Spatial Organization of the Genome in Identified Neurons of Memory Circuits

已识别的记忆回路神经元基因组的空间组织

• 批准号: 8080501
• 负责人: LEONID L MOROZ
• 金额: $ 17.36万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8080501
• 关键词: 3-Dimensional; Afferent Neurons; Animals; Aplysia; Appearance; Architecture; Award; Basic Science; Cell Nucleus; Cells; Cellular biology; Chromatin; Chromosome Territory; Chromosomes; Chromosomes, Human, 6-12 and X; Clinical Research; Code; Complex; Computational algorithm; Coupled; Cytosine; DNA; DNA Methylation; Data; Distant; Drug abuse; Epigenetic Process; Event; Gene Cluster; Gene Expression; Gene Expression Profile; Genes; Genome; Genome Mappings; Genomics; Gills; Global Change; Histones; Individual; Lead; Learning; Ligation; Location; Logic; Maps; Mediating; Memory; Memory Loss; Methodology; Methods; Methylation; Microdissection; Modeling; Molecular Conformation; Motor; Motor Neurons; Neurons; Neurosciences; Neurotransmitters; Nuclear; Nucleic Acid Regulatory Sequences; Nucleosomes; Output; Pathologic Processes; Pattern; Peripheral; Pharmaceutical Preparations; Phenotype; Positioning Attribute; Post-Translational Protein Processing; Process; Protocols documentation; Reflex action; Repression; Research; Sampling; Sensory; Serotonin; Signal Transduction; Synapses; System; Techniques; Testing; Withdrawal; Work; base; chromatin immunoprecipitation; demethylation; genome-wide; histone modification; long term memory; mammalian genome; memory process; novel; operation; polarized cell; programs; public health relevance; response

DESCRIPTION (provided by applicant): In every cell the genome operates as a three-dimensional integrative unit where different chromosomes occupy distinct territories or compartments within a nucleus but where the precise architecture and functional consequences of such organization remain elusive. We hypothesize that physical interactions between distant chromatin regions do occur in a neuron-specific manner and contribute to establishment of unique neuronal phenotypes and plasticity within a circuit. As a result, the preexisting three-dimensional (3-D) position coding can be a factor in genome-wide integration of the activity of thousands of genes, including establishing crucial epigenetic marks, and can be one of the mechanisms coordinating the complex transcriptional output of a cell. The major aims of this proposal are (1) to map long-range interactions of the cellular genome in synaptically coupled identified neurons and, (2) to characterize the dynamics of the 3-D reorganization of the nuclear genome following standard learning tests and synaptic stimulation. Here, using large accessible sensory, modulatory and motor neurons of the simpler defensive circuit in Aplysia, we will implement a novel Hi-C (chromosome conformation capture) approach to probe the 3-D architecture of the whole genome at the level of single neurons. The method is based on the combination of proximity-based ligation and selective capture of distinct anatomical regions of a nucleus with massive parallel sequencing. Thus, we will map interactive regions of the neuronal genome both in control conditions and following well established long-term plasticity tests (such as 5-HT applications). First, such spatial mapping of the genome conformation will allow us to unbiasedly characterize the location within a single nucleus of distinct mutually interacting chromatin compartments. Second, we will correlate their positions (e.g. central vs. peripheral) to the expression level of genes and their regulatory regions located within these compartments. Finally, we will correlate the expression level of selected genes with 5-cytosine methylation patterns (methylome) within gene regulatory regions, focusing upon components of 5-HT mediated signal transduction. This approach can be extended to other epigenetic marks (e.g. using chromatin immunoprecipitation for selective histone posttranslational modification events as activation and repression marks respectively) to probe mechanisms of integrative activity of neurons following synaptic inputs or drug administration. This paradigm can serve as a powerful proof-of-concept platform to characterize mechanisms of this most elusive cellular and genomic process leading to integrative activity of neurons, with broad implications to fundamental and clinical studies from drug abuse mechanisms to memory research. PUBLIC HEALTH RELEVANCE: Knowing the spatial organization of DNA methylation and transcriptional units within functionally characterized neurons is crucial for understanding the mechanisms of integrative activity of neurons. Indeed, in every cell the genome operates as a three-dimensional integrative entity where different chromosomes occupy distinct territories or compartments within a nucleus. Yet physical interactions between distant chromatin regions do occur to regulate gene activity, form epigenetic marks and coordinate complex transcriptional output of a cell. Here, we will characterize the 3-D architecture of long-range interactions of the cellular genome and its dynamics in uniquely identified neurons as they learn and remember. Information about positional coding within the nuclear genome is central to developing targeted therapies for the broad spectrum of pathological processes associated with drug abuse and memory loss.

描述（由申请人提供）：在每个细胞中，基因组作为一个三维整合单位运作，其中不同的染色体占据细胞核内的不同区域或区室，但这种组织的精确结构和功能结果仍然难以捉摸。我们假设，远距离染色质区域之间的物理相互作用确实以神经元特异性的方式发生，并有助于建立独特的神经元表型和回路内的可塑性。因此，预先存在的三维（3-D）位置编码可以是数千个基因的活性的全基因组整合的因素，包括建立关键的表观遗传标记，并且可以是协调细胞的复杂转录输出的机制之一。该建议的主要目的是（1）映射突触耦合识别的神经元中细胞基因组的长程相互作用，以及（2）表征标准学习测试和突触刺激后核基因组的3-D重组的动力学。在这里，使用大的可访问的感觉，调节和运动神经元的简单防御电路在Aaplasia，我们将实施一种新的Hi-C（染色体构象捕获）的方法来探测的3-D架构的整个基因组在单个神经元的水平。该方法是基于基于邻近的连接和选择性捕获不同的解剖区域的细胞核与大规模并行测序的组合。因此，我们将映射神经元基因组的相互作用区域，无论是在控制条件下，并遵循良好的长期可塑性测试（如5-HT应用）。首先，这样的空间映射的基因组构象将使我们能够无偏地表征在一个不同的相互作用的染色质室的单个核内的位置。其次，我们将它们的位置（例如中央与外周）与基因的表达水平及其位于这些隔室中的调控区相关联。最后，我们将选定的基因的表达水平与5-胞嘧啶甲基化模式（甲基化组）内的基因调控区域，专注于5-HT介导的信号转导的组件。这种方法可以扩展到其他表观遗传标记（例如，使用染色质免疫沉淀选择性组蛋白翻译后修饰事件作为激活和抑制标记分别），以探测突触输入或药物给药后神经元整合活性的机制。这种范式可以作为一个强大的概念验证平台，以表征这种最难以捉摸的细胞和基因组过程导致神经元整合活动的机制，对从药物滥用机制到记忆研究的基础和临床研究具有广泛的影响。 公共卫生相关性：了解功能特征神经元内DNA甲基化和转录单位的空间组织对于理解神经元整合活动的机制至关重要。事实上，在每个细胞中，基因组都是一个三维整合实体，不同的染色体占据细胞核内不同的区域或隔室。然而，遥远的染色质区域之间确实发生了物理相互作用，以调节基因活性，形成表观遗传标记，并协调细胞复杂的转录输出。在这里，我们将描述细胞基因组的远程相互作用的3-D架构及其在唯一识别的神经元中的动态，因为它们学习和记忆。关于核基因组内位置编码的信息对于开发与药物滥用和记忆丧失相关的广泛病理过程的靶向疗法至关重要。