Characterization of the Novel DNA Binding Activity of the BRG1 At-Hook-Bromodomain

Abstract

The packaging of eukaryotic DNA with histones into chromatin acts to both compact it into the nucleus as well as provide a mechanism for regulation of the genome. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes and dysregulation of these pathways is associated with a number of diseases. The BRG1/BRM associated factor (BAF) complex, is an ATP-dependent remodeling complex that plays an important role in gene regulation, several subunits of which are mutated in human cancers. The Brahma related gene 1 (BRG1) subunit of BAF, which provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. BDs are well-characterized readers of acetylated lysines on histones, and the BRG1-BD has been shown to bind H3K14ac. However, we have recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. In addition, we have demonstrated that an adjacent AT-hook contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity (Morrison et al., Nature Communications, 2017). Notably, the newly identified DNA binding interface harbors several cancer-associated mutations. Here we present our continued studies on this newly recognized composite DNA binding domain. We use systematic evolution of ligands by exponential enrichment (SELEX-seq) to generate a biophysical model of sequence specificity for the domain that we are exploring structurally using NMR spectroscopy. To determine the kinetic and thermodynamic basis of association we use biolayer interferometry (BLI). In addition, we are investigating the molecular mechanism of DNA binding and how it is altered in the context of the nucleosome. Together, our results are revealing the molecular details of how this novel DNA binding domain functions to navigate chromatin, and how dysregulation of the ATBD may impact BAF function.