Dai Liuhan, Johnson-Buck Alexander, Walter Nils G
Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI 48109, USA.
bioRxiv. 2024 Sep 24:2024.09.22.614380. doi: 10.1101/2024.09.22.614380.
DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.
DNA甲基化是表观遗传调控的基本要素，由MBD蛋白超家族控制，该超家族是一组“读取器”，它们共享一个高度保守的甲基化CpG结合域（MBD），并介导染色质重塑因子的募集、转录调控以及DNA和组蛋白修饰的协调。先前的研究已经描述了含MBD的蛋白质对含有5mCpG二核苷酸的5-甲基胞嘧啶（5mC）回文序列的结合亲和力和序列选择性，这些回文序列通常被称为单对称甲基化CpG位点。然而，对于MBD结合如何受到甲基化CpG位点的典型局部聚集以及例如在DNA复制和转录过程中遇到的DNA结构基序的影响，人们知之甚少。在这里，我们使用通过平衡泊松采样的单分子动力学（SiMKEPS）来测量人类蛋白质MBD1的MBD与具有多种甲基化CpG位点模式和不同结构基序的DNA的精确结合和解离速率常数。MBD结合受其DNA底物的两个主要特性促进：1）双链DNA中的串联（连续）对称甲基化CpG位点和单链DNA中的二级结构；以及2）DNA叉。基于我们的发现，我们提出了一个关于MBD蛋白如何促进转录沉默和活跃基因组区域之间表观遗传边界维持的机制模型。
