Exploiting proteomic mass spectrometry analysis to understand the roles of BAF chromatin remodelling complex subunit ARID1A

Abstract

AT-rich interactive domain-containing protein 1A (ARID1A) is the most frequently mutated subunit of BRG/Brahma-associated factors (BAF) chromatin remodelling complex across diverse cancer types. Although some roles of BAF complex subunits are known, specific roles of ARID1A and the biological importance of its interplay with other proteins in the normal state as well as in oncogenesis are not entirely understood. In this project, I investigated the role of ARID1A by extensive characterisation of its native protein interaction network using a range of mass spectrometry-based strategies. A comprehensive ARID1A interactome was compiled using AP-MS across various cell types, including human and mouse pluripotent cells and human-differentiated non-transformed cell lines in different conditions to identify core, stable, weak and transient interactors. Over 500 partners of ARID1A were identified, including known and previously reported potentially novel interactors. The ARID1A interactome was enriched in proteins involved in cellular processes such as chromatin remodelling, DNA damage, RNA splicing and paraspeckle proteins, in agreement with its previously reported functions. Interactome mapping following cellular perturbations revealed that PARP1 and ARID1A might have a liquid-liquid phase separation-dependent interaction. Chromatin profiling of cells lacking ARID1A or expressing a truncated version showed extensive changes in chromatin integrity and impact on various processes critical for tumorigenesis and cancer progression, such as cell division, splicing and metabolism. A combination of approaches was utilised to decipher the structural basis of ARID1A protein interactions. The first study using BN-PAGE-MS to resolve ARID1A interactome provided supplementary topological information for the BAF complex, suggesting the existence of stable BAF sub-complexes. From XL-MS experiments with cleavable crosslinker DSSO, I identified interaction surfaces between subunits of a novel Thrap3-Erh-Bclaf1 (TEB) complex co-enriched in ARID1A immunoprecipitates. Finally, a recently developed protein interaction screen on peptide matrices (PRISMA) was adopted to obtain the first ARID1A interactome map at amino acid resolution.