Defining the roles for the sequence-non-specific DNA-binding domains in mammalian SWI/SNF chromatin remodeling complex assembly and function - PROJECT SUMMARY/ABSTRACT The mammalian SWI/SNF (mSWI/SNF) complexes represent a diverse family of ATP-dependent chromatin remodeling complexes (CRCs) that play critical roles in the modulation of chromatin accessibility and gene expression. mSWI/SNF complexes are combinatorically assembled from 29 different gene products to generate three distinct 11-15-subunit classes termed BAF, pBAF, and ncBAF complexes. Importantly, mSWI/SNF genes are mutated in over 20% of human cancers and several neurodevelopmental disorders (NDDs), in several cases representing causative genomic abnormalities. While biochemical, structural, and genomics-based studies have begun to define subunit-specific contributions to overall mSWI/SNF complex function, the specific roles for DNA- binding and histone reader domains present on subunits remain largely unassigned. In particular, the functional contributions of sequence-non-specific DNA-binding domains, including the winged-helix (WH) domain of SMARCB1, the high-mobility group (HMG) domain of SMARCE1, and the ARID domain of ARID1A/B subunits, have not been identified and represent key ‘hubs’ of frequent single-residue mutations in human cancer and NDDs. I aim to define the role for these domains in mSWI/SNF complex function in vitro and in the human cell context via the assessment of disease-associated perturbations. During this F31 fellowship, I will determine the roles for sequence-non-specific DNA-binding domains in a) proper mSWI/SNF complex assembly and biochemical integrity; b) mSWISNF complex catalytic (ATPase) activity and nucleosome remodeling; and c) for genome-wide mSWI/SNF targeting and DNA accessibility generation in human cell line model systems. I will accomplish this by generating wild-type (WT) and DNA-binding-deficient disease-associated mutant variants of mSWI/SNF complexes and evaluating their biochemical assembly, stoichiometry, and catalytic and nucleosome remodeling functions. Further, I aim to determine the impact of DNA-binding domain perturbations on mSWI/SNF complex genomic targeting, DNA accessibility generation, and subsequent gene expression in human cell contexts. This research will elucidate key features of DNA-binding domain-mediated SWI/SNF complex assembly, integrity, activities, and chromatin localization, linking these functions to resulting DNA accessibility and gene expression programs in human cells in normal and disease states. The mechanisms governing chromatin remodeling complex activities during basic cellular processes and in human disease remain incompletely understood, and with the highly frequent mutations in these processes observed in human cancers and neurodevelopmental conditions, such studies are uniquely pertinent and represent a high-impact priority for the field at-large.
