Understanding the Role of Nuclear Architecture in Olfactory Receptor Choice

描述(申请人提供)：嗅觉受体(OR)选择，即数千个哺乳动物OR等位基因中的一个在转录上被激活，是一个知之甚少的过程。我们先前已经证明，在少数OR特异的异染色灶中，OR基因在染色体内和染色体间的核聚集有助于有效地沉默OR，保留了OR表达的单基因和单等位基因的性质。在这里，我们检验了一种假设，即复杂的染色体间关联也是单个OR等位基因转录激活的原因。利用高通量的表观遗传学和遗传学方法，我们确定了一组新的OR增强子，它们支持斑马鱼和小鼠嗅觉神经元显着部分的报告基因表达。我们的初步数据表明，这些增强子可能协同作用激活OR表达，从而在嗅觉神经元的核团中产生结构和功能上的奇异性。我们提出了一系列实验，将使用成像和Hi-C方法绘制和量化新识别的增强子的染色体间关联。此外，我们建议进行遗传功能丧失和功能获得实验，以测试OR增强剂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增强子不寻常的表观遗传学特征在其功能和核组织中的作用。我们的实验将为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.