Systematic functional analysis of non-coding sequences in transgenic zebrafish

描述(申请人提供)：近年来，多个脊椎动物基因组序列完成，并揭示了非编码序列的保守程度令人惊讶；非编码序列似乎至少是编码序列在人类和小鼠之间保守的两倍。守恒是功能约束的一个常用指标。我们假设许多保守的非编码序列参与了基因表达的调控，尽管目前我们缺乏工具来根据初级序列预测它们的功能。对这些序列的充分理解依赖于体内的功能测试，但通过小鼠转基因进行的全面分析成本高昂，而且不容易揭示基因调控的动态变化。我们建议将基于转座子的转基因技术应用到斑马鱼中，从功能上分析体内大量保守的非编码元件。我们首先将重点放在骨骼的形态发生中重要的基因，编码各种与发育和人类疾病有关的蛋白质产物，斑马鱼的同源基因已经被描述出来。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一起在斑马鱼胚胎中驱动报告基因表达的能力。这些实验将创建一个规模史无前例的单一数据集，将脊椎动物有机体的初级序列与调节功能联系起来。我们还建议测试对进化序列保守的依赖程度，以丰富调控功能。初步实验表明，标准计算方法遗漏了部分调控元件。因此，我们将构建一条跨越单个基因座的“平铺路径”，并测试该区间内的所有序列的调控功能，包括那些在灵长类以外没有明显保守的序列。在目标2中，我们将对目标1中分析的人类基因的斑马鱼同源基因进行平行分析。迄今为止，我们已经积累了大量人类增强子元件在斑马鱼中发挥适当功能的例子，尽管缺乏与直系同源区域的明显序列相似性。我们的目标是为其中一些元件确定具有相似功能的相应斑马鱼增强子；这些序列的比较可以用来改进对调节元件的计算预测。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应用，以促进现有转基因的原位重组。这项技术将大大提高AIMS 1和AIMS 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用途。我们建议的一个重要目标是剖析在单个器官系统的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，并产生一种试剂，这将具有广泛的适用性，以理解基因组测序工作产生的丰富的比较信息。 公共卫生相关性：对多个生物体的基因组序列的比较突显了基因编码区以外令人惊讶的序列保守程度，我们认为许多保守的序列参与了基因表达的调节。我们已经在斑马鱼身上开发了一种方法来测试DNA序列调控基因表达的能力，斑马鱼胚胎的光学清晰度、快速发育和丰富使我们能够在比其他模式生物(如小鼠)实际规模大得多的范围内进行这些实验。通过应用这种方法，我们的目标是建立一个范例并产生有助于我们理解基因组测序工作产生的丰富信息的试剂。

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.