CTCF depletion uncouples the role of enhancer-promoter interactions and higher-order chromatin hubs in gene regulation during cellular differentiation [Tri-C]. CTCF depletion uncouples the role of enhancer-promoter interactions and higher-order chromatin hubs in gene regulation during cellular differentiation [Tri-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: Tri-C experiments. Studies of multiway cis-element interactions during human B cell to macrophage transdifferentiation. Please note that processed data file generated from all replicates is 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(3D chromatin structures)相互作用以调控基因表达(gene expression)。目前，驱动此类结构形成的机制及其在细胞分化(cellular differentiation)过程中的功能尚未完全阐明。
本研究以极高分辨率解析了淋巴系-髓系分化系统(lymphoid-to-myeloid differentiation system)中基因组的结构-功能关联。我们证实调控蛋白结合、染色质相互作用形成与基因表达三者间存在紧密相关性。通过整合单等位基因拓扑结构(single-allele topologies)分析与计算建模(computational modeling)，我们发现组织特异性基因位点会组装为染色质枢纽(chromatin hubs)，其特征为多个增强子、启动子与CCCTC结合因子(CTCF)结合位点之间的协同相互作用。
CTCF耗竭会导致此类结构近乎完全丧失，这表明CTCF介导的相互作用为染色质枢纽的形成提供了支架。与之相反，CTCF耗竭对基因表达的影响相对温和，该现象可通过重编程的增强子-启动子相互作用加以解释。
综上，本研究证实增强子-启动子相互作用在细胞分化过程的基因调控中发挥指导性作用，且该作用不依赖于染色质枢纽内的协同相互作用。
整体实验设计：开展Tri-C实验，研究人类B细胞向巨噬细胞转分化(transdifferentiation)过程中的多顺式元件相互作用(multiway cis-element interactions)。请注意，所有生物学重复(replicates)所生成的处理后数据文件(processed data file)均关联至对应的*rep1样本记录。