EXPLORING CHROMATIN REMODELING THROUGH CHEMICALLY ENGINEERED DESIGNER CHROMATIN

Abstract

Brg/Brm Associated Factor (BAF) complexes are ATP-dependent chromatin remodelers that play an important role in governing genomic architecture and gene expression. These megadalton-sized molecular machines are found mutated in over 20% of human cancers. Therefore, understanding the mechanism of BAF-mediated remodeling holds promise for biomedical applications. In this thesis, we use chemically defined designer chromatins to decipher the mechanisms of BAF-mediated chromatin remodeling. First, using a DNA-barcoded nucleosome library containing various histone post-translational modifications (PTMs), mutations, and variants, we identified the chromatin features that affect the binding and remodeling activities of three BAF complexes. Importantly, we found that three BAF subfamilies, cBAF, PBAF, and ncBAF, have different preferences for different chromatin marks. Secondly, we investigated the role of nucleosome symmetry in chromatin remodeling activities. We developed an approach to synthesize asymmetric nucleosomes, which allows the orientation of a nucleosome to be tightly controlled relative to the underlying DNA sequence. Using this technology, we examined the functional consequence of asymmetry on BAF-mediated remodeling. Our results indicate that the asymmetric incorporation of cancer-associated histone mutations can reprogram the inherent activity of BAF chromatin remodeling to induce aberrant chromatin structure. Lastly, we introduced a FRET-based designer trinucleosome system to investigate a BAF-specific remodeling outcome, nucleosome eviction. Our current data suggest that BAF complex evicts histones from the nucleosome to which it is bound. We also found histone mutations at the nucleosome dimer-tetramer interface can specifically facilitate nucleosome eviction.