Multiplex Chromatin Interactions with Single Molecule Precision

Emerging 3D genome mapping efforts suggest complex chromosomal folding structures. However, the true multiplex nature of chromatin interactions has yet to be fully explored. Here, we describe a chromatin interaction analysis by droplet-based sequencing (ChIA-Drop). In ChIA-Drop, individual chromatin complexes are partitioned into droplets that contain a gel bead of DNA-barcoded primers, such that tethered chromatin DNA fragments are uniquely indexed and amplified for sequencing and mapping to demarcate multiplex chromatin contacts. Thus, ChIA-Drop can identify complex chromatin interactions with unprecedented single-molecule precision, which is not possible using methods that analyze pairwise contacts via proximity ligation. We demonstrate that multiplex chromatin interactions predominantly contribute to topologically associated domains with high heterogeneity, and that multivalent promoter-centered interactions provide a topological model for gene transcription. The microfluidics device (10X Genomics’ Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions.

新兴的三维基因组图谱绘制研究揭示了复杂的染色体折叠结构，但染色质相互作用的真正多重特性仍有待全面探索。本研究中，我们介绍了一种基于液滴测序的染色质相互作用分析技术（ChIA-Drop）。在ChIA-Drop技术中，单个染色质复合物被分配至包含DNA条形码引物凝胶微珠的液滴中，使锚定的染色质DNA片段获得唯一的索引标记，并进行扩增以用于测序及图谱绘制，从而界定多重染色质相互作用。因此，ChIA-Drop能够以前所未有的单分子精度识别复杂染色质相互作用，这是通过邻近连接分析成对相互作用的方法无法实现的。我们的研究表明，多重染色质相互作用主要促成了具有高度异质性的拓扑关联结构域，而以启动子为中心的多价相互作用为基因转录提供了一种拓扑模型。该技术使用的微流控设备（10X Genomics公司的Chromium系统）可生成包裹染色质复合物的乳液凝胶微珠（Gel beads in Emulsion, GEM），用于复合物特异性的DNA条形码标记与扩增。将所有GEM（液滴）产生的带有条形码的DNA扩增产物混合后，进行DNA测序与图谱绘制，以识别染色质相互作用。