Regulation of Odorant Receptor Gene Expression

描述（由申请人提供）：嗅觉系统能够识别数千种气味，从而维持人类的食物消耗。人类识别气味的能力依赖于鼻上皮细胞内嗅觉神经元内的气味受体。这种上皮细胞在基底干细胞群的持续细胞分裂下产生未成熟的嗅觉神经元，然后这些嗅觉神经元变成成熟的嗅觉神经元。气味受体（ORs）在未成熟的嗅觉神经元中克隆表达。哺乳动物基因组由约1500个OR基因组成;这些基因属于七跨膜受体超家族。然而，每个基因的每个等位基因被视为一个单独的实体，导致~3000个OR等位基因以克隆（单一）方式被嗅觉神经元表达。这种单一OR等位基因表达的能力是多步骤的方式。一个关键步骤是生产功能性OR蛋白。嗅觉神经元存在于上皮内的区域中，使得给定的嗅觉神经元（通常）在~100个OR基因（~200个等位基因）之间进行选择。选择过程分两步进行：从约200个等位基因中选择一个在未成熟神经元内表达（第一选择），然后产生OR蛋白，并对其进行功能性测试。不能产生七跨膜蛋白允许单一选择机制在同一未成熟的嗅觉神经元中选择另一个OR等位基因（第二选择）。如果选择的七跨膜OR不能与信号转导机制耦合，那么嗅觉神经元将无法成熟并死亡。OR基因表达的关键步骤是在未成熟神经元内最初选择一个OR等位基因（第一选择）。到目前为止，完全不知道这种“奇异选择”过程是如何在分子水平上发生的。对非常小的OR启动子区域（~300bp）的分析已经导致鉴定出可能起作用的两种转录因子：O/E-1型结合蛋白和同源结构域型结合蛋白。识别更多的候选DNA结合蛋白及其相互作用是必要的，以了解“奇异选择”的过程。该项目充分利用了小的调节区域用于产生或单一的选择在未成熟的嗅觉神经元。在目标1中，将定义最小OR控制区以鉴定其他候选转录因子。至少有10 - 15种不同的嗅觉神经元细胞类型，每种细胞注定表达约200种OR等位基因之一。在目标2中，通常在不同的嗅觉细胞类型中表达的两个最小OR启动子将用于鉴定另外的转录因子。这将有助于了解嗅觉上皮内的特定模式是如何获得的。在目标3中，候选同源结构域蛋白将在未成熟和/或成熟神经元内缺失，以确定其对OR基因表达的作用。在目标4中，我将尽一切努力促进这项工作成为出版物和更多的资金。 公共卫生相关性：基因表达是细胞在人体内正常运作所必需的。嗅觉非常依赖于嗅觉神经元，这些神经元将化学世界翻译到大脑。嗅觉神经元通过仔细表达气味受体来控制这种气味识别过程。嗅觉功能的丧失导致老年人饮食不良以及食物中毒的发生率较高。研究气味受体基因表达将有助于了解嗅觉系统是如何组合在一起并在整个成年期维持的。

DESCRIPTION (provided by applicant): The olfactory system is able to recognize thousands of odorants thus maintaining food consumption in humans. The ability to identify odors by humans is dependent on odorant receptors within olfactory neurons that line the nasal epithelium. This epithelium is under continuous cell division by a basal stem cell population that produces immature olfactory neurons, which then become mature olfactory neurons. Odorant receptors (ORs) are clonally expressed in immature olfactory neurons. The mammalian genome consists of ~1500 OR genes; these genes belong to the seven-transmembrane receptor superfamily. However, each allele of each gene is treated as a separate entity leading to ~3000 OR alleles to be expressed in a clonal (singular) manner by olfactory neurons. The ability for this singular OR allele expression is a multi-step manner. A critical step is the production of a functional OR protein. Olfactory neurons are found in domains within the epithelium such that a given olfactory neuron chooses (in general) between ~100 OR genes (~200 alleles). The selection process occurs in two-steps: one of ~200 alleles is chosen for expression (1st choice) within an immature neuron, followed by OR protein production, which is tested for functionality. Failure to produce a seven-transmembrane protein allows for the singular choice mechanism to choose another OR allele in that same immature olfactory neuron (2nd choice). If the chosen seven-transmembrane OR is unable to couple with the signal transduction machinery, then that olfactory neuron will fail to mature and die. The critical step in the expression of an OR gene is the initial choice of one OR allele within an immature neuron (1st choice). To date, it is completely unknown how this "singular choice" process occurs at a molecular level. Analysis of very small OR promoter regions (~300bp) have lead to the identification of two transcription factors that may play a role: an O/E-1 type binding protein and a homeodomain type biding protein. The identification of more candidate DNA binding proteins and their interactions is necessary to understand the "singular choice" process. This project fully exploits the small regulatory regions used to produce OR singular choice in immature olfactory neurons. In Aim 1, the most minimal OR control region will be defined in order to identify other candidate transcription factors. There are at least 10-15 different olfactory neuronal cell types each fated to express one of ~200 OR alleles. In Aim 2, two minimal OR promoters normally expressed in different olfactory cell types will be used to identify additional transcription factors. This will allow the understanding of how specific patterns within the olfactory epithelium are obtained. In Aim 3, a candidate homeodomain protein will be deleted within immature and/or mature neurons in order to determine its role on OR gene expression. In Aim 4, I will make every attempt to promote this work into publications and greater funding. PUBLIC HEALTH RELEVANCE: Gene expression is necessary for cells to function properly in the human body. The sense of smell is critically dependent on olfactory neurons that translate the chemical world to the brain. Olfactory neurons govern this odor recognition process through careful expression of odorant receptors. Loss of olfactory function leads to poor feeding in geriatric populations as well as higher incidence of food poisoning. Studying odorant receptor gene expression will give insights into how the olfactory system is put together and maintained throughout adulthood.