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 嗅觉是人类的一种基本感觉。尽管人类不依赖嗅觉生存，但嗅觉是 对身体和行为健康是必要的。嗅觉对挥发性化学物质的检测与鉴定 该系统建立在“每个神经元一个感受器”规则的基础上，即每个成熟的嗅觉神经元 表达来自一个等位基因的单一嗅觉受体(OR)基因。单一的表达方式对嗅觉至关重要 知觉，因为它既定义了OSN的接受场，也定义了它的轴突电路。 或者，基因的选择和独特的表达取决于核结构的精心设计的变化。 利用活细胞成像，我将阐明这种变化的核结构是如何导致基因变化的。 表情。在目标1中，我将成像内源转录因子(LHX2/EBF/LDB1)和活性 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，形成了一个激活中心， 这个中枢代表了转录因子的更大丰富，而不是简单地用 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，以确定与激活中心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 HUB通过单粒子跟踪招募LHX2/EBF/LDB1，超越结合位点的化学计量比 转录因子突变体。我会确定招募中的放大是否是由于 LHX2/EBF/LDB1的内在无序结构域，或者是由于蛋白质-蛋白质的协同作用。最后， 在目标3，这一提议的独立阶段，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对核蛋白的完整表征 调控OR基因表达的动力学。具体地说，我将从描述OR的交互开始 与LHX2/EBF/LDB1的增强子和OR mRNA，以及活细胞成像中主动表达的OR。 我决心领导一个学术机构的独立研究实验室，在 OR基因调控与光学显微镜的接口。我处于实现这一目标的最佳位置，工作 就像我和哥伦比亚大学的斯塔夫罗斯·隆瓦达斯博士一样。Lomvardas博士是嗅觉和基因组方面的专家 在K99比赛期间，我将从他那里接受先进测序技术的技术培训。 我还组建了一个顾问团队，由伊丽莎白·希尔曼博士、理查德·阿克塞尔博士、卡罗尔·A·梅森博士和 阿纳姆·格拉斯哥。这个顾问团队将指导我进行专业培训并过渡到独立。在……里面 除了阐明核组织如何决定转录特异性的一般原则外， 这一提议中的实验将使对调节分子相互作用的机械询问成为可能 单数或转录。