We have obtained important insight regarding each of our objectives.
Our first objective was to discover the driving force of loop formation. This activity is controlled by molecular devices such as cohesin that have so-called ATPase machineries at their basis. We found that these ATPase machineries display surprising asymmetric roles in the loop formation reaction. The activity of cohesin turns out to be controlled by a chemical modification, known as acetylation. This modification is placed on top of cohesin's ATPase, which we found limits the degree to which cohesin can enlarge loops. Unexpectedly, this role of acetylation is independent of a previously known pathway that protects against WAPL-mediated DNA release. We found that acetylation converts cohesin into a PDS5-bound state. We propose that the cohesin acetylation cycle enables the pausing and restart of the DNA looping machinery, and does so through a PDS5 braking mechanism (van Ruiten et al., Nature Structural & Molecular Biology, 2022).
Our second objective was to learn about cohesin's path on the DNA. Cohesin's trajectory on DNA is controlled by CTCF. Together with our collaborators, we found how CTCF controls cohesin. A specific part of CTCF turns out to bind to cohesin, which anchors cohesin on the DNA. CTCF does so by associating with a composite interface consisting of cohesin’s SA2 and SCC1 subunits. The binding of CTCF to this interface stabilizes cohesin at CTCF sites, which turns out to be essential for CTCF-anchored loops genome-wide. This interaction hereby plays a major role in the shaping of the DNA inside our cells (Li et al., Nature, 2020).
Importantly, other factors turn out to bind the same interface as CTCF to enable cohesin to control diverse chromosomal events, ranging from DNA replication to mitosis, transcription and DNA repair. The includes the cohesin protector SGO1 which, just like CTCF, deploys its YxF motif to engage cohesin and render cohesin resistant against WAPL-mediated release from DNA. By hereby stabilising cohesin specifically at centromeres, SGO1 thus allows chromosomes to manifest their archetypal X-shaped morphology. Cohesin complexes that build loops or that rather connect the sister chromatids are thus regulated through a universal mechanism (García-Nieto et al., Nature Structural & Molecular Biology, 2023).
Our third objective was to learn how loops are maintained after they have been formed. We had hypothesized that the Mediator complex might maintain cohesin-dependent loops. As explained above, we find that CTCF stabilizes such DNA loops (Li et al., Nature, 2020). We find that the Mediator CDK module in fact controls both the linear and the spatial epigenome by counteracting the amplification of heterochromatin domains. By preventing the formation of dense heterochromatin bodies, the Mediator CDK module maintains chromatin in a transcription-permissive state, and thus enables transcription across large segments of the genome (Haarhuis et al., Nature Communications, 2022).
