Transcriptional condensates, epigenetic editing and Rett Syndrome

摘要 Rett综合征（RTT）是一种出生后进行性神经发育障碍， 残疾和自闭症样综合征，表现在女童在幼儿期，是由突变的 X连锁DNA结合蛋白MeCP 2（甲基CpG结合蛋白2）。携带Mecp 2无效等位基因的小鼠 与患者的症状非常相似，是疾病的忠实模型。重要的是，发展 通过以下方法纠正突变型Mecp 2等位基因后，成人中的RTT样症状可以减缓甚至停止： 转基因介导的MeCP 2表达。 MeCP 2是神经元中最丰富的蛋白质之一，大多数致病突变聚集在神经元中。 DNA结合结构域（MBD）和转录抑制结构域（TRD）。然而， MeCP 2仍然是个谜，已经提出了两个主要的假设：（i）MeCP 2作为 转录或（ii）作为转录激活因子。显然，这些建议的功能都不能完全解释 MeCP 2缺陷或过表达导致RTT或MECA 2复制的复杂表型 综合征基于我们的初步证据，我们假设MeCP 2的主要功能可能是 通过冷凝物形成调节3D染色体结构。 常染色质和异染色质的组分都可以形成相分离的凝聚物， 提供了一种在细胞内划分和集中生化反应的机制， 通过由蛋白质的固有无序区（IDR）驱动的液-液相分离产生。MeCP2 蛋白质含有一个大的IDR，我们已经获得了初步证据，MeCP 2参与阶段- 分离的异染色质浓缩物。因此，除了MeCP 2作为基因的阻遏物或激活物的作用之外， 表达，该蛋白质可能在细胞生理学和疾病中具有更广泛和更复杂的作用。 在这个项目中，我们将定义MeCP 2对异色和常色凝聚体的贡献， 正常和突变的神经元，并分析RTT引起的突变对LLPS的影响。我们的目标是 深入了解MeCP 2的功能，作为设计新型治疗方法的基础。潜在的新 基于这一假设的治疗需要时间才能发展成应用。去探索一个更直接的 我们将使用表观遗传编辑作为治疗工具来激活位于 在不活跃的X染色体上最重要的是，表观遗传编辑将使MeCP 2表达恢复到完全相同的水平。 野生型水平，从而避免MeCP 2过表达的毒性后果。相反，其他战略 例如使用载体介导的MeCP 2转导将总是产生过表达MeCP 2的细胞， 因此将导致严重的副作用，如在患有MECP 2复制综合征的患者中所见。

Abstract Rett syndrome (RTT) is a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like syndromes that manifests in girls during early childhood, and is caused by mutation of the X-linked DNA binding protein MeCP2 (Methyl CpG-binding Protein 2). Mice carrying null alleles of Mecp2 closely mimic symptoms seen in patients and are faithful models of the disease. Importantly, development of RTT-like symptoms can be slowed or even halted in the adult following correction of a mutant Mecp2 allele by transgene-mediated MeCP2 expression. MeCP2 is one of the most abundant proteins in neurons, and most disease-causing mutations cluster in the DNA binding domain (MBD) and in the transcription repression domain (TRD). However, the function of MeCP2 remains enigmatic, with two major hypotheses having been proposed: (i) MeCP2 acts as repressor of transcription or (ii) as an activator of transcription. Clearly, none of these proposed functions can fully explain the complex phenotype of MeCP2 deficiency or overexpression leading to RTT or MECP2 Duplication Syndrome. Based on our preliminary evidence we postulate that MeCP2’s primary function may be to modulate the 3D chromosome architecture through condensate formation. Components of both euchromatin and heterochromatin can form phase-separated condensates, which provide a mechanism to compartmentalize and concentrate biochemical reactions within cells and are produced by liquid-liquid phase separation driven by intrinsically disordered regions (IDRs) of proteins. MeCP2 protein contains a large IDR and we have obtained preliminary evidence that MeCP2 is involved in phase- separated heterochromatin condensates. Thus, beyond MeCP2’s role as a repressor or activator of gene expression, the protein may have a much wider and more complex role in the cell physiology and disease. In this project we will define the contribution of MeCP2 to heterochromatic and euchromatic condensates in normal and mutant neurons and analyze the effect of RTT causing mutations on LLPS. Our goal is to gain insights into the function of MeCP2 as the basis for designing novel therapeutic approaches. Potential new therapies based on this hypothesis will take time to develop into applications. To explore a more immediate approach we will use epigenetic editing as a therapeutic tool to activate the inactive wt MECP2 allele located on the inactive X chromosome. Most importantly, epigenetic editing will restore MeCP2 expression to exactly wild type levels and thus avoid toxic consequences of MeCP2 overexpression. In contrast, other strategies such as using vector-mediated MeCP2 transduction will invariably produce cells that overexpress MeCP2 and thus will result in serious side effects as seen in patients with MECP2 duplication syndrome.