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. First, by examining the probability for short chromatin fragments to bend and form a cycle, we show that nuclear human chromatin is flexible at the kilobase scale, inconsistent with the widespread existence of 30-nm fibers in vivo. Next, we find that 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 inter-phase. 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, why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation, and why edges of enhanced contacts occur at some domains. Furthermore, loop extrusion leads naturally to the formation of chromosome territories. 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 wild-type. 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. Support or Funding Information This project is supported by NSF Grant PHY-1308264, NSF Grant PHY-1427654, NIH New Innovator Award 1DP2OD008540-01, Cancer Prevention Research Institute of Texas Scholar Award R1304, a McNair Medical Institute Scholar Award, and the President's Early Career Award in Science and Engineering, and funding from the Welch Foundation, International Business Machines, and Nvidia. (A)Summary of loop extrusion binding (i, ii), extruding (iii), and halting at motifs (iv). (B) 3D rendering of an extrusion globule. (C) Contact probability vs. distance within domains created in silico using loop extrusion. (D) Molecular simulations of loop extrusion based only on CTCF ChIP-Seq signals accurately recapitulate features observed in our Hi-C maps. (A) Results of CRISPR/Cas9-based editing experiments on chr 8. Simulations shown on left, experimental data shown on right. (B) Similar results on chr 1. (C) We disrupted two loops by inserting a single basepair. (D) Our data suggest that the region shown in (A) is typically found in one of two states. (E) Extrusion can explain the formation of exclusion domains.
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Mar 1, 2017
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Open Access