Identifying the mechanism of olfactory receptor gene regulation in olfactory neurons with live-cell imaging

通过活细胞成像识别嗅觉神经元中嗅觉受体基因调控的机制

• 批准号: 10722860
• 负责人: Joan M. Pulupa
• 金额: $ 12.54万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10722860
• 关键词: Adaptor Signaling Protein; Advisory Committees; Alleles; Alzheimer' s Disease; Architecture; Axon; Binding; Binding Sites; Cell Nucleus; Chemicals; DNA; DNA Binding; DNA receptor; Detection; Development; Disease; Distant; Down-Regulation; Elderly; Enhancers; Future; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Health; Heterochromatin; Human; Image; Imaging Techniques; Individual; Institution; Label; Laboratory Research; Lead; Location; Maintenance; Measurement; Measures; Mediating; Messenger RNA; Microscopy; Modality; Modeling; Molecular; Mutate; Mutation; Nature; Neurodegenerative Disorders; Neurons; Nuclear; Nucleoproteins; Olfactory Pathways; Olfactory dysfunction; Optics; Phase; Positioning Attribute; Process; Proteins; Receptor Gene; Repression; Research; Sensory; Smell Perception; Spatial Distribution; Specificity; System; Technology; Time; Training; Transcriptional Regulation; Universities; Volatilization; behavioral health; epidemiology study; experimental study; fascinate; insight; live cell imaging; molecular imaging; mortality; mutant; olfactory receptor; olfactory sensory neurons; particle; physical conditioning; postmitotic; programs; protein protein interaction; receptive field; receptor; receptor expression; recruit; stoichiometry; temporal measurement; transcription factor

Project Summary/Abstract Smell is a fundamental human sense. Even though humans do not rely on smell for survival, olfaction is necessary for physical and behavioral health. Detection and identification of volatile chemicals by the olfactory system is built upon the “one receptor per neuron” rule, whereby each mature olfactory sensory neuron expresses a single olfactory receptor (OR) gene from one allele. Singular expression is critical for olfactory perception, since it defines both the receptive field of the OSN and the circuitry of its axon. OR gene choice and singular expression is contingent on orchestrated changes in nuclear architecture. Using live-cell imaging, I will elucidate how this changing nuclear architecture leads to changes in gene expression. In Aim 1, I will image the endogenous transcription factors (Lhx2/Ebf/Ldb1) and the actively expressed OR DNA to determine how these transcription factors promote the expression of a single OR allele. My preliminary results suggest that these transcription factors come together to form an activating hub, and that this hub represents a greater enrichment of transcription factors than can be explained simply by the stoichiometry of enhancer binding sites. I will image the relationship between the actively expressed OR and heterochromatin markers to determine if association with the activating hub isolates this allele from heterochromatin. In Aim 2, I will investigate the nature of the biomolecular interactions that allow this activating hub to recruit Lhx2/Ebf/Ldb1 beyond the stoichiometry of binding sites through single-particle tracking of transcription factor mutants. I will determine if the amplification in recruitment is due to phase separation of the intrinsically-disordered domains of Lhx2/Ebf/Ldb1 or if it is due to cooperative protein-protein interactions. Finally, in Aim 3, the independent phase of this proposal, I will integrate the imaging of these transcription factors with imaging of the genome to extend my research towards a complete characterization of the nucleoprotein dynamics regulating OR gene expression. Specifically, I will begin by characterizing the interactions of OR enhancers and OR mRNA with Lhx2/Ebf/Ldb1 and the actively expressed OR using live-cell imaging. I am determined to lead an independent research laboratory at an academic institution, working at the interface of OR gene regulation and optical microscopy. I am optimally positioned to achieve this goal, working as I am with Dr. Stavros Lomvardas at Columbia University. Dr. Lomvardas is an expert in olfaction and genome organization, and during the K99, I will receive technical training from him in advanced sequencing technologies. I have also assembled an advisory team consisting of Drs. Elizabeth Hillman, Richard Axel, Carol A. Mason, and Anum Glasgow. This advisory team will guide me in professional training and transitioning to independence. In addition to elucidating general principles of how nuclear organization can dictate transcriptional specificity, the experiments in this proposal will enable a mechanistic interrogation of the molecular interactions that regulate singular OR transcription.

项目摘要/摘要 气味是一种基本的人类意识。即使人类不依赖气味来生存，嗅觉是 身体和行为健康所必需的。嗅觉检测和鉴定挥发性化学物质 系统建立在“每个神经元的一个受体”规则上，每个成熟的嗅觉神经元中 从一个等位基因表达一个单个嗅觉受体（OR）基因。奇异表达对于嗅觉至关重要 感知，因为它既定义了OSN的接受场及其轴突的电路。 或基因选择和奇异表达取决于核结构的精心策划变化。 使用活细胞成像，我将阐明这种不断变化的核结构如何导致基因变化 表达。在AIM 1中，我将成像内源性转录因子（LHX2/EBF/LDB1）和积极的 表达或DNA确定这些转录因子如何促进单个或等位基因的表达。 我的初步结果表明，这些转录因子汇集在一起​​形成一个激活中心，并且 该枢纽代表转录因子的富集，而不是简单地解释 增强子结合位点的化学计量法。我会想象主动表达之间的关系或 异染色质标记以确定与激活中心是否相关 异染色质。在AIM 2中，我将研究允许这种激活的生物分子相互作用的性质 通过单粒子跟踪的单粒子跟踪，募集LHX2/EBF/LDB1的集线器超出了结合位点的化学计量。 转录因子突变体。我将确定募集中的放大是否是由于相位分离 LHX2/EBF/LDB1的本质上排序的结构域，或者是由于合作蛋白 - 蛋白质相互作用所致。最后， 在本提案的独立阶段AIM 3中，我将将这些转录因子的成像整合到 基因组的成像将我的研究扩展到核蛋白的完整表征 调节或基因表达的动力学。具体来说，我将首先表征或 使用LHX2/EBF/LDB1的增强剂和/或mRNA以及主动表达或使用活细胞成像。 我决心领导一个学术机构的独立研究实验室，在 基因调节或光学显微镜的界面。我最适合实现这一目标，可以实现这一目标 就像我在哥伦比亚大学的Stavros Lomvardas博士一样。 Lomvardas博士是嗅觉和基因组专家 组织，在K99期间，我将在他的高级测序技术中获得他的技术培训。 我还组建了一个由DRS组成的咨询团队。伊丽莎白·希尔曼，理查德·阿克塞尔，卡罗尔·梅森和 阿努姆·格拉斯哥。这个咨询团队将指导我进行专业培训和过渡到独立性。在 除了阐明核组织如何决定转录特异性的一般原则， 该提案中的实验将使对调节的分子相互作用进行机械审查 单数或转录。