm1,two CpG islands in the Kdm3a,Brca1,and Kdm4b,and 3 CpG islands in the Btg1,indicating that the potential target genes of ATF4 were affected by methylation modification.PPI analysis indicated that there was strong interaction among Brca1,Btg1,Carm1,Ezh2,Kdm3a,Kdm4b and Prmt6.Furthermore,GO analysis of Sangerbox 3.0 online analysis website showed that the functions of Brca1,Btg1,Carm1,Ezh2,Kdm3a,Kdm4b,and Prmt6 mainly focused on histone methylation,histone modification,and covalent modification of chromatin.Conclusion ATF4 may be a potential regulator of methylation modification during macrophage inflammatory response.