Systematic functional analysis of non-coding sequences in transgenic zebrafish

描述（由申请人提供）：近年来完成了多个脊椎动物基因组序列，并揭示了非编码序列的惊人保守程度；在人和老鼠之间，非编码序列的保守性至少是编码序列的两倍。守恒是功能约束的常用指标。我们假设许多保守的非编码序列参与基因表达的调控，尽管我们目前缺乏从初级序列预测其功能的工具。对这些序列的全面了解依赖于体内功能测试，但通过小鼠转基因进行全面分析成本过高，而且无法轻易揭示基因调控的动态变化。我们建议在斑马鱼中应用基于转座子的转基因技术，对体内大量保守的非编码元件进行功能分析。我们将首先关注在骨骼形态发生中重要的基因，编码各种蛋白质产物，这些蛋白质产物与发育和人类疾病有关，斑马鱼的同源物已经被表征。在Specific Aim 1中，我们将鉴定与选定的人类基因相关的bbb200保守序列，并测试它们与最小启动子一起驱动斑马鱼胚胎中报告基因表达的能力。这些实验将创建一个空前规模的单一数据集，将初级序列与脊椎动物有机体的调节功能联系起来。我们还建议测试对进化序列保守的依赖在多大程度上丰富了调控功能。初步实验表明，标准计算方法遗漏了部分调控元件。因此，我们将在单个基因座上构建一条“平铺路径”，并在这段时间内测试所有序列的调控功能，包括那些在灵长类动物之外没有明显保护的序列。在Aim 2中，我们将对Aim 1中分析的人类基因的斑马鱼同源物进行平行分析。迄今为止，我们已经积累了许多人类增强子元件在斑马鱼中正常发挥作用的例子，尽管缺乏与同源区域的明显序列相似性。我们的目标是确定其中一些元素，相应的斑马鱼增强子具有类似的功能；这些序列的比较可以用来改进调节元件的计算预测。最后，我们将评估噬菌体&C31位点特异性重组酶在斑马鱼中的应用，以促进现有转基因基因的原位重组。这项技术将大大提高目标1和目标2中产生的转基因系的效用，并将广泛应用于斑马鱼的其他用途。我们提议的一个重要目标是剖析单个器官系统发育过程中控制关键基因表达的调控元件，并测试它们与人类疾病的可能相关性。然而，我们也旨在建立一个范例，并产生试剂，这将广泛适用于理解基因组测序工作中产生的丰富的比较信息。

DESCRIPTION (provided by applicant): Recent years have seen the completion of multiple vertebrate genome sequences, and revealed a surprising degree of conservation of non coding sequence; at least twice as much non-coding as coding sequence appears to be conserved between human and mouse. Conservation is a commonly used indicator of functional constraint. We hypothesize that many conserved non-coding sequences are involved in the regulation of gene expression, although we currently lack the tools to predict their function from primary sequence. A full understanding of these sequences depends on functional testing in vivo, but comprehensive analysis through mouse transgenesis is cost prohibitive and cannot readily reveal dynamic changes in gene regulation. We propose to apply transposon-based transgenesis in zebrafish, to functionally analyze a large number of conserved non-coding elements in vivo. We will initially focus on genes important in the morphogenesis of the skeleton, encoding a variety of protein products that have been implicated in development and human disease, and for which the zebrafish orthologues have been characterized. In Specific Aim 1, we will identify >200 conserved sequences associated with selected human genes, and test them for their ability, in conjunction with a minimal promoter, to drive reporter gene expression in zebrafish embryos. These experiments will create a single data set of unprecedented size, correlating primary sequence with regulatory function in a vertebrate organism. We also propose to test the degree to which reliance on evolutionary sequence conservation enriches for regulatory function. Preliminary experiments suggest that some fraction of regulatory elements is missed by standard computational approaches. Therefore, we will construct a "tiling path" across a single locus and test all sequences in the interval for regulatory function, including those showing no overt conservation beyond primates. In Aim 2, we will carry out parallel analyses on zebrafish orthologues of the human genes analyzed in Aim 1. We have accumulated numerous examples to date of human enhancer elements that function appropriately in zebrafish, despite lack of overt sequence similarity to orthologous regions. We aim to identify, for some of these elements, corresponding zebrafish enhancers with similar function; comparison of these sequences can be used to refine computational predictions of regulatory elements. In the last Aim, we will evaluate use of the phage &C31 site specific recombinase in zebrafish, to facilitate re-engineering of existing transgenes in situ. This technology will greatly enhance the utility of the transgenic lines generated in Aims 1 and 2, and will be broadly applicable in zebrafish for other purposes. An important goal of our proposal is to dissect the regulatory elements controlling expression of key genes during development of a single organ system, and test their possible relevance to human disease. However, we also aim to establish a paradigm, and generate reagents, that will have wide applicability to understanding the wealth of comparative information arising out of genome sequencing efforts. PUBLIC HEALTH RELEVANCE: The comparison of genome sequences from multiple organisms has highlighted the surprising degree of sequence conservation outside of gene coding regions, and we believe that many of the conserved sequences are involved in the regulation of gene expression. We have developed an approach in zebrafish to test the ability of DNA sequences to regulate gene expression, and the optical clarity, rapid development, and abundance of the zebrafish embryo allow us to perform these experiments on a scale much larger than practical in other model organisms such as the mouse. Through application of this approach, we aim to establish a paradigm and generate reagents that will aid our understanding of the wealth of information arising out of genome sequencing efforts.