Structural and biochemical insights into WAPLmediated cohesin release and regulation of genomelooping

Turnover of cohesin is tightly controlled by loader complexes and the key release factor WAPL, which is required for the regulation of vital genomic processes such as sister chromatid cohesion, chromosome segregation and chromatin looping. In this thesis, I sought to establish how the different domains and motifs of WAPL can bind to cohesin and carry out it’s important release function. I have shown that that WAPL binds to the cohesin subunits SA2-SCC1 and have determined the crystal structure of a middle region of WAPL (WAPL-M) bound to these subunits. Furthermore, I have established that WAPL binds in a bidentate manner to SA2-SCC1, with the WAPL-M being an important ‘anchor’ to allow the structured C-terminus of WAPL to engage a different site and compete with the CCCTC-binding factor (CTCF) for binding. The interaction of both WAPL-M and WAPL-C was confirmed to be crucial in regulating both cohesion and looping. I anticipate that competition between WAPL-C and other chromosomal regulators such as CTCF, regulates 3D genome folding and cohesin function for several chromosomal processes.

Having established the key interaction site, I aimed to investigate how WAPL interacts with meiotic cohesin complexes. I observed that REC8, which substitutes for SCC1 in meiotic cohesin complexes, lacked the key WAPL-M site required for WAPL binding to cohesin and I confirmed biochemically that REC8 residues at the WAPL-M site abolish binding. This may explain why REC8 cohesin complexes are resistant to WAPL mediated release and why cohesion in female primary oocytes is prolonged.