Disruption of mammalian SWI/SNF chromatin remodeler function in human disease

Abstract

The regulation of chromatin architecture and accessibility is central to the control of gene expression programs that govern cellular identity, cell fate and lineage commitment, differentiation, and responses to stimuli. Chromatin topology is controlled, in part, by ATP-dependent chromatin remodeling complexes (CRCs), groups of multi-subunit protein complexes that utilize energy generated from ATP hydrolysis to alter nucleosome-DNA contacts. This thesis is focused on the mammalian SWI/SNF (mSWI/SNF) or BAF family of CRCs, specifically the canonical BAF or cBAF complex. mSWI/SNF complexes are highly conserved molecular machines that assemble in a modular fashion. The cBAF complex, defined by the incorporation of the ARID1A or ARID1B paralog subunits within the core module, cooperates extensively with transcriptional coactivators and the transcriptional machinery to bind chromatin at distal enhancer sites and generate DNA accessibility by engaging nucleosomes and either sliding them along DNA or evicting the histone octamer completely. Perturbations in mSWI/SNF subunits have been strongly linked to transcriptional dysregulation in human disease, most prominently, in human cancer and neurodevelopmental disorders. In this thesis we have: (1) deciphered the contribution of the largest and most frequently mutated mSWI/SNF complex subunits, ARID1A and ARID1B, to cBAF activity; and (2) characterized the role of mSWI/SNF complexes in mediating SARS-CoV-2 infection in cells and to evaluate BAF inhibition as a host-directed anti-viral strategy against COVID-19. Specifically, we dissect the role of the N-terminal intrinsically disordered regions (IDRs) of ARID1A/B and the sequence non-specific ARID DNA-binding domain using a combination of genomic-, proteomic-, and microscopy-based approaches. We find that the IDRs of ARID1A/B drive biomolecular condensate formation of the cBAF complex in vitro and in cells. Inactivation of the ARID domain’s capability to bind DNA leads to mis localization of condensates. While the N-terminal IDR and the ARID domain of ARID1A are dispensable for the core enzymatic function of cBAF, they are essential for chromatin localization of the complex at distal enhancer sites. Furthermore, ARID1A/B IDRs form both homotypic (self-self) and heterotypic (self-nonself) interactions and establish an amino acid sequence-specific protein interaction network, which is required for the proper localization and activity of cBAF on chromatin. Replacing the ARID1A N-terminal IDR with IDRs derived from unrelated proteins such as FUS and DDX4 rescues condensation of cBAF but not its chromatin occupancy and protein interactions, underscoring the specificity of the ARID1A IDR region in cBAF complex function. In the second section of this work, we find that ATPase-competent cBAF plays a proviral role in SARS-CoV-2 infection by localizing to and maintaining DNA accessibility at the ACE2 gene locus and regulating expression of ACE2 in cells (ACE2 is the cellular receptor which binds to the Spike protein of SARS-CoV-2 and is essential for viral entry). These data are consistent with and begin to inform the results of several recent CRISPR/Cas9-based genome-wide screens which have revealed that inactivation of genes encoding mSWI/SNF subunits confers resistance to SARS-CoV-2 infection. Localization of cBAF at the ACE2 locus is directed by the HNF1A/B transcription factors. Importantly, small molecule inhibition of mSWI/SNF ATPase activity or degradation of the ATPase subunits SMARCA2/4 leads to downregulation of ACE2 expression and resistance to multiple variants of SARS-CoV-2, demonstrating the anti-viral effect of mSWI/SNF inhibition. Taken together, these data elucidate the complex molecular mechanisms governing BAF complex localization and function in cells. Specifically, we uncover the role of the N-terminal ARID1A/B IDR-mediated biomolecular condensate formation and protein network formation in genome-wide targeting and activity of the cBAF complex in cells and highlight mSWI/SNF as a host-directed anti-viral drug target against SARS-CoV-2. Overall, this thesis provides insights into the functionality of intrinsically disordered regions of a highly disease-relevant chromatin remodeler and underscores its role in coronavirus infection.