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.

项目总结/摘要 意识是人类的基本感觉。尽管人类并不依靠嗅觉生存，但嗅觉 身体和行为健康所必需的。嗅觉对挥发性化学物质的检测和识别 系统是建立在“一个神经元一个受体”的规则，即每个成熟的嗅觉感觉神经元 表达来自一个等位基因的单个嗅觉受体（OR）基因。奇异表情对嗅觉至关重要 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协调变化。 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， 表情在目标1中，我将对内源性转录因子（Lhx 2/Ebf/Ldb 1）和活性转录因子（Lhx 2/Ebf/Ldb 1）进行成像。 表达的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， 这个中心代表了比可以简单地用 增强子结合位点的化学计量。我将想象积极表达的OR和 异染色质标记物，以确定与激活枢纽的关联是否将该等位基因从 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 通过单粒子跟踪， 转录因子突变体我将确定是否在招聘扩增是由于相分离的 Lhx 2/Ebf/Ldb 1的内在无序结构域或是否由于协同蛋白质-蛋白质相互作用。最后， 在目标3（本提案的独立阶段）中，我将整合这些转录因子的成像， 基因组成像，以扩展我的研究，对核蛋白的完整表征 调节OR基因表达的动力学。具体来说，我将开始通过表征的互动或 增强子和OR mRNA与Lhx 2/Ebf/Ldb 1和使用活细胞成像的活性表达的OR。 我决心在一个学术机构领导一个独立的研究实验室， OR基因调控和光学显微镜的接口。我处于实现这一目标的最佳位置， 就像我和哥伦比亚大学的Stavros Lomvardas博士一样。隆瓦达斯博士是嗅觉和基因组方面的专家 在K99期间，我将接受他在先进测序技术方面的技术培训。 我还组建了一个顾问小组，成员包括伊丽莎白·希尔曼博士、理查德·阿克塞尔博士、卡罗尔·A·梅森和 Anum格拉斯哥。这个顾问团队将指导我进行专业培训和向独立过渡。在 除了阐明核组织如何决定转录特异性的一般原理外， 在这个提议中的实验将能够对调节分子相互作用的机制进行询问。 单数或转录。