Periodic Reporting for period 4 - CohesinLooping (Cohesin-mediated chromosomal looping: From linear paths to 3D effects)
Periodo di rendicontazione: 2022-10-01 al 2023-03-31
The human genome consists of a collection of DNA stretches known as chromosomes. DNA in essence is the code of life. DNA contains the genes that determine the behaviour of our cells, which together form an individual. Each cell in our body contains our entire genome. The genome is meters in length, while a cell is merely a few micrometers in diameter. These simple numbers sketch the momentous task of folding these long DNA threads so that they fit inside tiny cells. This feat becomes even more challenging if we consider that within this confided space, the DNA must be spatially organized in such a manner that the cell can perform its many vital roles.
Key to this spatial organization is a protein complex known as cohesin. Cohesin builds loops in the DNA, which provides structure to chromosomes. By building these loops, cohesin can bring together regulatory DNA elements inside the cell. Cohesin can hereby control the activity of genes by connecting enhancers to promotes, but cohesin can also enable recombination between DNA segments that lie along the same chromosome. An important example would be V(D)J recombination, which allows for the formation of antibodies. Cohesin is controlled on the DNA by a factor known as CTCF. CTCF binds to specific DNA elements, which are present at many sites along all our chromosomes. CTCF somehow prevents that cohesin can build loops beyond these sites. As such, CTCF in essence directs cohesin to connect the right DNA elements. Cohesin and CTCF hereby act in concert to shape our chromosomes and determine the behaviour of our cells. The activity of cohesin and CTCF is often disturbed in human disease. Cancer cells for example often harbour defective cohesin complexes, and CTCF binding elements are also often inactivated in cancer. This can then lead to the incorrect regulation of genes inside cells, and can result in the activation of the oncogenes that drive cancer. Correct functioning of the mechanism by which cohesin and CTCF together shape DNAs thus is essential for human health.
To gain insight into the mechanism by which cohesin shapes DNA, we formulated three main objectives:
1) What provides the force that allows cohesin to build loops?
2) What is the trajectory of cohesin on the DNA?
3) How are loops maintained after they have been formed?
Key to this spatial organization is a protein complex known as cohesin. Cohesin builds loops in the DNA, which provides structure to chromosomes. By building these loops, cohesin can bring together regulatory DNA elements inside the cell. Cohesin can hereby control the activity of genes by connecting enhancers to promotes, but cohesin can also enable recombination between DNA segments that lie along the same chromosome. An important example would be V(D)J recombination, which allows for the formation of antibodies. Cohesin is controlled on the DNA by a factor known as CTCF. CTCF binds to specific DNA elements, which are present at many sites along all our chromosomes. CTCF somehow prevents that cohesin can build loops beyond these sites. As such, CTCF in essence directs cohesin to connect the right DNA elements. Cohesin and CTCF hereby act in concert to shape our chromosomes and determine the behaviour of our cells. The activity of cohesin and CTCF is often disturbed in human disease. Cancer cells for example often harbour defective cohesin complexes, and CTCF binding elements are also often inactivated in cancer. This can then lead to the incorrect regulation of genes inside cells, and can result in the activation of the oncogenes that drive cancer. Correct functioning of the mechanism by which cohesin and CTCF together shape DNAs thus is essential for human health.
To gain insight into the mechanism by which cohesin shapes DNA, we formulated three main objectives:
1) What provides the force that allows cohesin to build loops?
2) What is the trajectory of cohesin on the DNA?
3) How are loops maintained after they have been formed?
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).
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).
The progress beyond the state of the art is considerable. The discovery that CTCF by binding cohesin locally stabilises cohesin on the DNA, and that this interaction is essential for CTCF anchored loops, has been transformative for the field. Cohesin and CTCF together control fundamental chromosomal processes such at the activity of genes, and the recombination that allows the formation of antibodies. Our discoveries thus have opened up new avenues of research for each of these fields of research.
We also made an unexpected finding regarding the role of the condensin II complex. This complex is structurally related to cohesin and can likewise build loops in the DNA. We found that condensin II surprisingly plays a major role in the distribution of the chromosomes inside our cells. Through a collaboration involving specialists in evolutionary genomics and computational modelling, we found that condensin II is a key determinant of nuclear architecture type across eukaryotic evolution. This unexpected result went significantly beyond the state of the art.
We also made an unexpected finding regarding the role of the condensin II complex. This complex is structurally related to cohesin and can likewise build loops in the DNA. We found that condensin II surprisingly plays a major role in the distribution of the chromosomes inside our cells. Through a collaboration involving specialists in evolutionary genomics and computational modelling, we found that condensin II is a key determinant of nuclear architecture type across eukaryotic evolution. This unexpected result went significantly beyond the state of the art.