Mechanisms underlying strikingly divergent responses of genetically distinct macrophages to IL-4 [HiChIP]

Mechanisms by which non-coding genetic variation influences gene expression remain only partially understood but are considered to be major determinants of phenotypic diversity and disease risk. To investigate these mechanisms with respect to signal-dependent gene expression, we evaluated effects of > 50 million SNPs and InDels provided by five inbred strains of mice on the responses of macrophages to the anti-inflammatory cytokine IL-4. Remarkably, of the > 600 genes observed to be induced >2-fold across the five strains after 24 hours of IL-4 treatment, only 26 genes reached this threshold in all strains and more than half of the induced genes were observed in only a single strain. By examining the effects of SNPs and InDels on transcription factor binding and enhancer activity under basal and IL-4 treatment conditions, we identified dominant collaborative roles of the signal-dependent transcription factors (SDTFs) STAT6, PPARg and EGR2 in driving late enhancer activation that were dependent on general macrophage lineage determining factors (LDTFs). As expected, SNPs and InDels that affected the relative affinities of SDTFs primarily influenced the ability of enhancers with similar basal activities to respond to IL-4. In contrast, SNPs and InDels that altered the relative binding affinities of macrophage LDTFs had divergent effects on basal and activated enhancer activity. Variants resulting in strong reductions in LDTF binding affinity were associated with low basal enhancer activity and failure to recruit SDTFs, whereas variants that increased LDTF binding affinities were associated with constitutively high levels of enhancer activity and a blunted response to SDTF recruitment. Together, these studies reveal mechanisms by which noncoding genetic variation influences absolute levels of enhancer activity and their dynamic responses to IL-4, thereby contributing to strain-specific patterns of gene expression and phenotypic diversity. Overall design: HiChIP-seq for BMDMs (bone marow-derived macrophages) from C57BL/6J mice with IL-4 treatment

非编码遗传变异影响基因表达的机制目前仍仅部分阐明，但被认为是表型多样性与疾病风险的主要决定因素。为研究信号依赖性基因表达相关的此类机制，我们针对5个近交品系小鼠提供的超5000万个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）与插入缺失变异（Insertion-Deletion, InDel），评估了其对巨噬细胞应答抗炎细胞因子IL-4的影响。值得注意的是，在经24小时IL-4处理后5个品系中均观察到诱导表达上调2倍以上的600余个基因中，仅26个基因在所有品系中均达到该阈值，且超过半数的诱导基因仅在单个品系中被检测到。通过分析基础条件与IL-4处理条件下SNP及InDel对转录因子结合与增强子活性的影响，我们发现信号依赖性转录因子（signal-dependent transcription factors, SDTFs）STAT6、PPARγ与EGR2，在依赖巨噬细胞谱系通用决定性因子（lineage determining factors, LDTFs）的晚期增强子激活过程中发挥主导性协同作用。正如预期，影响SDTF相对亲和力的SNP与InDel，主要影响具有相似基础活性的增强子对IL-4的应答能力。与之相反，改变巨噬细胞LDTF相对结合亲和力的SNP与InDel，则对基础与激活态增强子活性产生迥异影响。导致LDTF结合亲和力显著降低的变异，与低水平基础增强子活性及无法招募SDTF相关；而提升LDTF结合亲和力的变异，则与组成型高增强子活性及SDTF招募应答减弱相关。综上，本研究揭示了非编码遗传变异影响增强子活性绝对水平及其对IL-4动态应答的机制，进而促成品系特异性基因表达模式与表型多样性的形成。总体实验设计：对经IL-4处理的C57BL/6J小鼠骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）进行HiChIP测序（HiChIP-seq）。