The Molecular Basis of Specific DNA Binding by the BRG1 AT-hook and Bromodomain

Abstract

<jats:title>Abstract</jats:title><jats:p>The ATP-dependent BAF chromatin remodeling complex plays a critical role in gene regulation by modulating chromatin architecture, and is frequently mutated in cancer. Indeed, subunits of the BAF complex are found to be mutated in &gt;20% of human tumors. The mechanism by which BAF properly navigates chromatin is not fully understood, but is thought to involve a multivalent network of histone and DNA contacts. We previously identified a composite domain in the BRG1 ATPase subunit that is capable of associating with both histones and DNA in a multivalent manner. Mapping the DNA binding pocket revealed that it contains several cancer mutations. Here, we utilize SELEX-seq to identify the DNA specificity of this composite domain and NMR spectroscopy and molecular modelling to determine the structural basis of DNA binding. Finally, we demonstrate that cancer mutations in this domain alter the mode of DNA association.</jats:p>

Evolutionary Conservation of Structural and Functional Coupling between the BRM AT-Hook and Bromodomain

“…This may suggest that it is largely the overall charge, rather than sidechain-specific protein-DNA contacts, that is conserved. Notably, two Y residues in the ZA-loop, one of which we previously observed to form interactions in the major groove of DNA, 20 do show conservation.…”

“…We had previously found that the BRG1 AT-hook and BD span the minor and major grooves in binding to AT-rich DNA. 20 Our computational model of the complex suggested that a turn in the polypeptide at a linker proline (the C-terminal P of the PNPP element) is important for adopting this conformation. Here we find that the full PNPP element is important for promoting the conformation of the AT-L-BD in both the apo and DNA-bound states, and indeed all three prolines in the PNPP element of the linker are seen to be mutated in cancers.…”

“…We previously found that the BRG1/BRM AT-hook and BD form a composite DNA binding domain, 19 , 20 however the importance of this activity in BAF function is unclear. As conservation is a strong indicator of functional importance, we investigated the conservation of the DNA binding elements of this domain.…”

“…We found that this multi-component module has moderate preference for AT-rich DNA and that the AT-hook and BD span the minor and major grooves respectively. 20 These regions do not resolve in the recent BAF/nucleosome structures, but based on the position of the BRG1 C-terminus would clearly be positioned at the nucleosome interface near the H3 tail and entry DNA. 15 , 16 This suggests that they directly associate with the nucleosome substrate, however, the importance of DNA binding by the AT-hook and BD in BAF function has yet to be determined.…”

“… 17 – 19 In addition, we have recently shown that these BDs can bind to DNA. 19 , 20 BD binding to DNA is enhanced by the adjacent AT-hook, an intrinsically disordered DNA-binding motif. We found that this multi-component module has moderate preference for AT-rich DNA and that the AT-hook and BD span the minor and major grooves respectively.…”