Daan NORDERMEER - Université Paris Saclay

Topologically Associating Domains (TADs) cover regions in the genomes of higher eukaryotes where intra-domain contacts are increased over their surroundings. Mammalian TADs are enriched for the binding of the CTCF insulator protein at their boundaries, which can prevent the formation of enhancer-promoter loops between neighbouring domains. Mechanistically, TADs are shaped by a process of active Cohesin-mediated loop extrusion, resulting in intra-TAD chromatin compaction, followed by blocking of this process at the boundaries by the CTCF protein, creating inter-TAD separation. How optimal separation between neighboring TADs is achieved remains incompletely understood.

We recently reported that the separation between most TADs extends over zones spanning several tens of kilobases, where local interactions are enriched. Most extended TAD boundaries contain multiple sites of CTCF binding, indicating multiple instances of loop extrusion blocking. Indeed, using a Nano-C assay, a newly developed multi-contact 3C assay, we could show that individual CTCF binding sites are permeable sites of loop extrusion blocking. Within extended TAD boundaries, individual CCTF binding sites thus additively contribute to the loop extrusion blocking capacity.

Here, I will discuss collaborative efforts where we combined genomics data analysis and biophysical modeling approaches, to further characterize the extended nature of TAD boundaries. Using mouse cells as a model, we find that the span of TAD boundaries is highly variable. This variation is directly linked to the distribution of CTCF and other chromatin factors. Using a generalized random-crosslinked polymer model, we determined optimal chromatin configurations to reproduce different types of TAD boundaries. Moreover, we used these configurations to determine their impact on longer-range inter-TAD encounters. Interestingly, we found that narrow TAD boundaries are associated with a considerable increase in spurious inter-TAD contacts. This may be explained by the “reeling in” of neighboring TADs by the Cohesin complexes that may be blocked at the boundary in the middle. These increased spurious inter-TAD contacts were not observed at extended boundaries. As such, wider boundaries improve TAD insulation both by reducing loop extrusion readthrough and the reduction of spurious contacts with neighboring TADs. I will discuss how these effects, and the associated DNA-encoded patterns of CTCF binding, may be used for the fine-tuning of enhancer promoter loop formation and gene regulation.

Citation:

Chang LH, Ghosh S, et al. (2023) Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and Topologically Associating Domain boundaries. Nature Communications; 14:5615.

Contact : Daniel Jost