Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries

Topologically Associating Domains (TADs) separate vertebrate genomes into insulated regulatory neighborhoods that focus genome-associated processes. TADs are formed by Cohesin-mediated loop extrusion, with many TAD boundaries consisting of clustered binding sites of the CTCF insulator protein. Here we determined how this clustering of CTCF binding contributes to the blocking of loop extrusion and the insulation between TADs. We identified enrichment of three features of CTCF binding, whereby strongly bound and closely spaced binding sites are enriched around strong TAD boundaries. Using multi-contact Nano-C analysis in cells with normal and perturbed CTCF binding, we established that individual CTCF binding sites contribute to the blocking of loop extrusion, but in an incomplete manner. When clustered individual CTCF binding sites thus create a stepwise insulation between neighboring TADs. Based on these results, we propose a model whereby multiple instances of temporal loop extrusion blocking create strong isolation between TADs.

拓扑关联结构域（Topologically Associating Domains, TADs）可将脊椎动物基因组划分为彼此绝缘的调控邻域，以此聚焦基因组相关的生物学过程。TADs由黏连蛋白（Cohesin）介导的环挤压（loop extrusion）过程形成，多数TAD边界由成簇分布的CTCF绝缘子蛋白结合位点构成。本研究旨在明确CTCF结合的成簇特征如何参与环挤压阻断及TAD间的绝缘调控。我们鉴定出CTCF结合的三类富集特征：高亲和力结合且间距紧密的结合位点在强TAD边界区域显著富集。通过对正常及CTCF结合被扰动的细胞开展多接触Nano-C（multi-contact Nano-C）分析，我们证实单个CTCF结合位点可介导环挤压阻断，但该作用并不完全。因此，成簇分布的单个CTCF结合位点可在相邻TAD之间构建阶梯式的绝缘屏障。基于上述研究结果，我们提出一种模型：多个瞬时环挤压阻断事件可在TAD间形成强绝缘隔离。