An EGR1-dependent cascade modulates genome architecture at the CSF1R locus [Capture-C]

The organization and dynamics of chromatin are key to regulating gene expression during myeloid cell differentiation. Sequence-specific transcription factors initiate and maintain a complex network of enhancer-promoter contacts, which is supported by insulating elements and genome folding organizers such as CTCF and Cohesin. The spatial arrangement of enhancers and promoters, as well as their epigenetic state, drives cell and tissue-specific transcriptomes. Here we dissect the spatial, transcriptional, and epigenetic landscape of the CSF1R locus in monocytes and macrophages. CSF1R is a receptor tyrosine kinase that triggers the signaling cascade required for macrophage differentiation. Previous work showed that CSF1R expression is regulated by multiple enhancers, including the fms-intronic regulatory element (FIRE). Here, we find that a single EGR-1 binding motif dictates activation of CSF1R. We also discover that the CSF1R entire locus folds into a hub of gene regulation, affecting an extended network of myeloid and inflammatory genes. Globally, EGR1 may have an expanded role as a macrophage-specific boundary element, supporting enhancer-promoter looping at several genes. In sum, we describe a novel 3D chromatin network that is critical for macrophage development and function. Overall design: Capture HiC targeting CSF1R gene locus in primary human monocytes and macrophages and HL-60 wildtype and CRISPR/Cas9 mutant cell lines targeting the EGR1 binding site in FIRE enhancer of the CSF1R gene. HL-60 wildtype and mutant cells were either untreated or treated with phorbol 12-myristate 13-acetate (PMA) to induce monocytic differentiation.

染色质的组织与动态变化是髓系细胞分化过程中调控基因表达的核心机制。序列特异性转录因子启动并维持复杂的增强子-启动子互作网络，该网络得到绝缘子元件以及CTCF、黏连蛋白（Cohesin）等基因组折叠调控因子的支持。增强子与启动子的空间排布及其表观遗传状态，决定了细胞和组织特异性的转录组特征。
本研究解析了单核细胞与巨噬细胞中集落刺激因子1受体（CSF1R）基因座的空间、转录及表观遗传景观。CSF1R是一种受体酪氨酸激酶，可触发巨噬细胞分化所需的信号级联反应。既往研究表明，CSF1R的表达受多种增强子调控，其中包括fms内含子调控元件（FIRE）。
本研究发现，单个EGR-1结合基序决定了CSF1R的激活；同时还揭示，完整的CSF1R基因座折叠形成一个基因调控枢纽，影响髓系与炎症基因的扩展调控网络。
整体而言，EGR1或许可作为巨噬细胞特异性边界元件发挥更广泛的功能，支持多个基因的增强子-启动子环化互作。综上，本研究揭示了一个对巨噬细胞发育与功能至关重要的新型三维染色质调控网络。
实验设计：针对原代人单核细胞、巨噬细胞，以及靶向CSF1R基因FIRE增强子中EGR-1结合位点的HL-60野生型与CRISPR/Cas9突变体细胞系，开展捕获Hi-C（Capture Hi-C）实验。将HL-60野生型与突变体细胞分为两组，分别不予处理，或用12-肉豆蔻酰-13-乙酸佛波酯（PMA）处理以诱导单核细胞分化。