Enhancer hubs and loop collisions identified from single-allele topologies

Abstract

Chromatin folding contributes to the regulation of genomic processes such as gene activity. Existing conformation capture methods characterize genome topology through analysis of pairwise chromatin contacts in populations of cells but cannot discern whether individual interactions occur simultaneously or competitively. Here we present multi-contact 4C (MC-4C), which applies Nanopore sequencing to study multi-way DNA conformations of individual alleles. MC-4C distinguishes cooperative from random and competing interactions and identifies previously missed structures in subpopulations of cells. We show that individual elements of the β-globin superenhancer can aggregate into an enhancer hub that can simultaneously accommodate two genes. Neighboring chromatin domain loops can form rosette-like structures through collision of their CTCF-bound anchors, as seen most prominently in cells lacking the cohesin-unloading factor WAPL. Here, massive collision of CTCF-anchored chromatin loops is believed to reflect 'cohesin traffic jams'. Single-allele topology studies thus help us understand the mechanisms underlying genome folding and functioning.

Nanopore sequencing of DNA concatemers reveals higher-order features of chromatin structure

“…Recent technologies enabling the study of three-way 33 or higher-order chromatin interactions include chromosomal walks (c-walks) 34 , genome architecture mapping (GAM) 35 , split-pool recognition of interactions by tag extension (SPRITE) 36 , ChIA-drop 37 , Tri-C 38 , Tethered multiple 3C 39 , the concatemer ligation assay (COLA) 33 and Multicontact 4C (MC-4C) 40 . Among these assays, only a subset (Tri-C, SPRITE, COLA, GAM) have been shown to generate genome-wide maps in mammals, and all methods but SPRITE suffer from very rare representation of (<1%) of contacts with order >3 36 .…”

“…Previous studies combining targeted chromatin conformation capture with long-read sequencing have revealed that many of the products of proximity ligation are in fact DNA "concatemers" of multiple interacting loci 33,34,37,40,41 . We surmised that chromatin conformation capture could be paired with nanopore sequencing to efficiently assay the combinatorial chromatin structure in human cells ( Fig.…”

Dynamics of the 4D genome during lineage specification, differentiation and maturation in vivo

“…As cells differentiate further, these interactions are strengthened, concomitant with strong upregulation of gene activity. It has recently been shown for the globin loci that gene activation is associated with the formation of higher-order hub-like structures, in which multiple enhancers and promoters form simultaneous, specific interactions 23,31 . Our data suggest that these structures may only be formed in the final stage of differentiation, when chromatin accessibility and interactions between enhancers and promoters are strongest, and may be important to achieve maximal gene expression.…”

Multi-contact 3C data reveal that the human genome is largely unentangled

“…The method is based on conventional chromosome conformation capture (3C) 10 and the C-walk and multi-contact 4C approaches developed earlier [11][12][13] . 3C performed with the restriction enzyme DpnII produces long ligation products where multiple restriction fragments become ligated together to form DNA molecules that can be >6 kb long, and that are mostly linear (Supplemental Figure 1).…”