MeCP2-induced heterochromatin organization is driven by oligomerization-based liquid–liquid phase separation and restricted by DNA methylation

Heterochromatin is the highly compacted form of chromatin with various condensation levels hallmarked by high DNA methylation. MeCP2 is mostly known as a DNA methylation reader but has also been reported as a heterochromatin organizer. Here, we combine liquid–liquid phase separation (LLPS) analysis and single-molecule tracking with quantification of local MeCP2 concentrations <i>in vitro</i> and <i>in vivo</i> to explore the mechanism of MeCP2-driven heterochromatin organization and dynamics. We show that MeCP2 alone forms liquid-like spherical droplets via multivalent electrostatic interactions and with isotropic mobility. Crowded environments and DNA promote MeCP2 LLPS and slow down MeCP2 mobility. DNA methylation, however, restricts the growth of heterochromatin compartments correlating with immobilization of MeCP2. Furthermore, MeCP2 self-interaction is required for LLPS and is disrupted by Rett syndrome mutations. In summary, we are able to model the heterochromatin compartmentalization as well as MeCP2 concentration and heterogeneous motion in the minimal <i>in vitro</i> system.

异染色质（Heterochromatin）是染色质的高度压缩形式，具有不同程度的压缩状态，其标志性特征为高水平的DNA甲基化。甲基化CpG结合蛋白2（MeCP2）通常被视为DNA甲基化阅读器，同时也有研究表明其可作为异染色质组织者。本研究结合液-液相分离（liquid–liquid phase separation, LLPS）分析、单分子追踪技术，以及对体外（in vitro）和体内（in vivo）环境下MeCP2局部浓度的定量检测，以探究MeCP2介导的异染色质组装机制与动态变化过程。研究结果表明，单独存在的MeCP2可通过多价静电相互作用形成类液状球形液滴，并呈现各向同性的运动特性。拥挤环境与DNA可促进MeCP2的液-液相分离，并减慢其运动速率。然而，DNA甲基化会限制异染色质区室的扩张，这一过程与MeCP2的固定化密切相关。此外，MeCP2的自身相互作用是其发生液-液相分离的必要条件，且该相互作用可被雷特综合征（Rett syndrome）相关突变所破坏。综上，本研究可在最简体外实验体系中，构建异染色质区室化过程以及MeCP2浓度与异质性运动的模型。