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

基本信息

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
项目类别：
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

项目摘要

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，基因组作为三维综合单元起作用，其中不同的染色体占据了一个核内的不同领土或隔室，但是该组织的确切结构和功能后果仍然难以捉摸。我们假设遥远的染色质区域之间的物理相互作用确实以神经元特异性发生，并有助于在电路内建立独特的神经元表型和可塑性。结果，先前存在的三维（3-D）位置编码可能是全基因组整合数千个基因活性的因素，包括建立关键的表观遗传标记，并且可以是协同细胞复杂转录输出的机制之一。该提案的主要目的是（1）绘制突触耦合鉴定的神经元中细胞基因组的远程相互作用，以及（2）表征核基因组在标准学习测试和突触刺激之后核基因组的3-D重组的动力学。在这里，我们将使用Aplysia中更简单的防御电路的大型感觉，调节性和运动神经元，我们将在单个神经元的水平上实施一种新型的HI-C（染色体构象捕获）方法来探测整个基因组的3-D结构。该方法基于基于接近度的连接和选择性捕获具有大量平行测序的核的不同解剖区域的组合。因此，我们将在对照条件下以及经过良好建立的长期可塑性测试（例如5-HT应用）中绘制神经元基因组的互动区域。首先，基因组构象的这种空间映射将使我们能够公开表征不同相互相互作用染色质区室的单个核中的位置。其次，我们将将它们的位置（例如中心与周围）与基因的表达水平及其位于这些区室内的调节区域相关联。最后，我们将在基因调节区域内将所选基因的表达水平与5-氰甲基化模式（甲基化）相关联，重点是5-HT介导的信号转导的成分。该方法可以扩展到其他表观遗传标记（例如，分别将染色质免疫沉淀用于选择性组蛋白后，分别作为激活和抑制标记），以探测突触输入或药物给药后神经元积分活性的机制。该范式可以作为一个有力的概念验证平台，以表征这种最难以捉摸的细胞和基因组过程的机制，从而导致神经元的综合活性，对从药物滥用机制到记忆研究的基本和临床研究具有广泛的影响。公共卫生相关性：了解功能表征神经元内DNA甲基化和转录单元的空间组织对于理解神经元综合活性的机制至关重要。实际上，在每个细胞中，基因组作为三维综合实体起作用，其中不同的染色体占据了核内的不同领土或隔室。然而，确实发生了遥远染色质区域之间的物理相互作用，以调节基因活性，形成表观遗传标记并协调细胞的复杂转录输出。在这里，我们将表征细胞基因组远程相互作用的3-D结构及其在学习和记住时独特地识别的神经元中的动力学。有关核基因组中位置编码的信息对于开发针对与药物滥用和记忆丧失相关的广泛病理过程的有针对性疗法至关重要。