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.

项目概要/摘要 嗅觉是人类的基本感觉。尽管人类不依赖嗅觉生存，但嗅觉 身体和行为健康所必需的。嗅觉检测和鉴别挥发性化学物质 该系统建立在“每个神经元一个受体”规则的基础上，每个成熟的嗅觉感觉神经元 表达来自一个等位基因的单一嗅觉受体（OR）基因。单一表达对于嗅觉至关重要 感知，因为它定义了 OSN 的感受野及其轴突的电路。 OR 基因的选择和单一表达取决于核结构的精心安排的变化。 使用活细胞成像，我将阐明这种变化的核结构如何导致基因的变化 表达。在目标 1 中，我将对内源转录因子 (Lhx2/Ebf/Ldb1) 和主动转录因子进行成像 表达 OR DNA 以确定这些转录因子如何促进单个 OR 等位基因的表达。 我的初步结果表明这些转录因子聚集在一起形成一个激活中心，并且 该中心代表了比简单地解释的转录因子更丰富的转录因子 增强子结合位点的化学计量。我会想象主动表达的 OR 和 异染色质标记以确定是否与激活中枢的关联将该等位基因从 异染色质。在目标 2 中，我将研究允许这种激活的生物分子相互作用的性质 中心通过单粒子跟踪招募 Lhx2/Ebf/Ldb1 超出结合位点的化学计量 转录因子突变体。我将确定招募的放大是否是由于相分离造成的 Lhx2/Ebf/Ldb1 本质上无序的结构域，或者是否是由于蛋白质-蛋白质相互作用造成的。最后， 在目标 3（该提案的独立阶段）中，我将把这些转录因子的成像与 基因组成像将我的研究扩展到核蛋白的完整表征 动态调节 OR 基因表达。具体来说，我将首先描述 OR 的相互作用 增强子和 OR mRNA 与 Lhx2/Ebf/Ldb1 以及使用活细胞成像的活跃表达 OR。 我决心在学术机构领导一个独立的研究实验室，在 OR基因调控和光学显微镜的界面。我处于实现这一目标的最佳位置，正在努力工作 正如我和哥伦比亚大学的 Stavros Lomvardas 博士一样。 Lomvardas 博士是嗅觉和基因组方面的专家 在K99期间，我将接受他关于先进测序技术的技术培训。 我还组建了一个由博士组成的顾问团队。伊丽莎白·希尔曼、理查德·阿克塞尔、卡罗尔·A·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并过渡到独立。在 除了阐明核组织如何决定转录特异性的一般原则外， 该提案中的实验将能够对调节分子相互作用的机制进行询问 单数 OR 转录。