University of Iowa, Carver College of Medicine, Department of Biochemistry, Iowa City, IA 52242, United States.
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, United States; Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR 97403, United States.
J Mol Biol. 2021 Jul 9;433(14):166845. doi: 10.1016/j.jmb.2021.166845. Epub 2021 Feb 2.
The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.
BAF 染色质重塑复合物对于基因组调控至关重要。BAF 的中央 ATP 酶要么是 BRM，要么是 BRG1，两者都包含一个 C 端溴结构域，已知与乙酰化赖氨酸结合。我们最近的研究表明，除了与乙酰赖氨酸结合外，BRG1/BRM 溴结构域还可以通过与乙酰赖氨酸结合口袋相邻的富含赖氨酸/精氨酸的补丁与 DNA 结合。溴结构域侧翼是一个 AT 钩，由一个短的富含脯氨酸的接头隔开。我们之前发现 AT 钩和溴结构域可以以多价的方式与 DNA 结合。在这里，我们研究了这个复合模块的保守性，发现 AT 钩、接头和富含赖氨酸/精氨酸的溴结构域补丁是古老的，在大约 10 亿年的时间里保持保守。我们利用广泛的诱变、NMR 光谱学和荧光各向异性来剖析每个保守元件在该模块与 DNA 结合中的作用。我们的结果揭示了接头介导的 AT 钩和溴结构域之间的结构和功能偶联。溴结构域上的富含赖氨酸/精氨酸补丁和 AT 钩的保守元件对于与 DNA 具有强大的亲和力至关重要，而接头的保守元件对于整体 DNA 亲和力不是必需的，但对于在与 DNA 结合时保持 AT 钩和溴结构域的相对构象至关重要。这支持了 AT 钩和溴结构域的偶联作用对于 BAF 活性很重要。
