Molecular mechanisms of nucleosome remodeling by the BAF chromatin remodeling complexes - ABSTRACT Genes encoding subunits of mammalian SWI/SNF (mSWI/SNF or BAF) chromatin remodeling complexes are mutated in over 20% of human cancers. Mutations impacting BAF subunits also cause neurodevelopment disorders, such as Coffin-Siris syndrome. Mammalian BAF complexes exist as three major complexes -- cBAF, PBAF, and ncBAF, which differ based on their distinct subunit compositions. Among these subunits, ARID1A, a cBAF-specific subunit, is the most frequently mutated gene (~9 % of all types of cancer). The majority of ARID1A mutations are frameshift or nonsense mutations, causing loss of function. Previous studies have suggested that ARID1A facilitates the formation of open chromatin domains, but a biochemical role for ARID1A in BAF remodeling activity has not been identified. We have recently established the in vitro reconstitution system of cBAF, PBAF, and ncBAF, and we compared their biochemical activities. In addition, we reconstituted a variety of cBAF subcomplexes, defining roles for several subunits in high affinity nucleosome binding and nucleosome sliding activity. Remarkably, we found that the ARID1A subunit of cBAF is largely dispensable for nucleosome binding, nucleosome sliding, and ATPase activity, but ARID1A is required for cBAF to transfer histone octamers between DNA templates. Our study revealed the novel biochemical function of ARID1A in cBAF-mediated chromatin remodeling and suggests that dysregulation of histone octamer transfer activity of BAF complexes may be relevant to cancer formation (Moro et al., Nucleic Acids Research, In press). Based on these recent findings by our group, we hypothesize that histone octamer transfer activity is a key biochemical activity of BAF complexes for chromatin regulation and cancer formation. The overall objective of this proposed research is to elucidate the molecular mechanisms of histone octamer transfer activity of BAF complexes and how histone octamer transfer activity of BAF complexes regulates chromatin organization and gene expression, by exploiting a powerful combination of biochemical and biophysical techniques including cryo-electron microscopy (EM), and genomics approaches in embryonic stem cells (ESCs). This proposal has two specific aims. Aim1 will dissect the molecular mechanisms of histone octamer transfer activity of BAF complexes. We have discovered that ARID1A is required for the histone octamer transfer activity of cBAF, but the molecular mechanism by which AIRD1A mediates histone octamer transfer activity is unclear. To obtain mechanistic insights into this, we will perform cryo-EM analysis of cBAF complex that lacks ARID1A (∆ARID1 complex). Furthermore, we found that the not only cBAF, but also PBAF and ncBAF, exhibit histone octamer transfer activity, suggesting that histone octamer transfer activity may be also a key biochemical activity of PBAF and ncBAF. We will elucidate the molecular mechanisms of the histone octamer transfer activity of PBAF and ncBAF. We showed that BAF subcomplex reconstituted with 8 core subunits shared among all BAF complexes lacks histone octamer transfer activity, demonstrating that PBAF- and ncBAF-specific subunit(s) is required for the histone octamer transfer activity of each complex. To identify subunit(s) required for the histone octamer transfer activity of PBAF and ncBAF, we will eliminate each of PBAF- and ncBAF-specific subunits from each complex using our reconstitution system and investigate the biochemical activities of the purified subcomplexes. Since PBAF and ncBAF-specific subunits (ARID2, PBRM1, BRD7, GLTSCR1, and BRD9) are frequently mutated in various cancers, this study will certainly uncover novel molecular functions of these subunits. We will also define the roles for the histone octamer transfer activity of BAF complexes in chromatin accessibility in ESCs using various genomics analyses, such as ATAC-seq and CUT&RUN. Aim2 will define the role for the SMARCE1 subunit in histone octamer transfer ac
