Analysis of sub-kilobase chromatin topology reveals nano-scale regulatory interactions with variable dependence on cohesin and CTCF

Enhancers and promoters predominantly interact within large-scale topologically associating domains (TADs), which are formed by loop extrusion mediated by cohesin and CTCF. However, it is unclear whether complex chromatin structures exist at sub-kilobase-scale and to what extent fine-scale regulatory interactions depend on loop extrusion. To address these questions, we present an MNase-based chromosome conformation capture (3C) approach, which has enabled us to generate the most detailed local interaction data to date and precisely investigate the effects of cohesin and CTCF depletion on chromatin architecture. Our data reveal that cis-regulatory elements have distinct internal nano-scale structures, within which local insulation is dependent on CTCF, but which are independent of cohesin. In contrast, we find that depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Together, our data show that loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression. Overall design: Tiled-Micro-Capture-C (Tiled-MCC) combines Micro-Capture-C (MCC) library preparation with oligonucleotide capture enrichment for deep, targeted sequencing of chromatin interactions within regions of interest. Details are described in the Methods section of the paper.

增强子（enhancer）与启动子（promoter）主要在大规模拓扑关联结构域（topologically associating domains, TADs）内发生相互作用，而此类结构域由黏连蛋白（cohesin）与CTCF介导的环挤出（loop extrusion）过程形成。然而，目前尚不清楚亚千碱基尺度下是否存在复杂的染色质结构，且精细尺度的调控相互作用在多大程度上依赖于环挤出过程。为解答上述疑问，本研究开发了一种基于微球菌核酸酶（MNase）的染色质构象捕获（chromosome conformation capture, 3C）技术，借此生成了截至目前最为精细的本地染色质相互作用数据，并得以精准探究黏连蛋白与CTCF耗竭对染色质架构的影响。本研究数据显示，顺式调控元件（cis-regulatory elements）具有独特的内部纳米尺度结构，此类结构的局部绝缘效应依赖于CTCF，却与黏连蛋白无关。与之相反，本研究发现黏连蛋白耗竭会导致长距离增强子-启动子相互作用出现轻微减弱，而CTCF耗竭则可引发调控接触网络的重连。综合来看，本研究数据表明，环挤出过程并非增强子-启动子相互作用所必需，但可提升此类相互作用的稳定性与特异性，并助力基因表达的精准调控。整体实验设计：平铺微染色质捕获-构象捕获（Tiled-Micro-Capture-C, Tiled-MCC）技术将微染色质捕获-构象捕获（Micro-Capture-C, MCC）的文库制备流程与寡核苷酸捕获富集步骤相结合，可对目标区域内的染色质相互作用进行深度靶向测序。具体细节详见本文的方法部分。