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. 2018 Feb 28;46(4):1648-1660.
doi: 10.1093/nar/gkx1123.

Transcription-induced supercoiling as the driving force of chromatin loop extrusion during formation of TADs in interphase chromosomes

Dusan Racko  1   2   3 , Fabrizio Benedetti  1   4 , Julien Dorier  1   4 , Andrzej Stasiak  1   2
Affiliations

Transcription-induced supercoiling as the driving force of chromatin loop extrusion during formation of TADs in interphase chromosomes

Dusan Racko et al. Nucleic Acids Res. .

Abstract

Using molecular dynamics simulations, we show here that growing plectonemes resulting from transcription-induced supercoiling have the ability to actively push cohesin rings along chromatin fibres. The pushing direction is such that within each topologically associating domain (TAD) cohesin rings forming handcuffs move from the source of supercoiling, constituted by RNA polymerase with associated DNA topoisomerase TOP1, towards borders of TADs, where supercoiling is released by topoisomerase TOPIIB. Cohesin handcuffs are pushed by continuous flux of supercoiling that is generated by transcription and is then progressively released by action of TOPIIB located at TADs borders. Our model explains what can be the driving force of chromatin loop extrusion and how it can be ensured that loops grow quickly and in a good direction. In addition, the supercoiling-driven loop extrusion mechanism is consistent with earlier explanations proposing why TADs flanked by convergent CTCF binding sites form more stable chromatin loops than TADs flanked by divergent CTCF binding sites. We discuss the role of supercoiling in stimulating enhancer promoter contacts and propose that transcription of eRNA sends the first wave of supercoiling that can activate mRNA transcription in a given TAD.

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Figures

Figure 1.
Figure 1.
Active swivel that mimics a joint action of RNA polymerase with associated TOP1 introduces negative supercoiling into modelled chromatin fibre. (A) A snapshot of starting, thermally equilibrated configuration of a circular, coarse-grained chromatin fibre with the length corresponding to 120 kb and with one active swivel (shown in red). The active swivel is not working yet. (B and C) Snapshot of configurations obtained after the active swivel performed about 5 and 10 rotations, respectively. The inset shows ideograms of active swivels and indicates their locations. Segments of modelled chromatin fibre that flank the active swivel are forced to swivel with respect to each other. The direction of active swivelling is such that it introduces negative supercoiling, which results in formation of right-handed, interwound plectonemes. In all our figures, to visualize better the structure of plectonemically wound supercoiled regions, the chromatin fibres are shown with the diameter corresponding to 0.3σ instead of 1σ.
Figure 2.
Figure 2.
Consequences of limiting axial rotation of chromatin by cohesin handcuffs. (A–C) Modelling of TADs in which supercoiling can freely diffuse and be dissipated at TADs borders. (A) Starting, equilibrated configuration of modelled TAD with the active swivel ready for action and two sites where supercoiling can be dissipated. (B and C) two snapshots taken after 5 and 10 rotations of the active swivel, respectively. (D–F) Modelling of TADs in which free diffusion of supercoiling is strongly limited by cohesin handcuffs. (D) Starting, equilibrated configuration of modelled TAD, such as shown in A, but having in addition cohesin handcuffs placed between the active swivel and the TADs borders where supercoiling can be dissipated. (E and F) Two snapshots taken after 5 and 10 rotations of the active swivel, respectively. Notice accumulation of supercoiling in the chromatin portion between active swivels and cohesin handcuffs. Insets show ideograms and locations of active and passive swivels. Passive swivels are presented as sharp conical tips in contact with opposing surface. Torsional stress can freely dissipate by swivelling occurring at passive swivels. As mentioned in the legend to Figure 1, for better visualization of supercoiled regions chromatin fibres are shown with the diameter corresponding to 0.3σ instead of 1σ. However, the modelled cohesin handcuffs are presented with the diameter of 1σ, corresponding to the diameter they had during simulations.
Figure 3.
Figure 3.
Supercoiling-driven chromatin loop extrusion. (A–D) Simulation snapshots illustrating how a growing plectonemic region pushes cohesin handcuffs away from the source of supercoiling and towards borders of the modelled TAD. This process eventually brings the two borders of the modelled TAD into close physical proximity (D). Schematic maps, shown above each simulated configuration, illustrate the progress of chromatin loop extrusion process. The loops spanned by cohesin handcuffs grow till reaching the size of the entire TAD. The ends of red arcs correspond to positions of individual cohesin rings forming the handcuff. The linear maps of simulated circular construct are centred at the active swivel. Maps also show position of the two borders of the modelled TAD, with passive swivels and semi-transparent regions accounting for action of type II DNA topoisomerases. Supplementary Movie S2 presents the entire simulation from which the snapshots were taken to compose the figure.
Figure 4.
Figure 4.
Asymmetric loop extrusion is also efficient in bringing together borders of individual TADs. (A–C) Simulation snapshots illustrating the progression of supercoiling-induced chromatin loop extrusion when the source of supercoiling is significantly closer to one than to the other border element of the modelled TAD. CTCF proteins bound to CTCF binding sites at borders of TADs are modelled as large beads that cannot pass through cohesin rings. Notice that once one of the cohesin rings forming the handcuff is blocked in its progression by CTCF border element (B) the second ring can still move and eventually reaches the second CTCF border element (C). Therefore, both border elements of modelled TAD, with bound there CTCF proteins can be brought together. As in Figure 3, the schematic maps shown above each snapshot, illustrate the progress of chromatin loop extrusion.
Figure 5.
Figure 5.
Pushing of cohesin handcuffs by supercoiling can also explain why the orientation of CTCF binding sites determines the stability of chromatin loops forming TADs. (A) Transcribing RNA polymerase in association with TOP1 induces formation of negative supercoils. Cohesin handcuffs load near a crossing caused by negative supercoiling. (B) The growing plectoneme pushes cohesin handcuffs irrespectively of the orientation of CTCF sites at TADs borders. (C) When CTCF binding sites are convergent, the C terminal parts of bound CTCF protein can contact cohesin rings and this interaction stabilizes cohesin handcuffs and formed chromatin loops. On contact maps, such TADs form triangles with strong tips. (D) When CTCF binding sites are divergent, the C terminal part of bound CTCF protein is unavailable for contacts with cohesin rings. Without these stabilizing interactions cohesin rings dissociate from chromatin and this permits rapid relaxation of accumulated torsional stress by TOP2B that is normally associated with the N terminal part of bound CTCF. On contact maps, such TADs form triangles without strong tips.
Figure 6.
Figure 6.
Transcription of enhancers provides the first wave of supercoiling needed for chromatin loop extrusion forming TADs, in which promoters of developmentally regulated genes can contact their partner enhancers. (A) Transcription of an enhancer is initiated by the presence of some transcription factor but is independent of contacts with another enhancer. Cohesin handcuffs load at one of the crossings resulting from supercoiling generated during eRNA transcription. (B) The growing plectoneme pushes cohesin handcuffs towards borders of the TAD with CTCF binding sites in a convergent orientation at its border. (C) Once the growing chromatin loop spans the entire TAD the promoter of developmentally regulated gene can interact with its partner enhancer and start transcription.
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