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

Abstract

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.

The Mediator complex regulates enhancer-promoter interactions

“…However, since the MCC viewpoints are very narrow and focused on gene promoters, it remains unclear how large-scale 3D genome architecture is changed and how interactions between other c is -regulatory elements are impacted by Mediator depletion. We therefore used the Tiled-MCC approach, in which MCC library preparation is combined with an enrichment strategy based on capture oligonucleotides tiled across large genomic regions of interest [14], to investigate changes in genome architecture in DMSO- and dTAG-treated HCT-116 MED14-dTAG cells in a broader context. We focused on the MYC locus (3.3 Mb; Figure 3), MTAP locus (1.55 Mb; Supplementary Figure 4), HMGA2 locus (990 kb; Supplementary Figure 5) and ITPRID2 locus (900 kb; Supplementary Figure 6).…”

“…To further analyze the impact of Mediator depletion on chromatin architecture, we leveraged the ability of Tiled-MCC to directly identify ligation junctions and resolve localized nano-scale interaction patterns [14]. We focused our analyses on ligation junctions in regions containing super-enhancers, genes and boundary elements in the MYC, MTAP, HMGA2 , and ITPRID2 loci (Figure 5, Supplementary Figures 8-10).…”

“…It has previously been shown that regions containing CTCF-binding sites form characteristic architectural patterns, in which phased nucleosomes surrounding the CTCF motif form a grid-like structure, which is associated with strong insulation between the regions upstream and downstream of the CTCF-binding site [14,36]. We observe these patterns at the intergenic CTCF-binding sites in the loci we investigated and do not see any changes upon depletion of Mediator (Figure 5, right matrix, Supplementary Figures 8-10).…”

“…However, the molecular mechanism remains unclear. Furthermore, depletion of Cohesin causes a relatively subtle reduction in enhancer-promoter interaction strength [14]. This suggests that these interactions are not solely dependent on loop extrusion and that other mechanisms are involved in their formation.…”

Bioinformatics analysis of multi-omics data elucidates the primary function of Oct4 in gene regulation and pluripotency maintenance

“…Whether loop extrusion by cohesin also plays a direct role for enhancer-promoter interactions or not still remains unclear. A recent study has demonstrated that depletion of cohesin leads to only modest reduction of long-range enhancer-promoter interactions, indicating that cohesin associated loop extrusion is not essential for the maintenance of enhancer-promoter interactions (Aljahani et al, 2021).…”