Chromatin insulator mechanisms ensure accurate gene expression by controlling overall 3D genome organization

Abstract

Chromatin insulators are DNA-protein complexes that promote specificity of enhancer-promoter interactions and maintain distinct transcriptional states through control of 3D genome organization. In this review, we highlight recent work visualizing how mammalian CCCTC-binding factor acts as a boundary to dynamic DNA loop extrusion mediated by cohesin. We also discuss new studies in both mammals and Drosophila that elucidate biological redundancy of chromatin insulator function and interplay with transcription with respect to topologically associating domain formation. Finally, we present novel concepts in spatiotemporal regulation of chromatin insulator function during differentiation and development and possible consequences of disrupted insulator activity on cellular proliferation.

Figure 1.. Consequences of cohesin collision with CTCF during loop extrusion in mammals

A. Cohesin associates with chromatin and begins to extrude DNA and form a loop, promoting contact between an enhancer and promoter and subsequent transcription burst. The STAG1/2 subunit of cohesin encounters the YDF motif of CTCF at the N-terminus. B. Possible outcomes of collision are dependent on the amount of DNA tension created by loop extrusion. (i) At low tension, bypass of CTCF can occur. (ii) At higher tension, blocking is favored. (iii) Also at higher tension, cohesin can backtrack and/or loop slippage can occur. This outcome could result in repeated enhancer-promoter contact within the extruded loop. Red stop signs indicate extended blockage of translocation.

Figure 2.. Chromatin insulators and active transcription cooperate to promote 3D genome organization in Drosophila

A. Pairs of chromatin insulator binding sites not corresponding to future TAD borders are found in proximity in embryos at nuclear cycle 12. At this stage, zygotic transcription has not yet been fully established. Population averaged representations are shown. B. The major wave of zygotic transcription occurs during nuclear cycle 14, and TADs and TAD borders become clearly established on a population average. TAD borders are mainly located at highly transcribed housekeeping genes and chromatin insulator binding sites. Transcription at TAD borders may promote 3D interactions between TAD borders through formation of small scale compartments. Population averaged representations are shown; multi-way insulator-dependent interactions are not frequently observed in single cells.

Figure 3.. Redundancy and context-dependency of chromatin insulator function in TAD border function in Drosophila embryos

A. Wildtype positions of chromatin insulator proteins, highly transcribed gene promoters, and 3D interactions as displayed by Hi-C representation including insulator-dependent sub-TAD interactions nested within TADs. TAD borders are indicated by vertical hash marks. Blue dots indicate intra-TAD insulator-dependent interactions. B. Loss of CTCF can lead to disruption of CP190 recruitment and mainly affects TAD borders of highly co-occupied sites. White arrow indicates loss of TAD border, and translucent color of insulator proteins indicates reduced binding. C. Loss of BEAF-32 can lead to disruption of CP190 recruitment and mainly affects TAD borders where only BEAF-32 and CP190 bind together.

Figure 4.. Simplified model for sequential collinear activation of HoxD genes through CTCF-dependent stalling of cohesin loop extrusion

A. Cohesin-dependent loop extrusion begins downstream of inactive Hoxd4 (green). B. Cohesin stalls at CTCF bound with N-terminus facing cohesin, and only Hoxd4 is activated at this stage. C. Extrusion continues until another CTCF is encountered and allows for additional activation of Hoxd8 (purple).