Periodic Reporting for period 4 - CohesinMolMech (Molecular mechanisms of cohesin-mediated sister chromatid cohesion and chromatin organization)
Período documentado: 2021-04-01 hasta 2021-09-30
Eukaryotic genomes are large compared to the cells they are contained in, consisting of 2 meters DNA in humans and 20 or more meters in some species such as salamanders. At the various stages of the lifetime of cells, their genomes need to be transcribed, replicated, repaired, recombined, condensed and segregated with high speed and precision. For many of these processes it is crucial that the genome is folded correctly. A key molecule mediating this genome organization is cohesin, a large ring-shaped ATPase complex initially discovered for its essential role in sister chromatid cohesion and chromosome segregation. Our work indicates that in addition to mediating cohesion in proliferating cells, cohesin has a universal role in all cells in forming chromatin loops. Whereas cohesin is thought to mediate cohesion as a passive topological linker, several observations imply that cohesin forms chromatin loops actively by a mysterious extrusion mechanism. The major aims of this project are to understand how cohesin interacts with DNA to perform its functions in sister chromatid cohesion and chromatin organization and to obtain insight into the mechanism by which cohesin forms chromatin loops. Addressing these questions will contribute to understanding genome organization, function and inheritance, and may help to explain why cohesin subunits are among the most frequently mutated tumor suppressor genes in human cancers.
Prior work by us and others had shown that cohesin is required to form chromatin loops and that the position of these loops in mammalian genomes are defined by a DNA binding protein called CTCF which binds to specific sites in these genomes. It had been further proposed that cohesin forms chromatin loops by an active extrusion mechanism. According to this hypothesis cohesin would form a loop by extruding DNA or chromatin fibers until it encounters CTCF bound to DNA. CTCF would function as a boundary for cohesin, thereby stall the loop extrusion process and thus specify which genomic loci function as “anchors” for the loop.
Two key questions emerged from these results and hypothesis:
(i) How does CTCF function as a boundary cohesin complexes?
(ii) How does cohesin form chromatin loops?
Two key questions emerged from these results and hypothesis:
(i) How does CTCF function as a boundary cohesin complexes?
(ii) How does cohesin form chromatin loops?
During the first reporting period of this project we have focused on addressing these questions. For this purpose we have partially reconstituted cohesin translocation along DNA, DNA compaction by cohesin and CTCF’s boundary function by using recombinant human cohesin and CTCF. We have analyzed their interactions with DNA and each other at the single molecule level by imaging and force measurement techniques. The results from these experiments obtained so far indicate that cohesin can indeed actively bend and compact DNA and that CTCF functions as a boundary for this process. Interestingly, CTCF has an unusual asymmetry which enables it to block cohesin complexes translocating along DNA if they arrive from one side but not if they arrive from the other. Previous analyses of genome folding had predicted but not yet shown that such an asymmetric boundary function must exist. We expect that these studies will provide mechanistic insight into how cohesin forms chromatin loops and how CTCF controls and specifies this process.