Deciphering mechanisms for olfactory receptor choice in single cells

项目总结 值得注意的是，染色体避免了纠缠，因为它们被组织成各种空间域，支持 核内功能多样。在这些功能中，基因组的3D组织对于 限制基因表达的模式。这一点也许最好的证明是嗅觉现象。 哺乳动物嗅觉上皮的受体(OR)选择。嗅觉的嗅觉感觉神经元 上皮细胞随机选择~1400个OR基因中的1个稳定表达。所选的OR定义OSN 接受域，因此是OSN身份的关键要素。自发现OR基因库以来~30年 以前，OR选择的机制仍然没有定义。然而，我们实验室的研究表明，OR 表达依赖于核结构的分化依赖的改变。在嗅觉干细胞中， 异染色质脱离核周，导致核倒置。或基因 随后在新形成的异染色质核心附近组织成多染色体隔室。 最后，称为希腊群岛(GIs)的网络OR基因特异性增强子通过单个OR等位基因组装， 形成一个“或增强子中心”，以支持或转录。虽然所有这三个事件对于OR来说都是不可或缺的 表达，OR-增强子中心的形成可能是最重要的。已确定63个大兵； 然而，在任何细胞中，只有一个子集参与OR-Enhenger Hub，与 活跃的OR等位基因。围绕OR基因的地理信息系统的独特组织被认为是架构 单个转录活性OR的足迹。然而，我们实验室的单细胞基因组数据表明 在原子核的其他地方存在着拓扑上相同的结构。这些复合体由相同的 作为活性枢纽的增强子结构还含有转录沉默的OR基因。然而，额外的单曲- 细胞实验表明，这些结构可能通过潜在的生化特征来区分。在这 我们的目标是首先表征区分活性和非活性增强子中心的染色质属性， 然后测试这些结构如何组装并获得不同功能特性的可能机制。 我们设计了一种单细胞基因组策略来破译染色质标记和结合转录因子 这区分了增强子亚型，重点是那些解释GI与活性OR基因关联的亚型。或 发育早期的转录是已知的GI招募、强化或选择的趋向性信号。因此， 我们将对突变到扰动阶段的小鼠的基因组结构和功能分子特征进行分析 研究OR基因表达途径，以确定OR-增强子轮毂组装的决定因素。我们预料到 这些实验将揭示一种生物学计划，通过该计划，基因组的协调空间重排 伴随着对染色质的图案化修饰，基因在神经系统的一个独特细胞中表达。

PROJECT SUMMARY Remarkably, chromosomes avoid entanglement as they are organized into various spatial domains that support diverse functions in the nucleus. Among these functions, 3D organization of the genome is uniquely essential for constraining patterns of gene expression. This is perhaps best demonstrated by the phenomenon of olfactory receptor (OR) choice in the mammalian olfactory epithelium. Olfactory sensory neurons (OSNs) of the olfactory epithelium stochastically choose 1 of ~1400 OR genes to stably express. The chosen OR defines an OSN’s receptive field and is thus a crucial element of OSN identity. Since the discovery of the OR gene pool ~30 years ago, a mechanism for OR choice remains undefined. However, research from our lab has revealed that OR expression depends on differentiation-dependent alterations to nuclear architecture. In olfactory stem cells, heterochromatin detaches from the nuclear periphery leading to inversion of the nucleus. OR genes subsequently organize into multichromosomal compartments near the newly formed heterochromatic core. Lastly a network OR-gene specific enhancers known as Greek Islands (GIs), assemble over a single OR allele, forming an “OR-Enhancer Hub”, to support OR transcription. While all three events are indispensable for OR expression, the formation of the OR-Enhancer Hub is perhaps the most important. 63 GIs have been identified; however, in any cell, only a subset participate in the OR-Enhancer Hub, forming highly specific interactions with the active OR allele. The unique organization of GIs around an OR gene was considered to be the architectural footprint of the single transcriptionally active OR. However, single-cell genomics data from our lab demonstrate that topologically identical structures exist elsewhere in the nucleus. These complexes are defined by the same enhancer constitution as the active hub yet contain transcriptionally silent OR genes. However, additional single- cell experiments suggest these structures might be distinguished by underlying biochemical features. In this proposal we aim to first characterize the chromatin properties that separate active from inactive enhancer hubs, then test possible mechanisms for how these structures might assemble and gain different functional properties. We have devised a single-cell genomic strategy to decipher the chromatin marks and bound transcription factors that differentiate enhancer subtypes, focusing on those that explain GI association to the active OR gene. OR transcription early in development is a known tropic signal for GI recruitment, reinforcing OR choice. Therefore, we will perform analysis of genome structure and functional molecular features in mice mutated to perturb stages of the OR gene expression pathway to identify determinants of OR-Enhancer hub assembly. We anticipate that these experiments will reveal a biological scheme by which the coordinated spatial rearrangement of the genome alongside patterned modifications to chromatin guide gene expression in a unique cell of the nervous system.