Understanding the Role of Nuclear Architecture in Olfactory Receptor Choice

描述（由申请人提供）：嗅觉受体（OR）的选择，是数千个可用的哺乳动物OR等位基因中的一个的转录激活，是一个知之甚少的过程。我们之前已经证明，OR基因的核内和染色体间聚集在少数OR特异性异色灶中有助于有效的OR沉默，保持OR表达的单基因和单等位基因性质。在这里，我们研究了复杂的染色体间关联也负责单个OR等位基因的转录激活的假设。利用高通量表观遗传学和遗传学方法，我们鉴定了一组新的OR增强子，它们支持斑马鱼和小鼠嗅感觉神经元中报告基因的显著部分表达。我们的初步数据表明，这些增强剂可能协同作用，激活OR表达，在嗅觉感觉神经元的核中产生结构和功能上的奇异性。我们提出了一系列的实验，将利用成像和Hi-C方法绘制和量化新发现的增强子的染色体间关联。此外，我们提出了基因丧失和功能获得实验，以测试OR增强子是否协同作用于单个OR等位基因的激活。最后，我们试图研究OR增强子在其功能和核组织中的不寻常表观遗传特征的贡献。我们的实验将为细胞核结构在基因表达中的作用提供新的见解，并将揭示体内细胞多样性产生的分子机制。

DESCRIPTION (provided by applicant): Olfactory receptor (OR) choice, the transcriptional activation of one out of thousands of available mammalian OR alleles, is a poorly understood process. We previously demonstrated that the nuclear intra- and inter- chromosomal aggregation of OR genes in a few, OR-specific, heterochromatic foci contributes to the efficient OR silencing, preserving the monogenic and monoallelic nature of OR expression. Here, we examine the hypothesis that complex interchromosomal associations are responsible also for the transcriptional activation of a single OR allele. Using high throughput epigenetic and genetic approaches we identified a set of novel OR enhancers that support reporter expression in significant fractions of olfactory sensory neurons in zebrafish and mice. Our preliminary data suggest that these enhancers might work in concert for the activation of OR expression, creating a structural and functional singularity in the nuclei of olfactory sensory neurons. We propose a series of experiments that will map and quantify the interchromosomal associations of the newly identified enhancers using imaging and Hi-C approaches. Moreover, we propose genetic loss-, and gain-of- function experiments that will test whether OR enhancers act synergistically towards the activation of a single OR allele. Finally, we seek to investigate the contribution of the unusual epigenetic signature of OR enhancers in their function and nuclear organization. Our experiments will provide novel insight into the role of nuclear architecture in gene expression and will uncover molecular mechanisms involved in the generation of cellular diversity in vivo.