CTCF depletion uncouples the role of enhancer-promoter interactions and higher-order chromatin hubs in gene regulation during cellular differentiation [Micro-Capture-C]. CTCF depletion uncouples the role of enhancer-promoter interactions and higher-order chromatin hubs in gene regulation during cellular differentiation [Micro-Capture-C]

Enhancers and promoters interact in 3D chromatin structures to regulate gene expression. The mechanisms that drive the formation of these structures and their function during cellular differentiation are incompletely understood. Here, we study the structure-function relationship of the genome in a lymphoid-to-myeloid differentiation system at very high resolution. We demonstrate a close correlation between binding of regulatory proteins, formation of chromatin interactions, and gene expression. By integrating analysis of single-allele topologies and computational modeling, we show that tissue-specific gene loci are organized into chromatin hubs, characterized by cooperative interactions between multiple enhancers, promoters, and CTCF-binding sites. Depletion of CTCF leads to a near-complete loss of these structures, which indicates that CTCF-mediated interactions provide a scaffold for chromatin hub formation. In contrast, the effects of CTCF depletion on gene expression are relatively mild and can be explained by rewired enhancer-promoter interactions. Together, our results demonstrate an instructive role for enhancer-promoter interactions in gene regulation during cellular differentiation, which does not depend on cooperative interactions in chromatin hubs. Overall design: Micro-Capture-C experiments. Studies of cis-element interactions during human B cell to macrophage transdifferentiation. Please note that processed data files generated from all replicates are linked to the corresponding *rep1 sample records.

增强子（enhancer）与启动子（promoter）通过三维染色质结构相互作用以调控基因表达。目前，驱动此类结构形成及其在细胞分化过程中发挥功能的分子机制尚未被完全阐明。本研究以极高分辨率在淋巴系-髓系分化系统中探究基因组的结构-功能关联。我们证实，调控蛋白的结合、染色质相互作用的形成与基因表达三者之间存在紧密关联。通过整合单等位基因拓扑结构分析与计算建模手段，我们发现组织特异性基因位点会被组装为染色质枢纽（chromatin hub），其特征为多个增强子、启动子以及CTCF结合位点（CTCF-binding site）之间存在协同相互作用。CTCF（CCCTC结合因子）的敲低会导致此类结构近乎完全消失，这表明CTCF介导的相互作用为染色质枢纽的形成提供了骨架支撑。与之相反，CTCF敲低对基因表达的影响相对温和，这可通过重连的增强子-启动子相互作用加以解释。综上，我们的研究结果证实，在细胞分化过程中，增强子-启动子相互作用对基因调控具有指导性作用，且该作用并不依赖于染色质枢纽内的协同相互作用。整体实验设计：微捕获-染色质构象捕获（Micro-Capture-C）实验；针对人类B细胞向巨噬细胞转分化过程中顺式作用元件相互作用的研究。请注意，所有生物学重复所生成的预处理数据文件，均与对应的*rep1样本记录相关联。