Single-molecule Investigation of the Interaction Between MeCP2 and Chromatin
MeCP2 与染色质相互作用的单分子研究
基本信息
- 批准号:10606104
- 负责人:
- 金额:$ 4.68万
- 依托单位:
- 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:2022-09-01 至 2025-08-31
- 项目状态:未结题
- 来源:
- 关键词:AddressAffectAffinityBehaviorBindingBinding ProteinsBiophysicsCell NucleusChromatinChromosomesCollaborationsComplexCryoelectron MicroscopyCustomDNADNA MethylationDataDefectDiffuseDiffusionDiseaseEnvironmentExhibitsFemaleFluorescenceFluorescence MicroscopyGene ExpressionGenesHeterogeneityHistonesHumanIndividualInterventionInvestigationKineticsLinker DNAMapsMass Spectrum AnalysisMethodsMethyl-CpG-Binding Protein 2MethylationMolecular ConformationMotionMutateMutationNeuraxisNeuronsNucleosomesPatternPost-Translational Protein ProcessingPreparationProteinsReaderReportingResolutionRett SyndromeSamplingSiteSpecificityStochastic ProcessesStructureTechnologyTestingTimeTrainingVisualizationbasebiophysical propertieschromatin proteincrosslinkdensityfluorescence imagingimprovedinsightloss of functionloss of function mutationmotor regressionnervous system disordernoveloptical trapsparticlepreferenceprotein complexreal-time imagessingle moleculestructural biologytemporal measurement
项目摘要
PROJECT SUMMARY/ABSTRACT
The methyl-CpG binding protein 2 (MeCP2) is a highly abundant chromatin-binding protein that recognizes
methylated DNA to coordinate the expression of thousands of genes essential for neuronal function. Mutations
in MeCP2 cause Rett syndrome, a severe neurological disorder affecting one in every 10,000 females and
characterized by psychiatric and motor regression at 6-18 months. Although several treatments are available for
improving some isolated features of Rett syndrome, there are currently no approved therapies that directly
address the underlying defects of MeCP2 loss of function. One primary reason for the lack of MeCP2-targeted
interventions is an inadequate understanding of how MeCP2 reads methylated DNA within hierarchically
organized chromosomes. MeCP2 is known to bind to methylated DNA with a higher affinity than unmethylated
DNA, but how it navigates the main structural form of packaged DNA in the nucleus, nucleosomes, to reach its
canonical methyl-DNA substrate remains unclear. Over 80% of the MeCP2 protein is structurally disordered,
resulting in extensive conformational heterogeneity and binding plasticity that could underlie the regulatory
capacity of the protein, but simultaneously hamper detailed mechanistic characterization of the protein’s
chromatin-binding behavior. To this end, I used a single-molecule platform combining fluorescence microscopy
with optical trapping to directly observe the real-time trajectory and dynamics of individual MeCP2 on DNA and
nucleosomes. This approach enabled the visualization of how MeCP2 navigates the chromatin landscape
harboring methylated CpG sites. I discovered that MeCP2 exhibits long-range, diffusive behavior on bare DNA,
whereas methylation drastically suppresses such motions. Unexpectedly, I also found that MeCP2 preferentially
and stably binds nucleosomes irrespective of methylation status, suggesting that nucleosomes may serve to
regulate the availability of MeCP2 for its canonical methyl-reader activity. Based on my preliminary data, I
hypothesize that nucleosomes regulate the availability of MeCP2 for methyl-DNA recognition and biophysically
modify MeCP2 dynamics and binding configurations on DNA. To test this hypothesis, I will 1) characterize the
dynamics of MeCP2 on DNA with and without methylation, 2) examine the binding and biophysical interaction of
MeCP2 with nucleosomes wrapped with methylated or unmethylated DNA and investigate how Rett mutations
impact this interaction, and 3) determine the structural basis of the MeCP2-nuclesome interaction. Successful
completion of the proposed aims will provide novel insights into how MeCP2 biophysically navigates through a
complex chromatin environment to reach its canonical methyl-DNA substrate at unprecedented spatial and
temporal resolution. My report will establish an experimental framework for systematic interrogation of Rett
syndrome mutations and their impact on fundamental MeCP2-nucleosome and DNA interactions and dynamics.
项目摘要/摘要
甲基-CpG结合蛋白2(MeCP 2)是高度丰富的染色质结合蛋白,其识别
甲基化的DNA协调成千上万的基因的表达神经元功能所必需的。突变
MeCP 2导致Rett综合征,这是一种严重的神经系统疾病,每10,000名女性中就有一人受到影响,
其特征在于在6-18个月时精神和运动退化。虽然有几种治疗方法可用于
虽然Rett综合征的一些孤立特征得到改善,但目前还没有批准的治疗方法直接
解决MeCP 2功能丧失的潜在缺陷。缺乏MeCP 2靶向的一个主要原因是
干预措施是对MeCP 2如何在分层结构中读取甲基化DNA的理解不足。
有组织的染色体已知MeCP 2与甲基化DNA的结合亲和力高于未甲基化DNA
DNA,但它如何导航的主要结构形式的包装DNA在细胞核,核小体,以达到其
典型的甲基DNA底物仍不清楚。超过80%的MeCP 2蛋白质是结构无序的,
导致广泛的构象异质性和结合可塑性,这可能是调控的基础。
蛋白质的能力,但同时妨碍蛋白质的详细机制表征
染色质结合行为为此,我使用了单分子平台结合荧光显微镜
利用光学捕获直接观察DNA上单个MeCP 2的实时轨迹和动力学,
核小体这种方法使MeCP 2如何导航染色质景观的可视化成为可能
具有甲基化的CpG位点。我发现MeCP 2在裸DNA上表现出长距离扩散行为,
而甲基化显著抑制这种运动。出乎意料的是,我还发现MeCP 2优先
并稳定地结合核小体,而不管甲基化状态如何,这表明核小体可能有助于
调节MeCP 2的典型甲基阅读活性的可用性。根据初步数据,我
假设核小体调节MeCP 2对于甲基-DNA识别和生物药理学可用性
改变MeCP 2动力学和DNA上的结合构型。为了验证这一假设,我将1)描述
在有和没有甲基化的情况下,2)检查MeCP 2在DNA上的结合和生物物理相互作用,
MeCP 2与核小体包裹甲基化或未甲基化的DNA,并研究如何Rett突变
影响这种相互作用,和3)确定MeCP 2-核体相互作用的结构基础。成功
所提出的目标的完成将为MeCP 2如何通过生物途径导航提供新的见解。
复杂的染色质环境,以前所未有的空间和
时间分辨率我的报告将建立一个系统审讯雷特的实验框架
综合征突变及其对基本MeCP 2-核小体和DNA相互作用和动力学的影响。
项目成果
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Gabriella N. L. Chua的其他文献
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