Dissecting the regulatory strategies of NFkB RelA target genes in the inflammatory response reveals differential transactivation logics [MEF]

NFkB RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (ChIPseq) and functional (RNAseq in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a novel primary-cell genetic complementation assay. All endogenous target genes required that RelA makes DNA-base-specific contacts, and none could be activated by the DNA binding domain alone. However, endogenous target genes differed widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic timecourse data we reveal gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent vs. context-independent control of endogenous RelA-target genes. While some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NFkB RelA core response. Total RNA extracted from primary mouse embryonic fibroblasts stimulated with IL-1b, LPS, and TNF.

NFκB RelA（NFkB RelA）是炎症应答基因的强效转录激活因子。我们通过整合物理表征数据集（染色质免疫共沉淀测序，ChIP-seq）与功能数据集（基因敲除样本的RNA测序，RNA-seq），严格界定了一组直接RelA靶基因。随后，我们在一种新型原代细胞遗传互补实验中通过检测RelA变体，解析了各靶基因的调控策略。所有内源性靶基因均要求RelA形成DNA碱基特异性接触，且仅靠DNA结合结构域无法激活任意靶基因。不过，不同内源性靶基因在利用两个转录激活结构域的模式上存在显著差异。通过对动态时间进程数据开展模型辅助分析，我们揭示了转录激活结构域1（TA1）与转录激活结构域2（TA2）的基因特异性协同与冗余效应。鉴于翻译后修饰可调控TA1的活性，而辅激活因子的固有亲和力决定TA2的活性，这种差异化的TA调控逻辑表明，内源性RelA靶基因存在环境依赖型与环境非依赖型两类调控机制。部分炎症起始因子似乎需要共刺激型TA1激活，而炎症消退因子则属于NFκB RelA核心应答的组成部分。实验所用总RNA提取自经白细胞介素1β（IL-1b）、脂多糖（LPS）及肿瘤坏死因子（TNF）刺激的原代小鼠胚胎成纤维细胞。