Maurici Nicole, Phan Tien M, Henty-Ridilla Jessica L, Kim Young C, Mittal Jeetain, Bah Alaji
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, United States.
Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843, United States.
J Phys Chem B. 2025 Jun 12;129(23):5728-5743. doi: 10.1021/acs.jpcb.5c02741. Epub 2025 May 11.
Chromatin organization controls DNA's accessibility to regulatory factors to influence gene expression. Heterochromatin, or transcriptionally silent chromatin enriched in methylated DNA and methylated histone tails, self-assembles through multivalent interactions with its associated proteins into a condensed, but dynamic state. Liquid-liquid phase separation (LLPS) of key heterochromatin regulators, such as heterochromatin protein 1 (HP1), plays an essential role in heterochromatin assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most studied member of the methyl-CpG-binding domain (MBD) family of proteins, has been recently shown to undergo LLPS in the absence and presence of methylated DNA. These studies provide a new mechanistic framework for understanding the role of methylated DNA and its readers in heterochromatin formation. However, the details of the molecular interactions by which other MBD family members undergo LLPS to mediate genome organization and transcriptional regulation are not fully understood. Here, we focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent roles in gene regulation. Using an integrated computational and experimental approach, we uncover the homotypic and heterotypic interactions governing MBD2 and MBD3 phase separation and DNA's influence on this process. We show that despite sharing the highest sequence identity and structural homology among all the MBD protein family members, MBD2 and MBD3 exhibit differing residue patterns resulting in distinct phase separation mechanisms. Understanding the molecular underpinnings of MBD protein condensation offers insights into the higher-order, LLPS-mediated organization of heterochromatin.
染色质组织控制DNA对调控因子的可及性，从而影响基因表达。异染色质，即富含甲基化DNA和甲基化组蛋白尾巴的转录沉默染色质，通过与其相关蛋白的多价相互作用自组装成一种浓缩但动态的状态。关键异染色质调节因子（如异染色质蛋白1，HP1）的液-液相分离（LLPS）在异染色质组装和功能中起重要作用。甲基-CpG结合蛋白2（MeCP2）是甲基-CpG结合结构域（MBD）蛋白家族中研究最多的成员，最近已证明其在有无甲基化DNA的情况下都会发生LLPS。这些研究为理解甲基化DNA及其识别蛋白在异染色质形成中的作用提供了一个新的机制框架。然而，其他MBD家族成员通过LLPS介导基因组组织和转录调控的分子相互作用细节尚未完全了解。在这里，我们聚焦于两种MBD蛋白，MBD2和MBD3，它们在基因调控中具有不同但相互依赖的作用。我们采用综合计算和实验方法，揭示了控制MBD2和MBD3相分离以及DNA对该过程影响的同型和异型相互作用。我们表明，尽管MBD2和MBD3在所有MBD蛋白家族成员中具有最高的序列同一性和结构同源性，但它们表现出不同的残基模式，导致不同的相分离机制。理解MBD蛋白凝聚的分子基础有助于深入了解异染色质的高阶、LLPS介导的组织形式。
