Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds)DNA-binding activity in addition to their normal histone assocaiatd function. Here, we highlight the advantages of using protein-observed 19F NMR (PrOF NMR)  to characterize a new interaction a dsDNA with the N-terminal bromodomain of the Bromodomain and Extra-Terminal (BET) protein, BRD4. These studies are further complemented with <sup>1</sup>H-<sup>15</sup>N HSQC NMR, an electrophoretic mobility shift assay (EMSA), and competitive-inhibition assays.  Through these studies we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated-histone (KAc) binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. In this talk, we will further describe higher affinity interactions using tandem N-terminal bromodomains of both BRD4 and BRDT indicating a new role of the large unstructured linker in between the bromodomains in chromatin recognition. Together, these studies highlight PrOF NMR as a rapid structure-based tool to characterize biomolecular interactions.  Our findings help to establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin recognition mechanisms of the BRD4 protein for regulating gene expression.

Alternative mechanisms for DNA engagement by BET bromodomain-containing proteins

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ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.