Systematic functional analysis of non-coding sequences in transgenic zebrafish

描述（由申请人提供）：近年来已经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;在人和小鼠之间，非编码序列似乎是编码序列保守的至少两倍。守恒性是一种常用的功能约束指标。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一级序列预测其功能的工具。对这些序列的全面了解取决于体内功能测试，但通过小鼠转基因进行全面分析成本过高，并且不能轻易揭示基因调控的动态变化。我们拟应用转座子转基因技术在斑马鱼体内对大量保守的非编码元件进行功能分析。我们将首先关注骨骼形态发生中重要的基因，这些基因编码与发育和人类疾病有关的各种蛋白质产物，并且斑马鱼的直系同源物已被鉴定。在具体目标1中，我们将确定与选定的人类基因相关的>200个保守序列，并测试它们与最小启动子结合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前所未有的规模的单一数据集，将脊椎动物有机体中的一级序列与调节功能相关联。我们还建议测试在何种程度上依赖进化序列保守丰富的监管功能。初步的实验表明，一些分数的调控元件是错过了标准的计算方法。因此，我们将在一个单一的基因座上构建一个“平铺路径”，并测试该区间内所有序列的调节功能，包括那些在灵长类动物之外没有明显保守性的序列。在目标2中，我们将对目标1中分析的人类基因的斑马鱼直系同源物进行平行分析。迄今为止，我们已经积累了许多在斑马鱼中适当发挥作用的人类增强子元件的例子，尽管缺乏与orthopathic区域的明显序列相似性。我们的目标是确定，对于这些元素中的一些，相应的斑马鱼增强子具有相似的功能，这些序列的比较可以用来完善调控元件的计算预测。在最后一个目标中，我们将评估噬菌体C31位点特异性重组酶在斑马鱼中的使用，以促进现有转基因的原位再工程。该技术将大大提高目的1和2中产生的转基因品系的实用性，并将广泛应用于斑马鱼的其他目的。我们的建议的一个重要目标是剖析在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，我们的目标也是建立一个范例，并产生试剂，这将有广泛的适用性，以了解丰富的比较信息所产生的基因组测序工作。 公共卫生相关性：来自多种生物体的基因组序列的比较突出了基因编码区之外的序列保守性的惊人程度，并且我们相信许多保守序列参与基因表达的调控。我们已经开发了一种在斑马鱼中测试DNA序列调节基因表达的能力的方法，斑马鱼胚胎的光学清晰度，快速发育和丰富度使我们能够在比其他模式生物（如小鼠）大得多的规模上进行这些实验。通过这种方法的应用，我们的目标是建立一个范例，并产生试剂，这将有助于我们的基因组测序工作所产生的信息财富的理解。

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.