UNDERSTANDING THE ROLES OF HETEROGENOUS SWI/SNF COMPLEXES IN REGULATION OF DIFFERENTIAL GENE EXPRESSION
SWI/SNF is a multi-subunit chromatin remodeling complex that is evolutionarily conserved from yeast to human. In humans, it is known to exist in 3 distinct assemblies and influence expression of genes involved in signal transduction, cell cycle, adhesion, cell morphology, cell fate determination DNA repair, and stress response, by altering chromatin architecture. Deletions in SWI/SNF subunits are known to have deleterious consequences on cells with about 20% of all known cancers harboring loss of protein mutations in SWI/SNF. This thesis focusses on understanding how heterogeneity in SWI/SNF complexes confer advantage to cells in the absence of ARID1A, one of the most mutated SWI/SNF subunits.
In chapter I, we review the structure and composition of SWI/SNF across different organisms. We discuss factors that govern the recruitment of SWI/SNF to specific sites on DNA. We then review the mechanism of SWI/SNF action and its role in driving cancers in human. Afterwards, consider the non- nuclear roles of SWI/SNF in the cell. Finally, we discuss the therapeutic intervention approaches that are currently being engaged in treatment of cancers bearing SWI/SNF mutations.
In chapter II, we try to ascertain dependency on other complexes of SWI/SNF in the absence of ARID1A, an event that is common in cancers driven by mutations in SWI/SNF. We show that cells lacking ARID1A display increased abundance of PBRM1 and BRD9. While we show that lack of BRG1 is lethal in cells lacking ARID1A, we cannot identify which complex is predominantly responsible for mounting response in the absence of ARID1A. We next turn our attention to studying vulnerabilities in SWI/SNF complexes in cells lacking ARID1A under conditions of hypoxia, a physiological condition in most cancers marked by low oxygen tension. We show that the complex composition of SWI/SNF is altered with increased abundance of PBAF and ncBAF members in ARID1A deficient cells subjected to hypoxia. Further, we revalidate that the catalytic subunit BRG1 is critical for cell survival and regulating gene expression in hypoxia. We however could not pinpoint vulnerabilities within the complex specific subunits. Finally, we employ multicellular tumorsphere model (MTS), a cell culture model that mimics in vivo conditions within tumors more accurately, to identify vulnerabilities in ARID1A deficient cell. We confirm that ARID1B continues to be a vulnerability in the absence of ARID1A, in an MTS model. We show that there is a lot if fluidity in utilization of SWI/SNF complexes between inner and outer layers of MTS both in the presence and absence of ARID1A. We also identify PBRM1 as a new susceptibility within the SWI/SNF complex. Levels of ARID1B and PBRM1 are elevated on loss of ARID1A. Further, depletion of ARID1B and PBRM1 protein levels by siRNA mediated knockdown, decreases survival of tumorspheres, reduces proliferation and reduces induction of hypoxia regulated gene EPO and PAI1. Additionally, we demonstrate that inhibition of PBRM1 binding to DNA by means of an inhibitor sensitizes MTSs lacking ARID1A to cell death. Our study suggests PBRM1 as a new vulnerability in ARID1A deficient cancers, which could aid in development of necessary therapeutic interventions. This thesis focusses
In chapter III, we venture into exploring non-nuclear roles of SWI/SNF in the cell. We use western blotting and immunofluorescence to show that a subset of SWI/SNF complex the core complex exists in the cytoplasm. Further, we demonstrate that this core complex is lost from cytoplasm when cells are subjects to hypoxia. Additionally, we use Immunoprecipitation and Mass Spectrometry to show that INI1 directly interacts with ERC1 in the cytoplasm. Additionally, we have strong evidence to indicate that loss of INI1 from cytoplasm causes changes to cytoskeletal structure by recruiting ERC1 to cell cortex and promoting mesenchymal phenotype in epithelial cells. Overall, our study indicates that INI1 has roles in the influencing cell edge dynamics of the cell which is independent of its role in the nucleus as a chromatin remodeler. The study opens new avenues and necessitates more investigations into roles of INI1 in influencing cell migration and thereby metastasis, which is the leading cause of deaths in cancer.
Overall, my work furthers our knowledge and understanding of SWI/SNF and identifies new vulnerabilities in cells lacking ARID1A. It also suggests possible non-nuclear roles of SWI/SNF in maintaining cytoskeletal structure, both of which can be exploited in development of necessary therapeutic interventions against cancers, the leading cause of death worldwide