Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

Abstract

We recently used in situ Hi-C to create kilobase-resolution 3D maps of mammalian genomes. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that the observed contact domains are inconsistent with the equilibrium state for an ordinary condensed polymer. Combining Hi-C data and novel mathematical theorems, we show that contact domains are also not consistent with a fractal globule. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during polymer condensation leads to formation of an anisotropic "tension globule." In the other, CCCTC-binding factor (CTCF) and cohesin act together to extrude unknotted loops during interphase. Both models are consistent with the observed contact domains and with the observation that contact domains tend to form inside loops. However, the extrusion model explains a far wider array of observations, such as why loops tend not to overlap and why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The convergent rule correctly predicts the affected loops in every case. Moreover, the extrusion model accurately predicts in silico the 3D maps resulting from each experiment using only the location of CTCF-binding sites in the WT. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.genome architecture | molecular dynamics | CTCF | chromatin loops | CRISPR

Two independent modes of chromatin organization revealed by cohesin removal

“…Cohesin and CTCF co-localize at TAD boundaries and the bases of Hi-C peaks 7,12 , but their roles are not fully defined. The recently proposed loop extrusion model 14,15 yields predictions that are consistent with experiments. In this model, TADs emerge from the progressive extrusion of chromatin loops by a protein complex (e.g.…”

“…2h). Together, these analyses and the observed effects of Nipbl deletion indicate that cohesin plays a central role in the local compaction of chromosomes, and support that this effect is mediated by the production of dynamic populations of extruded chromatin loops between boundary elements, which forms TAD and corner peak patterns in interphase Hi-C maps 14,15 .…”

“…This major reorganization of chromatin architecture is also reflected in the curves of contact frequency P(s) as a function of genomic distance s 3,29 . In control samples, like in other mammalian cells 14,15 , P(s) curve has two regimes: a more shallow decay for s<200Kb ( P(s)~s −0.7 ), and a steeper scaling for 200Kb < s < 3Mb ( P(s)~s −1.2 ). Loss of chromatin-associated cohesin leads to disappearance of the first regime, producing a single decay of contact probability across the whole range (Fig.…”

“…CTCF sites). Recent experimental manipulation of CTCF motifs 15–17 and CTCF depletion 18 support the proposed role of CTCFs as boundary elements, but cohesin’s role in interphase chromatin organization and the process of loop extrusion remains ambiguous, as experimental depletions of cohesin have shown limited impact on chromatin organisation 19–21 …”

Extruding Loops to Make Loopy Globules?

“…An important hint in this direction came from a high resolution Hi-C study (8), where~10 4 pair contacts were identified as loop anchors, and their sequence motifs were found to be antiparallel, oriented along the genome such as to face one another. Now, in hindsight, these motifs are beautifully interpreted as natural stop signs for the loop extruding proteins (5,6).…”