Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function.

Abstract

Background: The evolutionarily conserved cohesin complex is a pleiotropic regulator of chromosome structure and function, participating in sister chromatid cohesion, transcriptional regulation of genes, DNA replication, and DNA repair. Cohesin uses ATP hydrolysis to dynamically extrude DNA loops that bring together cis-regulatory elements and thus regulate gene expression. Some DNA loops are anchored by the binding of CTCF insulator proteins which can stall extruding cohesin complexes, however many DNA loops that connect enhancers and promoters lack CTCF and it is unclear how cohesin is stabilized at these cis-regulatory sites. While cohesin has been found to co-purify with a number of proteins, some of which regulate cohesin function, our current knowledge of cohesin activity is incomplete. Identification of transient or less stable interactions between cohesin and chromatin-associated proteins is crucial for understanding regulation of gene expression and chromosome structure.

Results: Here we utilize a TurboID proximity labeling and mass spectrometry approach for identifying cohesin-interacting proteins. We identify > 400 cohesin-interacting proteins in NIH-3T3 cells, including previously known and potentially novel cohesin interactors. Among the cohesin interactors were chromatin remodeling complexes and histone-modifying complexes. Interactions between seven of these chromatin regulating complexes and cohesin were confirmed with co-immunoprecipitations performed in multiple cell lines. The SWI/SNF complex was found to co-purify with cohesin and SWI/SNF co-occupied enhancers and promoters with cohesin. To investigate the functional relevance of the cohesin-SWI/SNF interaction, we assessed whether the binding of cohesin to the genome is regulated by SWI/SNF or vice versa. Acute small molecule perturbations of SWI/SNF altered the amount of both SWI/SNF and cohesin on chromatin, particularly affecting cohesin binding to CTCF sites.

Conclusions: This work represents the most comprehensive investigation of cohesin-interacting proteins to date. These results identify a physical link between cohesin and a vast number of chromatin-associated proteins inside of cells, including chromatin remodeling complexes and histone-modifying complexes. Furthermore, these results indicate SWI/SNF activity stabilizes cohesin on chromatin particularly at insulator sites. These cohesin interactome data are a resource for future studies aimed at characterizing the functional interactions between cohesin and numerous chromatin-associated proteins in regulating chromosome structure and gene control.