Dissecting functional roles of MeCP2 condensates in neurons with chemogenetic tools

使用化学遗传学工具剖析 MeCP2 凝聚物在神经元中的功能作用

基本信息

批准号：
10669793
负责人：
Yin Shen
金额：
$ 20.19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10669793
关键词：
ATAC-seq Acceleration Address Affect Binding Binding Proteins Biochemical Biological Biological Process Biology CRISPR/Cas technology Cell Line Cell Nucleus Cell physiology Cells Cellular Assay Chromatin Chromatin Structure DNA Sequence Alteration DNA-Binding Proteins Defect Disease Engineering Etiology Euchromatin Gene Expression Gene Expression Regulation Genetic Genetic Transcription Genome Heterochromatin Hi-C Human Impairment In Situ In Vitro Kinetics Light Link Liquid substance Membrane Methods Methyl-CpG-Binding Protein 2 Molecular Monitor Mus Mutation Nervous System Neuronal Differentiation Neurons Nuclear Nuclear Proteins Patients Phase Phenotype Physical condensation Pluripotent Stem Cells Property Proteins Regulation Reporting Rett Syndrome Role Structure System Technology Testing Transcriptional Regulation Ubiquitin cell type chromatin remodeling embryonic stem cell epigenome excitatory neuron functional genomics genetic regulatory protein human embryonic stem cell induced pluripotent stem cell insight interest molecular phenotype mouse model mutant nervous system disorder new technology novel overexpression protein expression protein function recruit screening small molecule stem cell differentiation therapeutic target tool transcription factor transcriptome transcriptome sequencing transgene expression

项目摘要

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.

项目摘要通过染色质调节蛋白和转录因子形成核冷凝物是一种新的范式解释基因组分配和基因调节机制。最近的研究表明疾病 - DNA结合蛋白（例如MECP2）的相关突变与异常冷凝水有关形成并破坏异染色质结构域。但是，最新的相分离研究核蛋白仅限于体外生化研究和细胞中的过表达研究形成点或核体，不足以剖析内源性的分子功能疾病中的蛋白质冷凝物。为了解决这些问题，此R21应用程序的目的是开发用于操纵，监测和测试神经元中内源性MECP2的新型化学发生工具。具体而言，我们将开发出散热以溶解MECP2冷凝物和火花以恢复来自分化神经元中RETT综合征患者的MECP2突变体的凝结。通过测序分析，我们将进一步研究MECP2冷凝物在染色质调节，染色质结构，和基因调节。我们的研究将为研究核的生物学功能提供新技术神经系统中的冷凝水，并有助于揭开MECP2冷凝水之间的因果关系和RETT综合征病因。