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.

摘要 最近单细胞技术的爆炸性增长揭示了细胞的多样性和复杂性 哺乳动物的神经系统。神经元由内在因素驱动发展其独特的特征，但也 必须适应当地的环境特点才能生产出功能电路。这就提出了一个问题：如何 在可变输入的背景下，神经元维持或恢复到稳定的细胞状态？有证据表明这一点 这一过程需要通过DNA甲基化和甲基结合蛋白MeCP2进行转录调控。这个 5-甲基胞嘧啶(MC)及其氧化形式5-羟甲基胞嘧啶(HMC)的水平在 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 在这些标记中，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在神经元中存在，在Rett综合征中常见突变。此外，Tet酶的突变导致了 最近发现将MC转化为HMC与神经系统疾病有关，这表明HMC可能 在大脑的基因调控中发挥关键的表观遗传学作用。尽管如此，我们对MC为什么 而且HMC模式对于单个细胞类型以及它们在大脑中的功能是独一无二的。我们最近的结果 另一些研究表明，HMC扮演着双重角色，既是MC抑制基因的上下文特异性激活因子，又是一种 神经元发育过程中的稳定抑制因子。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。与此同时，发育研究表明，神经元特异性的积聚 一种独特的非CG甲基化(MCH)以及HMC和MeCP2发生在出生后时期， 当神经元整合到回路中并完成其最终成熟为特定的功能亚型时。这 导致了一个有趣的假设，即MCH、HMC和MeCP2对于建立和维护 在微电路中有不同的、专门的神经元亚型。然而，理解MC的一个关键障碍是， HMC和MeCP2的S在大脑中的功能，以及对这一假说的检验，是缺乏可用的方法来 同时分析MC、HMC和基因组范围内单个细胞的基因表达。在此，我们建议 开发一种新的实验和计算方法来执行MC、HMC和基因表达的一体化 单细胞水平的分析。我们将把这种方法应用于视觉中的小白蛋白阳性中间神经元。 大脑皮层确定MC和HMC的模式在出生后神经元亚型指定和探索如何 在这些细胞中，MeCP2的破坏或挽救会影响最高细胞分辨率的基因调控。 总之，这些研究将为MeCP2在大脑中的功能提供关键的见解，同时开发一种新的 可用于全面评估独特的神经元甲基组及其对 正常和疾病状态下的转录。