The bovine alveolar macrophage (bAM) DNA methylome is resilient to infection with Mycobacterium bovis

Whole genome bisulfite sequencing (WGBS) was used to assess the effect of M. bovis infection on the bAM DNA methylome (5-methylcytosine). The methylomes of bAM infected with M. bovis (n = 8) were compared to those of non-infected control bAM (n = 8) at 24 hours post-infection (hpi). No differences in DNA methylation (CpG or non-CpG) were observed between control and infected bAM. Analysis of DNA methylation at proximal promoter regions uncovered >250 genes harbouring intermediately methylated (IM) promoters (average methylation = 33–66%). Gene ontology (GO) analysis, focusing on genes with low, intermediate or highly methylated promoters, revealed that genes with IM promoters were enriched for immune-related GO categories; this enrichment was not observed for genes in the high or low methylation groups. Targeted analysis of two non-imprinted genes in the IM category, C1QB and IL2RA, confirmed the WGBS observation. This study is the first in cattle to examine genome-wide DNA methylation at single nucleotide resolution in an important bovine cellular host-pathogen interaction model and provides evidence for intermediate promoter methylation in bAM. Overall design: Examination of CpG methylation in control and Mycobacterium bovis infected bovine macrophages, 24 hours post infection本研究采用全基因组亚硫酸氢盐测序（Whole genome bisulfite sequencing, WGBS）技术，评估牛分枝杆菌（Mycobacterium bovis, M. bovis）感染对牛肺泡巨噬细胞（bovine alveolar macrophage, bAM）DNA甲基化组（5-甲基胞嘧啶）的影响。将感染M. bovis的bAM样本（样本量为8）与未感染的阴性对照bAM样本（样本量为8）在感染后24小时（hours post-infection, hpi）进行甲基化组比较。对照组与感染组bAM的DNA甲基化水平（CpG或非CpG位点）未观察到显著差异。对近端启动子区域的DNA甲基化分析显示，超过250个基因的启动子呈现中等甲基化（intermediately methylated, IM）状态，平均甲基化水平为33%~66%。针对启动子呈低、中、高甲基化状态的基因开展基因本体（Gene Ontology, GO）富集分析，结果表明携带中等甲基化启动子的基因显著富集于免疫相关GO条目，而高、低甲基化组基因未出现此类富集现象。对中等甲基化类别中的两个非印记基因C1QB与IL2RA进行靶向验证，结果证实了WGBS的测序发现。本研究是牛领域首个在重要宿主-病原菌互作细胞模型中以单核苷酸分辨率检测全基因组DNA甲基化的研究，为牛肺泡巨噬细胞的启动子中等甲基化现象提供了实验证据。整体实验设计：检测对照组与牛分枝杆菌感染组牛肺泡巨噬细胞在感染后24小时的CpG甲基化水平。