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The mechanism by which cohesin controls gene expression

Final Report Summary - COHESIN CONTROL (The mechanism by which cohesin controls gene expression)

How cells retain, lose, and regain developmental plasticity, a topic of key medical importance, is poorly understood due to ignorance of the molecular mechanisms regulating gene expression. It has become apparent that cohesin is a ring-shaped protein complex capable of inter-connecting DNAs and translocating large distances along chromatin. In proliferating cells, cohesin’s primary function is to connect sister chromatids during DNA replication, holding them together until the onset of anaphase. However, cohesin is present in most quiescent cells and it is becoming increasingly clear that it also regulates gene expression. Our aim was to observe individual genes in real time and in three-dimensional space and to analyse the immediate consequences of altering the activity of regulatory factors. The finding that live cell imaging of salivary gland polytene chromosomes failed to reveal major changes in transcription within a 30 minute window, following TEV protease injection, led to a major realignment of our experimental priorities. Instead of live cell imaging, we have turned to measuring the effect of Rad21 cleavage by TEV protease on polymerase occupancy throughout the genome using PRO-seq, and the distribution throughout the genome using ChIP-seq of various forms of RNA polymerase and histone modifications. We have been able to use ChIP-seq to measure the actual distribution of cohesin across the polytene chromosome genome. These experiments have revealed that an important consequence of inactivating cohesin in salivary glands is the induction of transcription from ectopic sites (that are normally occupied by cohesin) and that this effect may be the cause of changes in the rate of transcription from bona fide start sites nearby. Our data raise the possibility that cohesin has an important role in preventing initiation of transcription from enhancers and thereby promoting initiation at neighbouring transcription start sites.
With the advent of CRISP-R we shifted our focus on modifying endogenous cohesin subunit genes in tissue culture cells (and in some cases mouse zygotes) in a manner that permitted us either to label, via photo-activation, subsets of the chromosomal cohesin pool, or to inactivate cohesin and its regulators by inserting TEV protease cleavage sites, or indeed to introduce cysteine pairs to cross-link in vivo all three interfaces of the cohesin ring.
Hi-C studies have shown that mammalian chromosomes are compartmentalized into Topologically Associating Domains (TADs). It turns out that TADs are likely a property that emerges from the continuous extrusion of loops by cohesin complexes. However, loop extrusion has not been visualized in vivo, and thus, its mechanics and energetics are unknown. In order to understand we needed to discern the precise nature of cohesin’s association with DNA. We found that in order to establish and maintain sister chromatid cohesion, newly replicated sister DNAs are co-entrapped within cohesin rings. Interestingly, we find that cohesin is able to associate with DNA in a non-topological manner and this interaction is likely sufficient to organize DNA into chromomsome-like threads. We suggest that both topological and non-topological modes of chromatin association depend on changes in cohesin’s Smc1/3 hinge domain that respond to changes in the state of its ATPase. Cohesin’s ability to hydrolyze ATP even upon association with DNA is likely important for its ability to extrude loops. The cohesin loader Scc2 (Nipbl) stimulates cohesin's ABC-like ATPase and is essential for loading cohesin onto chromosomes. We find that Scc2 binds dynamically to chromatin through an association with cohesin. Scc2's movement within chromatin is consistent with a 'stop-and-go' or 'hopping' motion. We suggest that Scc2 moves rapidly from one chromosomal cohesin complex to another, performing a function distinct from loading, perhaps stimulating ATP hydrolysis by cohesin in order to promote loop extrusion.