A complex CTCF binding code defines TAD boundary structure and function

Topologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundarie. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary. We report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals clustering not only among CTCF ChIP-seq peaks but frequently also within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites (CBS) additively contribute to TAD insulation. The clustering of CBS may counter against the dynamic DNA-binding kinetics of CTCF, which urges a re-evaluation of current models for the blocking of loop extrusion. Our work thus reveals an unanticipatedly complex CTCF binding code at TAD boundaries that expands the regulatory potential for TAD structure and function and may help to better explain how non-coding structural variation can influence gene regulation, DNA replication, recombination and repair.

拓扑关联结构域（TADs）将脊椎动物基因组划分为亚兆碱基级的功能邻域，为基因调控、DNA复制、重组与修复等生命过程提供结构基础。TADs由黏连蛋白（Cohesin）介导的环挤出过程形成，该过程会压缩结构域内的DNA，随后由CCCTC结合因子（CTCF）绝缘子蛋白在结构域边界处阻断环挤出进程。CTCF以依赖结合方向的方式阻断环挤出，现有实验与计算机模拟研究均认为，单个静态CTCF结合位点即可形成稳定的TAD边界。本研究发现，小鼠细胞内的绝大多数TAD边界属于模块化实体，CTCF结合位点在延伸的基因组区间内呈现聚集特征。经优化的染色质免疫共沉淀测序（ChIP-seq）分析显示，CTCF结合峰不仅彼此间存在聚集现象，且常出现在单个结合峰内部。我们利用新开发的多接触纳米C检测法（multi-contact Nano-C assay）证实，单个CTCF结合位点（CBS）可通过加和效应协同增强TAD的绝缘功能。CTCF结合位点的聚集现象或许能够抵消CTCF动态的DNA结合动力学特性，这要求学界重新评估当前的环挤出阻断模型。综上，本研究揭示了TAD边界处存在此前未被认知的复杂CTCF结合编码机制，该机制拓展了TAD结构与功能的调控潜力，或有助于更清晰地阐释非编码结构变异如何影响基因调控、DNA复制、重组与修复过程。