Single-cell approaches to probe the function of the unique neuronal epigenome

单细胞方法探测独特神经元表观基因组的功能

• 批准号: 10440762
• 负责人: John R Edwards
• 金额: $ 19.69万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10440762
• 关键词: Adult; Affect; Binding; Binding Proteins; Brain; Cell Shape; Cells; Cerebral cortex; CpG dinucleotide; Cues; Cytosine; DNA Methylation; DNA Methylation Regulation; DNA analysis; DNA methylation profiling; Data; Defect; Development; Disease; Early Onset Alzheimer Disease; Ensure; Enzymes; Epigenetic Process; Explosion; Frontotemporal Dementia; Future; Gene Activation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Transcription; Individual; Intellectual functioning disability; Interneurons; Intrinsic factor; Joints; Knock-out; Machine Learning; Maintenance; Maps; Measures; Mediating; Methods; Methyl-CpG-Binding Protein 2; Methylation; Molecular; Mus; Mutate; Mutation; Neonatal; Nervous system structure; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Oxides; Parvalbumins; Pathology; Pathway interactions; Pattern; Play; Process; Property; Proteins; Reader; Regulation; Regulatory Pathway; Repression; Research Personnel; Resolution; Rett Syndrome; Role; Signal Transduction; Techniques; Technology; Testing; Therapeutic; Time; Tissue-Specific Gene Expression; Transcription Regulatory Protein; Transcriptional Regulation; Variant; Visual Cortex; Writing; autism spectrum disorder; autistic; base; bisulfite sequencing; cell type; demethylation; epigenome; gene function; genome-wide; genomic profiles; insight; mammalian genome; methylome; nervous system disorder; neurodevelopment; neuron development; neuronal circuitry; neuroprotection; new technology; novel; novel strategies; novel therapeutic intervention; novel therapeutics; oxidation; postnatal; postnatal development; postnatal period; programs; promoter; response; single cell analysis; trait; transcriptome; transcriptome sequencing; transcriptomics

Abstract The recent explosion of single cell techniques has revealed striking cellular diversity and complexity in the mammalian nervous system. Neurons are driven to develop their unique features by intrinsic factors, but also must adapt to local environmental features to produce functional circuits. This raises the question: how do neurons maintain or return to stable cellular states in the context of variable inputs? Evidence suggests that this process requires transcriptional regulation by DNA methylation and the methyl-binding protein MeCP2. The levels of 5-methylcytosine (mC) and its oxidized form, 5-hydroxymethylcytosine (hmC), dramatically change in the developing mammalian brain in response to intrinsic cues and environmental input, and the genomic profiles of these marks appear to show unique patterns in different cell-types. MeCP2 modulates the effects of mC and hmC in neurons and is commonly mutated in Rett syndrome. Further, mutations in the TET enzymes responsible for converting mC to hmC have been recently associated with neurological disorders, indicating that hmC may play a key epigenetic role in gene regulation in the brain. Despite this, we have little understanding of why mC and hmC patterns are unique to individual cell-types and how they function in the brain. Recent results from us and others indicate that hmC plays a dual role as a context-specific activator of mC repressed genes and as a stable repressor during neuronal development. Emerging evidence further indicates MeCP2 as an important reader of mC and hmC signals. Meanwhile, developmental studies have shown that a neuron-specific buildup of a unique form of non-CG methylation (mCH), as well as hmC and MeCP2 occurs during the postnatal period, when neurons integrate into circuits and complete their final maturation into specific functional subtypes. This leads to the intriguing hypothesis that mCH, hmC, and MeCP2 are critical for the establishment and maintenance of diverse, specialized neuronal subtypes within microcircuits. However, a critical barrier in understanding mC, hmC, and MeCP2’s function in the brain, and to testing this hypothesis, is the lack of available methods to simultaneously analyze mC, hmC, and gene expression genome-wide in individual cells. Here we propose to develop a novel experimental and computational approach to perform integrative mC, hmC, and gene expression analysis at the single-cell level. We will apply this method to parvalbumin positive interneurons in the visual cortex to determine the patterns of mC and hmC during postnatal neuronal subtype specification and probe how disruption or rescue of MeCP2 in these cells impacts gene regulation at the highest levels of cellular resolution. Together these studies will provide key insights into the function of MeCP2 in the brain, while developing a new technology that can be used to comprehensively assess the unique neuronal methylome and its impact on transcription in normal and disease states.

抽象的 最近单细胞技术的爆炸性发展揭示了惊人的细胞多样性和复杂性 哺乳动物的神经系统。神经元受到内在因素的驱动而发展其独特的特征，但也 必须适应当地的环境特征来生产功能电路。这就提出了一个问题：如何 神经元在可变输入的情况下维持或返回稳定的细胞状态？有证据表明，这 该过程需要 DNA 甲基化和甲基结合蛋白 MeCP2 的转录调节。这 5-甲基胞嘧啶 (mC) 及其氧化形式 5-羟甲基胞嘧啶 (hmC) 的水平显着变化 发育中的哺乳动物大脑对内在线索和环境输入的反应，以及基因组图谱 这些标记似乎在不同的细胞类型中显示出独特的模式。 MeCP2 调节 mC 和 hmC 存在于神经元中，并且在 Rett 综合征中常见突变。此外，TET 酶的突变负责 最近发现将 mC 转化为 hmC 与神经系统疾病有关，表明 hmC 可能 在大脑基因调控中发挥关键的表观遗传作用。尽管如此，我们对为什么 mC 知之甚少 hmC 模式对于个体细胞类型及其在大脑中的功能而言是独特的。我们最近的结果 等人表明 hmC 发挥双重作用，作为 mC 抑制基因的特定环境激活剂和作为 神经元发育过程中的稳定阻遏物。新出现的证据进一步表明 MeCP2 是一种重要的 mC 和 hmC 信号的读取器。同时，发育研究表明，神经元特异性的积累 一种独特形式的非 CG 甲基化 (mCH) 以及 hmC 和 MeCP2 在产后发生， 当神经元整合到电路中并最终成熟为特定的功能亚型时。这 引出了一个有趣的假设：mCH、hmC 和 MeCP2 对于建立和维持至关重要 微电路内不同的、专门的神经元亚型。然而，理解 mC 的一个关键障碍是 hmC 和 MeCP2 在大脑中的功能，并且为了检验这一假设，缺乏可用的方法来 同时分析单个细胞中的 mC、hmC 和全基因组基因表达。在此我们建议 开发一种新的实验和计算方法来执行综合 mC、hmC 和基因表达 单细胞水平的分析。我们将将此方法应用于视觉中的小白蛋白阳性中间神经元 皮层确定出生后神经元亚型规范期间 mC 和 hmC 的模式并探讨如何 这些细胞中 MeCP2 的破坏或拯救会影响细胞分辨率最高水平的基因调控。 这些研究将共同​​提供关于 MeCP2 在大脑中的功能的关键见解，同时开发一种新的 可用于全面评估独特的神经元甲基化及其对神经元的影响的技术 正常和疾病状态下的转录。