Cohesin is a ring-shaped complex composed of four subunits: SMC1, SMC3, RAD21 and STAG. Thanks to its ability to entrap DNA within its lumen, cohesin performs two essential functions: 3D genome organization and sister chromatid cohesion. On the one hand, cohesin folds the genome through the formation of dynamic chromatin loops and this folding impacts gene expression but also DNA replication and recombination. On the other hand, cohesion facilitates faithful DNA repair by homologous recombination and proper chromosome segregation. In somatic vertebrate cells, the STAG subunit can be either STAG1 or STAG2, giving rise to the two coexisting cohesin-STAG1 and cohesinSTAG2 variants. Despite their overall similarity, both complexes are not functionally redundant. To better understand their different contributions and regulation, during this Thesis we have dissected the initial step of cohesin loading onto chromatin for the two cohesin variants in human cells. When cells exit mitosis and chromosomes decondense, cohesin is loaded on chromatin in order to establish genome organization. NIPBL together with MAU2 form the loading complex that is thought to be essential for cohesin loading and sliding along DNA extruding loops. These loops are stabilized when cohesin encounters a barrier, most often CTCF. Using a new flow cytometry assay to measure chromatin-bound proteins, we have found that cohesin can associate with chromatin when the loading complex is strongly reduced. Moreover, the two cohesin complexes respond in opposite ways to low NIPBL levels. While cohesin-STAG1 levels on chromatin increase and the complex accumulates at CTCF-bound positions, same as in control cells, cohesin-STAG2 decreases on chromatin and does not accumulate at any genomic loci. We propose a model in which NIPBL is not required for chromatin association of cohesin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 binds chromatin and becomes stabilized at CTCF sites even under low NIPBL levels, although genome folding is severely impaired. These results add to our understanding of the different behavior of cohesin-STAG1 and cohesin-STAG2, and provide a new perspective on the role of NIPBL on cohesin dynamics. Moreover, they can help understand the pathophysiology of Cornelia de Lange Syndrome, a rare disease caused in most cases by mutations in NIPBL

This thesis has been supported by a FPI fellowship awarded to Dácil Alonso Gil (BES2017-080051) and research grants from MINECO BFU2016-79841-R and PID2019106499RB-I00

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 18-05-2023