NF?B dynamics determine the stimulus-specificity of epigenomic reprogramming in macrophages

The epigenome defines the cell type, but also possesses plasticity to tune gene expression in the context of extracellular cues. This tuning is evident in immune sentinel cells such as macrophages, which respond to pathogens and cytokines with phenotypic shifts driven by epigenomic reprogramming. Recent studies indicate that this reprogramming arises from the activity of transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF?B). NF?B binds not only to available enhancers but may also produce de novo enhancers in previously silent areas of the genome. Here, we show that NF?B reprograms the macrophage epigenome in a stimulus-specific manner, in response only to a subset of pathogen-derived stimuli. The basis for these surprising differences lies in the stimulus-specific temporal dynamics of NF?B activity. In response to different stimuli, NF?B enters the nucleus with variable speed, amplitude, and duration, and may oscillate between the nucleus and cytoplasm. These dynamical features combine to specify the identity and dose of a given stimulus. We demonstrate through live cell imaging, mathematical modeling, and genetic perturbations that NF?B promotes open chromatin and formation of de novo enhancers most strongly when its dynamics are non-oscillatory. These de novo enhancers result in the activation of additional response genes. We propose a mechanistic paradigm in which the temporal dynamics of transcription factors are a key determinant of their capacity to control epigenomic reprogramming, thus enabling the formation of stimulus-specific memory in innate immune sentinel cells. Overall design: Sequencing data from murine bone marrow-derived macrophages, comparing different TLR stimuli and using genetic perturbations of NF?B signaling dynamics. H3K4me1 ChIP-seq (41 samples), ATAC-seq (36 samples), mRNA-seq (19 samples).

表观基因组（epigenome）不仅定义了细胞类型，还具备可塑性，可在细胞外信号的背景下调控基因表达。这种调控在免疫哨兵细胞（如巨噬细胞）中表现显著：巨噬细胞可在表观基因组重编程驱动的表型转变下，响应病原体与细胞因子的刺激。近期研究表明，此类重编程源于转录因子的活性，其中包括活化B细胞κ轻链增强子结合蛋白（nuclear factor kappa-light-chain-enhancer of activated B cells，NF-κB）。NF-κB不仅可结合已有的增强子，还能在基因组此前处于沉默状态的区域中从头（de novo）生成增强子。本研究证实，NF-κB以刺激特异性的方式重编程巨噬细胞的表观基因组，仅对部分病原体源性刺激产生响应。上述令人意外的差异的核心成因，在于NF-κB活性的刺激特异性时间动态学特征。在不同刺激条件下，NF-κB进入细胞核的速度、强度与持续时间各不相同，且可在细胞核与细胞质之间发生振荡。这些动态特征共同作用，可确定特定刺激的类型与剂量。本研究通过活细胞成像、数学建模与遗传扰动实验证明：当NF-κB的动态特征为非振荡模式时，其对染色质开放以及de novo增强子生成的促进作用最为显著。此类de novo增强子会激活更多的应答基因。本研究提出了一套机制范式：转录因子的时间动态学特征是其调控表观基因组重编程能力的关键决定因素，从而使先天免疫哨兵细胞能够形成刺激特异性的免疫记忆。实验整体设计：本研究采集小鼠骨髓来源巨噬细胞的测序数据，对比不同Toll样受体（TLR）刺激条件，并利用NF-κB信号动态学的遗传扰动实验。检测涵盖三类测序：H3K4me1染色质免疫共沉淀测序（ChIP-seq，41个样本）、转座酶可及性测序（ATAC-seq，36个样本）以及信使RNA测序（mRNA-seq，19个样本）。