Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, USA.
Toxicology Program, North Carolina State University, Raleigh, North Carolina, USA.
J Biol Chem. 2022 Oct;298(10):102428. doi: 10.1016/j.jbc.2022.102428. Epub 2022 Aug 28.
The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.
甲基-CpG 结合域蛋白 2 和 3（MBD2 和 MBD3）为核小体重塑和去乙酰化酶（NuRD）复合物提供结构和 DNA 结合功能。这两种蛋白质形成不同的 NuRD 复合物，对甲基化 DNA 具有不同的结合亲和力和选择性。先前的研究表明，MBD2 与单个甲基化 CpG 二核苷酸具有高亲和力和选择性结合，而 MBD3 则没有。然而，NuRD 复合物在基因组中包含许多 CpG 二核苷酸（CpG 岛）的区域发挥作用。因此，在这项工作中，我们研究了 MBD2 和 MBD3 在更具生物学相关性的 DNA 模板上的结合和扩散，这些模板包含大的 CpG 岛或有限的 CpG 位点。我们使用单分子和生物物理分析的组合表明，MBD2 和 MBD3 都可以自由且快速地扩散穿过未甲基化的富含 CpG 的 DNA。相比之下，我们发现大的 CpG 岛的甲基化会捕获 MBD2，导致其在 CpG 岛上稳定且明显的静态结合，而 MBD3 则继续自由扩散。此外，我们证明了这两种蛋白质都能使 DNA 弯曲，而甲基化则增强了这种弯曲。总之，这些研究支持了一种模型，即 MBD2-NuRD 强烈定位于并浓缩甲基化的 CpG 岛，而 MBD3-NuRD 可以独立于甲基化状态自由移动核小体。
