Targeted deletion of an Nr4a1­ associated enhancer ablates Ly6Clow monocytes while protecting pleiotropic gene function in macrophages [RNA-seq]

Mononuclear phagocytes are a diverse cell family that occupy all tissues and assume numerous functions to support tissue and systemic homeostasis. Our ability to investigate the roles of individual subsets is limited by an absence of approaches to ablate gene function within specific sub-populations. Using Nr4a1-dependent Ly6Clow monocytes as a representative cell type we show that enhancer deletion addresses these limitations. Combining ChIP-Seq and molecular approaches we identify a single, conserved, sub-domain within the Nr4a1 enhancer that is essential for Ly6Clow monocyte development. Mice lacking this enhancer lack Ly6Clow monocytes but retain Nr4a1 gene expression in macrophages during steady state and in response to LPS. Nr4a1 is a key negative regulator of inflammatory gene expression and decoupling these processes allows Ly6Clow monocytes to be studied without confounding influences. Enhancer targeting possesses greater specificity than cre recombinase-mediated gene deletion, providing a route to generate loss-of-function models in closely related cell types. Overall design: Paired End mRNA sequencing of FACS purified primary murine MDP, cMoP, Ly6Chi and Ly6Clow monocytes from the bone marrow and Ly6Chi and Ly6Clow monocytes from the peripheral blood

单核吞噬细胞（mononuclear phagocytes）是一类高度异质性的细胞家族，广泛分布于所有组织中，通过执行多种功能维持组织及全身稳态。我们对单个亚群功能的研究能力受限于缺乏可在特定细胞亚群内敲除基因功能的实验手段。本研究以依赖Nr4a1的Ly6C低表达单核细胞（Ly6Clow monocytes）作为代表性细胞类型，证实增强子敲除可解决上述研究局限。结合染色质免疫共沉淀测序（ChIP-Seq）与分子生物学实验手段，我们在Nr4a1增强子区域内鉴定出一个单一且保守的亚结构域，该结构域是Ly6C低表达单核细胞发育所必需的。缺失该增强子的小鼠无法产生Ly6C低表达单核细胞，但在稳态及脂多糖（lipopolysaccharide, LPS）刺激条件下，其巨噬细胞仍可正常表达Nr4a1基因。Nr4a1是炎症基因表达的关键负调控因子，将该调控过程与增强子靶向策略解偶联后，可在不受混杂因素干扰的前提下开展Ly6C低表达单核细胞的相关研究。与Cre重组酶介导的基因敲除技术相比，增强子靶向策略具有更高的特异性，为在密切相关的细胞类型中构建功能丧失型实验模型提供了全新路径。实验设计：对经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化的原代小鼠骨髓来源的MDP（巨噬细胞与树突状细胞前体，macrophage and dendritic cell progenitor）、cMoP（常见单核细胞前体，common monocyte progenitor）、Ly6C高表达（Ly6Chi）及Ly6C低表达单核细胞（Ly6Clow monocytes），以及外周血来源的Ly6C高表达（Ly6Chi）与Ly6C低表达单核细胞（Ly6Clow monocytes）进行双端mRNA测序。