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

다국어 초록 (Multilingual Abstract)

The packaging of eukaryotic DNA with histones into chromatin acts to compact DNA into the nucleus and provides a mechanism for regulation of DNA-templated processes. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes. The BRG1/BRM associated factor (BAF) complex, is an ATP-dependent chromatin remodeling complex that plays a role in gene regulation. 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, it was recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. My studies have demonstrated that an adjacent AT-hook motif contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity. The systematic evolution of ligands by exponential enrichment (SELEX-seq) was applied to generate a biophysical model of sequence specificity for the BRG1 ATBD. I used biolayer interferometry (BLI) to determine the kinetic and thermodynamic basis of association of the ATBD with DNA and NMR spectroscopy to study the molecular basis of this association. These studies culminated in a structural model that exhibits the AT-hook embedded in the minor groove of DNA and tethering the BD to the major groove of DNA. Together my results dissected the contribution of the BRG1-ATBD to the targeting of BAF to chromatin, and these findings are paving the way to investigate the functional implications of the DNA binding activity of the BRG1 ATBD in the context of BAF.