Spatial Organization of the Genome in Identified Neurons of Memory Circuits

描述(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不同的区域或隔间，但这种组织的确切结构和功能后果仍然难以捉摸。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有的方式发生，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)位置编码可以在全基因组范围内整合数千个基因的活性，包括建立关键的表观遗传标记，并且可以是协调细胞复杂转录输出的机制之一。这一建议的主要目的是(1)绘制突触耦合识别神经元中细胞基因组的远程相互作用图，(2)描述标准学习测试和突触刺激后核基因组三维重组的动力学。在这里，我们将利用海兔较简单的防御回路中大量可获得的感觉、调节和运动神经元，实现一种新的Hi-C(染色体构象捕获)方法，在单个神经元的水平上探索整个基因组的三维结构。该方法基于基于邻近的结扎和通过大规模并行测序选择性捕获核的不同解剖区域的组合。因此，我们将绘制在控制条件下和在公认的长期可塑性测试(如5-羟色胺应用)下的神经元基因组的相互作用区域。首先，基因组构象的这种空间映射将使我们能够公正地描述不同的相互作用的染色质隔间在单个核内的位置。其次，我们将它们的位置(例如，中央与外围)与位于这些间隔内的基因及其调节区的表达水平相关联。最后，我们将选择基因的表达水平与基因调控区域内的5-胞嘧啶甲基化模式(甲基组)相关联，重点关注5-羟色胺介导的信号转导的组成部分。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分别作为激活和抑制标记的选择性组蛋白翻译后修饰事件)，以探索突触输入或给药后神经元整合活动的机制。这一范式可以作为一个强大的概念验证平台来描述这一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意义。 公共卫生相关性：了解具有功能特征的神经元内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，基因组作为一个三维一体化实体运作，其中不同的染色体占据一个核内不同的区域或隔间。然而，远距离染色质区域之间的物理相互作用确实发生了，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，我们将描述细胞基因组远程相互作用的3-D结构及其在唯一识别的神经元学习和记忆时的动态。有关核基因组内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靶向治疗至关重要。

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.