Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein critical for normal brain function, and both depletion and overexpression of MeCP2 lead to severe neurodevelopmental disease, Rett syndrome (RTT) and MECP2 multiplication disorder, respectively. However, the molecular mechanism by which abnormal MeCP2 dosage causes neuronal dysfunction remains unclear. As MeCP2 expression is nearly equivalent to that of core histones and because it binds DNA throughout the genome, one possible function of MeCP2 is to regulate the 3D structure of chromatin. Here, to examine whether and how MeCP2 levels impact chromatin structure, we used high-resolution confocal and electron microscopy and examined heterochromatic foci of neurons in mice. Using models of RTT and MECP2 triplication syndrome, we found that the heterochromatin structure was significantly affected by the alteration in MeCP2 levels. Analysis of mice expressing either MeCP2-R270X or MeCP2-G273X, which have nonsense mutations in the upstream and downstream regions of the AT-hook 2 domain, respectively, showed that the magnitude of heterochromatin changes was tightly correlated with the phenotypic severity. Postnatal alteration in MeCP2 levels also induced significant changes in the heterochromatin structure, which underscored importance of correct MeCP2 dosage in mature neurons. Finally, functional analysis of MeCP2-overexpressing mice showed that the behavioral and transcriptomic alterations in these mice correlated significantly with the MeCP2 levels and occurred in parallel with the heterochromatin changes. Taken together, our findings demonstrate the essential role of MeCP2 in regulating the 3D structure of neuronal chromatin, which may serve as a potential mechanism that drives pathogenesis of MeCP2-related disorders.

SIGNIFICANCE STATEMENT Neuronal function is critically dependent on methyl-CpG binding protein 2 (MeCP2), a nuclear protein abundantly expressed in neurons. The importance of MeCP2 is underscored by the severe childhood neurologic disorders, Rett syndrome (RTT) and MECP2 multiplication disorders, which are caused by depletion and overabundance of MeCP2, respectively. To clarify the molecular function of MeCP2 and to understand the pathogenesis of MECP2-related disorders, we performed detailed structural analyses of neuronal nuclei by using mouse models and high-resolution microscopy. We show that the level of MeCP2 critically regulates 3D structure of heterochromatic foci, and this is mediated in part by the AT-hook 2 domain of MeCP2. Our results demonstrate that one primary function of MeCP2 is to regulate chromatin structure.

甲基-CpG 结合蛋白 2 (MeCP2) 是一种对正常脑功能至关重要的核蛋白，MeCP2 的消耗和过表达分别导致严重的神经发育疾病、Rett 综合征 (RTT) 和MECP2增殖障碍。然而，异常 MeCP2 剂量导致神经元功能障碍的分子机制仍不清楚。由于 MeCP2 的表达几乎等同于核心组蛋白的表达，并且因为它在整个基因组中结合 DNA，MeCP2 的一个可能功能是调节染色质的 3D 结构。在这里，为了检查 MeCP2 水平是否以及如何影响染色质结构，我们使用高分辨率共聚焦和电子显微镜检查了小鼠神经元的异色病灶。使用 RTT 和MECP2模型triplication 综合征，我们发现异染色质结构受到 MeCP2 水平改变的显着影响。对表达 MeCP2-R270X 或 MeCP2-G273X 的小鼠的分析分别在 AT-hook 2 域的上游和下游区域具有无义突变，表明异染色质变化的幅度与表型严重程度密切相关。MeCP2 水平的出生后改变也引起异染色质结构的显着变化，这强调了正确 MeCP2 剂量在成熟神经元中的重要性。最后，对 MeCP2 过表达小鼠的功能分析表明，这些小鼠的行为和转录组改变与 MeCP2 水平显着相关，并且与异染色质变化同时发生。综合起来，

意义声明神经元功能严重依赖于甲基-CpG 结合蛋白 2 (MeCP2)，这是一种在神经元中大量表达的核蛋白。严重的儿童神经系统疾病、Rett 综合征 (RTT) 和MECP2增殖障碍强调了 MeCP2 的重要性，这些疾病分别由 MeCP2 的消耗和过量引起。阐明MeCP2的分子功能，了解MECP2的发病机制相关疾病，我们通过使用小鼠模型和高分辨率显微镜对神经元核进行了详细的结构分析。我们表明，MeCP2 的水平严格调节异色病灶的 3D 结构，这部分是由 MeCP2 的 AT-hook 2 域介导的。我们的结果表明，MeCP2 的一个主要功能是调节染色质结构。