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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE80031 | GEO | 2016/11/02

SECONDARY ACCESSION(S): PRJNA317669

REPOSITORIES: GEO

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Project description:Objective: Monocytes are crucially involved in inflammatory processes. In mice, different subsets of monocytes can be distinguished by the expression of Ly6C, a surface marker that is highly expressed on inflammatory monocytes (Ly6C high) and to a lesser extent on patrolling monocytes (Ly6C low). Our previous study revealed an aggravated accumulation of inflammatory Ly6C high monocytes in atherosclerotic-prone mice bearing a deficiency in suppressor of cytokine signaling (Socs)-1. In the present study, we performed a genome-wide analysis of Socs-1-dependent gene regulation in Ly6C high and Ly6C low monocytes. Methods and Results: Monocyte subsets were isolated from socs-1+/+ rag2‒/‒ ldlr–/‒ and socs-1‒/‒ rag2‒/‒ ldlr-‒/‒ mice with subsequent identification of differential regulated genes using illumina MouseWG-6 v2.0 Expression BeadChip microarrays (45,200 transcripts). Principal component analysis illustrates a distinct separation of gene expression profiles in Ly6C high and especially Ly6C low monocytes from Socs-1 deficient mice. Additionally, microarray data revealed 46 genes to be differentially regulated in a Socs-1 dependent manner in both monocytic subsets by 2-fold or more, some of which were validated by qPCR (p<0.05, n=4-6). Among them, two principal regulators of monocyte differentiation, C-X3-C chemokine receptor 1 (Cx3cr1) and colony stimulating factor 1 receptor (Csf1r), were identified to be Socs-1 dependently regulated. Especially for Csf1r a co-expression network for correlating Socs-1 dependent gene expression and protein interaction could be compiled. Furthermore, in silico analysis of a transcription factor (TF) network correlating with Socs-1 dependent mRNA expression data revealed a cluster of TF that includes Nr4a1 as well as several ets-domain proteins that may interact and thus regulate gene expression, inter alia regression of Csf1r. Conclusion: By genome-wide analysis, we could identify Cx3cr1 and Csf1r as two essential receptors mediating monocyte differentiation that are subjected to Socs-1 dependent transcription factor regulation.

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

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