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

Chromosome conformation capture (3 C) techniques have captured largescale 3D genome architecture. Here the authors present their Tiled-MCC approach for generation of 3 C data across megabase-scale loci at very high (up to 20 bp) resolution, which allowed them to observe nano-scale chromatin structures and investigate how these structures depend 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 (20 bp resolution) 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.

Automated summary

Chromosome conformation capture techniques have provided insights into the 3D architecture of the genome. In this study, the authors present a high-resolution approach for generating 3C data and investigate the role of cohesin and CTCF in chromatin structure. They find that enhancers and promoters interact within large-scale topologically associating domains mediated by cohesin and CTCF. Additionally, they show that cis-regulatory elements have distinct nano-scale structures that depend on CTCF, while depletion of cohesin affects longer-range enhancer-promoter interactions.