Function and regulation of chromatin insulators in dynamic genome organization

Abstract

Chromatin insulators are DNA-protein complexes that play a crucial role in regulating chromatin organization. Within the past two years, a plethora of genome-wide conformation capture studies have helped reveal that insulators are necessary for proper genome-wide organization of topologically associating domains, which are formed in a manner distinct from that of compartments. These studies have also provided novel insights into the mechanics of how CTCF/cohesin-dependent loops form in mammals, strongly supporting the loop extrusion model. In combination with single-cell imaging approaches in both Drosophila and mammals, the dynamics of insulator-mediated chromatin interactions are also coming to light. Insulator-dependent structures vary across individual cells and tissues, highlighting the need to study the regulation of insulators in particular temporal and spatial contexts throughout development.

Figure 1.

Cohesin is required to position TAD boundaries at CTCF/cohesin-occupied loci. Variable locations of TAD-like structures and their boundaries are observed using high resolution imaging across individual cells, with the highest frequency of boundaries at CTCF/cohesin-occupied sites. Upon cohesin depletion, the location of TAD boundaries is randomized. This effect would be visualized as overall loss of TADs upon cohesin depletion in ensemble Hi-C studies.

Figure 2.

TADs and compartments are not hierarchical structures. CTCF/cohesin-dependent TADs can force interactions between different compartment types. Loss of TADs strengthens compartments genome-wide.

Figure 3.

The loop extrusion model underlying CTCF/cohesin-mediated chromatin organization. The cohesin complex loads onto chromatin, perhaps preferentially at superenhancers, and progressively pushes chromatin through its ring-like structure to extend the loop until the complex encounters a CTCF molecule positioned in the correct orientation. Extrusion can occur in one or both directions. This process requires ATP, and cohesin ATPase activity may be involved.

Figure 4.

In vivo imaging at the single-cell level to visualize insulator-mediated regulation of transcription activity. A) Without an insulator, the distal reporter (green spot) remains transcriptionally inactive. Transcriptional activity (blue spot) at the endogenous eve locus shows spatial separation. B) Presence of an insulator sequence increases the stability of the loop and pairing of the reporter with the endogenous locus. Sustained proximity is required to activate transcription of the reporter (red spot).