MC4C: Locus-Specific Enhancer Hubs And Architectural Loop Collisions Uncovered From Single Allele DNA Topologies

Chromatin folding is increasingly recognized as a regulator of genomic processes such as gene activity. Chromosome conformation capture (3C) methods have been developed to unravel genome topology through the analysis of pair-wise chromatin contacts and have identified many genes and regulatory sequences that, in populations of cells, are engaged in multiple DNA interactions. However, pair-wise methods cannot discern whether contacts occur simultaneously or in competition on the individual chromosome. We present a novel 3C method, Multi-Contact 4C (MC-4C), that applies Nanopore sequencing to study multi-way DNA conformations of tens of thousands individual alleles for distinction between cooperative, random and competing interactions. MC-4C can uncover previously missed structures in sub-populations of cells. It reveals unanticipated cooperative clustering between regulatory chromatin loops, anchored by enhancers and gene promoters, and CTCF and cohesin-bound architectural loops. For example, we show that the constituents of the active -globin super-enhancer cooperatively form an enhancer hub that can host two genes at a time. We also find cooperative interactions between further dispersed regulatory sequences of the active proto-cadherin locus. When applied to CTCF-bound domain boundaries, we find evidence that chromatin loops can collide, a process that is negatively regulated by the cohesin release factor WAPL. Loop collision is further pronounced in WAPL knockout cells, suggestive of a “cohesin traffic jam”. In summary, single molecule multi-contact analysis methods can reveal how the myriad of regulatory sequences spatially coordinate their actions on individual chromosomes. Insight into these single allele higher-order topological features will facilitate interpreting the consequences of natural and induced genetic variation and help uncovering the mechanisms shaping our genome.

染色质折叠已逐渐被证实为基因表达等基因组进程的关键调控因子。研究人员已开发出染色质构象捕获（Chromosome Conformation Capture, 3C）技术，通过分析成对染色质相互作用来解析基因组拓扑结构，并在细胞群体中鉴定出诸多参与多种DNA相互作用的基因与调控序列。然而，成对相互作用分析方法无法区分在单条染色体上，这些相互作用是同时发生，还是处于竞争状态。本研究提出一种新型3C技术——多接触4C（Multi-Contact 4C, MC-4C），该技术借助纳米孔测序（Nanopore sequencing），可对数万个单个等位基因的多维度DNA构象展开研究，以此区分协同作用、随机作用与竞争性相互作用。MC-4C能够揭示细胞亚群中此前未被发现的基因组结构。该技术还揭示了增强子与基因启动子锚定的调控性染色质环，以及结合CTCF与黏连蛋白（cohesin）的结构性染色质环之间，存在此前未被发现的协同簇集现象。例如，研究证实活跃的β-珠蛋白（β-globin）超级增强子的组成成分可协同形成一个增强子枢纽，该枢纽能够同时容纳两个基因。本研究还在活跃的原钙粘蛋白（proto-cadherin）基因座的远端分散调控序列之间，发现了协同相互作用。当将MC-4C应用于结合CTCF的结构域边界时，研究人员观测到染色质环可发生碰撞，这一过程受到黏连蛋白释放因子WAPL的负向调控。在WAPL敲除细胞中，染色质环碰撞现象更为显著，这提示存在‘黏连蛋白交通拥堵’的情况。综上，单分子多接触分析技术能够揭示海量调控序列如何在单条染色体上实现空间层面的功能协同。对这些单个等位基因高阶拓扑特征的深入解析，将有助于阐释自然与诱导性遗传变异所带来的影响，并助力阐明塑造人类基因组的分子机制。