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, 15 (9), 2038-49

Formation of Chromosomal Domains by Loop Extrusion

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Formation of Chromosomal Domains by Loop Extrusion

Geoffrey Fudenberg et al. Cell Rep.

Abstract

Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations-including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments-and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.

Figures

Fig 1
Fig 1. Loop extrusion as a mechanism TAD formation
A. Examples of Hi-C contact maps at 5kb resolution showing TADs from four chromosomal regions (GM12878 in-situ MboI (Rao et al., 2014)), highlighting TADs (purple lines) and interaction peaks (blue circles). B. Model of LEF dynamics (fig. S1): LEFs shown as linked pairs of yellow circles, chromatin fiber in grey. From top to bottom: extrusion, dissociation, association, stalling upon encountering a neighboring LEF, stalling at a BE (red hexagon). C. Schematic of LEF dynamics (Movie-M1, Movie-M2). D. Conformation of a polymer subject to LEF dynamics, with processivity 120kb, separation 120kb. Left: shows LEFs (yellow), and chromatin (grey), for one conformation, where darker grey highlights the combined extent of three regions of sizes (180kb, 360kb, 720kb) separated by BEs. Right: the progressive extrusion of a loop (black) within a 180kb region. E. Simulated contact map for processivity 120kb, separation 120kb.
Fig 2
Fig 2. Quantitative analysis of loop extrusion
A. Experimental P(s) (shaded areas) versus simulated P(s) for the 100 best-fitting parameter-sets (lines, one per parameter-set) within TADs (purple) and between TADs (green). Experimental P(s) calculated from 2kb contact maps and normalized to one at 4kb; shaded area shows 10th and 90th percentiles at each genomic distance. Simulated P(s) shown with vertical offsets from fitting (fig. S2). B. Goodness-of-fit versus LEF processivity and separation for the 100 best-fitting parameter-sets (from 6912 total parameters-sets). Circle areas represent the number of parameter-sets among the top-100, while color quantifies the best-fit at each processivity-separation pair; a value of 1 indicates a perfect fit. C–F. Simulated contact maps for the indicated processivity-separation pairs.
Fig 3
Fig 3. TADs formed by LEFs consist of dynamically forming, growing and dissociating loops
A. Illustration of how TADs formed by loop extrusion result from averaging the dynamic positions of loop-bases over many cells, including configurations with nested (cell 6) and consecutive (cell 4) loops (fig. S1). B. left: simulated contact map, as in 1E, is an average of many single-cell maps. right: simulated single-cell contact maps (18kb resolution, green circles show LEF positions). C. Conformation of a polymer subject to LEF dynamics with processivity 120kb, and separation 120kb. Three neighboring regions between BEs of sizes (180kb, 360kb, 720kb) colored in (green, pink, blue). Contacts from an ensemble of such conformations are averaged together to form a contact map.
Fig 4
Fig 4. Simple Strong Loops are not TADs
A. Experimental P(s) (shaded areas) versus simulated P(s) (solid lines) for a parameter-set with a strong loop between neighboring BEs, calculated as in Fig 2A. Here, the fit is relatively poor, (1.4137, rank 2208 out of 6912), and loops are not completely permanent, with BEs in contact 27% of the time for 180kb TAD and 14% for the 720kb TAD. B. Simulated contact map for a simple strong loop with processivity 960kb, separation 960kb. C. Illustration of how a single loop present in many cells leads to strong corner-peaks between neighboring BEs.
Fig 5
Fig 5. TADs require long range insulation
A. Illustration of a genomic region with an insulating element (red hexagon), a promoter (blue square) and an enhancer (green oval) in 1D (Supplemental note) B. Illustration and contact map for a model of BEs as large bulky objects (e.g. bound by proteins or RNA). Each BE is bound by 3 polymer chains of length 10. C. As above, for a model of BEs as a stiff region of chromatin (10 monomers of stiffness 6). D. As above, for a model with direct BE-to-BE attraction (attraction strength 3).
Fig 6
Fig 6. CTCF as a directional boundary element
A. Inward-oriented CTCF sites at TAD boundaries are consistent with loop extrusion and a directional boundary function of CTCF (fig. S6). B. Accumulation of LEFs at BEs for simulations with processivity 120kb and separation 120kb. C. Distributions of CTCF, Smc3, and Rad21 ChIP-seq peak summits in the vicinity of the 4000 strongest motif-associated CTCF binding peaks (orientation indicated by blue arrow). D. Same, but for the weakest 4000 motif-associated CTCF binding sites. E. Asymmetry in ChIP-seq peaks around the strongest 4000 motif-associated CTCF ChIP peaks. Each dot represents an ENCODE GM12878 ChIP-seq track. The y-axis shows the number of peaks within +/− 200bp of a CTCF motif. The x-axis shows the difference between the number of factors on the right and on the left of the motif, i.e. asymmetry of the factor relative to a CTCF motif. F. Same, but for the weakest 4000 motif-associated CTCF ChIP peaks.
Fig 7
Fig 7. Complex TAD architectures from loop extrusion
A. Schematic of LEF dynamics with directional BEs. B–C. Directional BE strength profile (the sum of BEs occupancies within a 12kb bin) for regions simulated in (d–g). D–G. Simulated contact maps for regions of human chr14, GM12878 cell type, for models with orientation specific BEs of varying permeability. Maps are compared with experimental maps for the same regions at the same 12kb resolution (fig. S7). LEF processivity is 120kb (d,e) and 360kb (e,f).

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