Dissecting functional roles of MeCP2 condensates in neurons with chemogenetic tools

项目摘要 染色质调节蛋白和转录因子形成核凝集物是一种新的研究范式 解释基因组分割和基因调控机制。最近的研究表明，疾病-- DNA结合蛋白的相关突变，如MeCP2，与异常凝集液相关 异染色质结构域的形成和破坏。然而，目前对相分离的最新研究 核蛋白仅限于体外生化研究和细胞中的过度表达研究，表明 点状或核体的形成，不足以剖析内源的分子功能。 蛋白质在疾病中凝聚。为了解决这些问题，此R21应用程序的目标是开发 用于操纵、监测和测试神经元内源性MeCP2凝聚物的新型化学发生工具。 具体地说，我们将开发火花技术来溶解MeCP2凝析油，并开发火花技术来恢复 Rett综合征患者MeCP2突变体在分化神经元中的凝聚。通过测序 分析，我们将进一步研究MeCP2缩合物在染色质调节、染色质结构、 和基因调控。我们的研究将为研究核的生物学功能提供新的技术 神经系统中的冷凝物，有助于揭开MeCP2冷凝物之间的因果关系的神秘面纱 和Rett综合征病因学。

Project Summary Nuclear condensate formation by chromatin regulatory proteins and transcription factors is a new paradigm for explaining genome partitioning and gene regulation mechanisms. Recent studies showed that disease- associated mutations of DNA-binding protein, such as MeCP2, are associated with aberrant condensate formation and disrupting the heterochromatin domains. However, the most current studies of phase-separation by nuclear proteins are limited to in vitro biochemical studies and overexpression studies in cells showing the formation of puncta or nuclear bodies, which are insufficient to dissect the molecular function of endogenous protein condensates in disease. To address these issues, the objective of this R21 application is to develop novel chemogenetic tools for manipulating, monitoring, and testing endogenous MeCP2 condensates in neurons. Specifically, we will develop SPARK-OFF to dissolve MeCP2 condensates and SPARK-ON to restore condensation for MeCP2 mutants from Rett Syndrome patients in differentiated neurons. Through sequencing analysis, we will further investigate the roles of MeCP2 condensates in chromatin regulation, chromatin structure, and gene regulation. Our study will provide new technologies for studying the biological function of nuclear condensates in the nervous system and help demystify the causal relationship between MeCP2 condensates and Rett Syndrome etiology.