Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox

斑马鱼基因组调控序列的计算与功能诠释 斑马鱼拥有越来越多的工具，可以产生转基因、基因敲除和 基因敲除和突变体资源加上其高通量和成本效益正在迅速成为 药物筛选和基因相关研究的主要动物模型。然而，与其他脊椎动物的基因组一样， 斑马鱼基因组的大部分（97%）由非基因序列组成， 仍然是未知的。基因调控是这些区域的一个重要功能， 指示基因何时何地开启或关闭。然而，与我们知道基因组的基因不同， 基因调控序列中的位置，它们的代码，以及它们内部核苷酸变化的后果 我们没有这些知识这方面的知识是非常重要的，具有广泛的临床和分子 支持这些序列的数据是发展，进化，多样性和疾病的重要驱动力。 在这项提议中，我们将联合收割机先进的计算工具与高通量斑马鱼功能 研究来注释这个非编码领域。使用和改进多个脊椎动物基因组比对， 产生了一组前所未有的166，693个斑马鱼保守非编码元件（CNE），其中至少 在人类基因组中有8，805个区域具有直接直系同源物。对其中一部分的初步研究 序列使用斑马鱼转基因增强子测定，发现这些序列中的41%作为 在受精后24至48小时，利用这种转基因检测，我们的目标是筛选200个 一年的增强子活动。这些序列将从我们的大型CNE集合中选择， 其增强子活性和组织时间点特异性将使用复杂的计算 工具和社区要求的序列。这种特性不仅允许功能注释 这些序列，但也将产生一个新的和极其重要的工具包基因调控元件， 它可以在精确的位置和精确的发育时间点驱动任何感兴趣的基因的表达。在 此外，我们还将使用注释的调控景观来发现具有潜在重要意义的新基因。 发展功能。这将通过分析表达模式和功能来进行 由于敲除位于丰富调控区的特征较少的基因而产生的后果， 重要的发育基因调节器的存在。将使用额外的计算技术 发现在新的组织环境中受到严格调控的基因，以及目前没有的途径， 在我们发现它们丰富的背景下进行研究。在这个提议中产生的所有数据，包括计算数据和 功能，将通过专用的Web浏览器提供给社区 (http：//zebrafish.stanford.edu/）以及集成到ZFIN、Ensembl和UCSC基因组浏览器中。 结合起来，我们的工作将推进斑马鱼作为注释和表征 脊椎动物基因组的非编码部分。

Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox Zebrafish with its growing arsenal of tools that allow the generation of transgenics, gene knockdowns and knockouts, and mutant resources coupled with its high-throughput and cost efficiency is quickly becoming the major animal model for drug screens and gene related studies. However, as with other vertebrate genomes, the majority of the zebrafish genome (97%) is made up of non-genic sequences whose functional necessity remains largely unknown. One vital function that is clearly embedded in these regions is gene regulation, instructing genes when and where to turn on or off. However, unlike genes where we know their genomic location, their code, and the consequences of nucleotide changes within them, in gene regulatory sequences we don't have that knowledge. This knowledge is extremely vital, with a wide variety of clinical and molecular data supporting these sequences to be an important driver for development, evolution, diversity, and disease. In this proposal, we will combine advanced computational tools with high-throughput zebrafish functional studies to annotate this noncoding terrain. Using and refining multiple vertebrate genome alignments we have generated an unprecedented set of 166,693 zebrafish conserved noncoding elements (CNEs), with at least 8,805 regions having a direct ortholog in the human genome. Preliminary studies for a portion of these sequences using a zebrafish transgenic enhancer assay, find 41% of these sequences to function as enhancers at 24 to 48 hours post fertilization. Taking advantage of this transgenic assay we aim to screen 200 sequences a year for enhancer activity. These sequences will be selected from our large CNE set, sequences whose enhancer activity and tissue-timepoint specificity will be predicted using sophisticated computational tools, and community requested sequences. This characterization will not only allow the functional annotation of these sequences, but will also generate a novel and extremely important toolkit of gene regulatory elements that can drive expression of any gene of interest at precise locations and precise developmental time points. In addition, we will also use the annotated regulatory landscape to discover novel genes with potential important developmental function. This will be carried out by analyzing the expression patterns and functional consequences due to knockdown of less characterized genes that lie in rich regulatory regions, a common sign for the existence of important developmental gene regulators. Additional computational techniques will be used to discover genes under tight regulation in novel tissue contexts, as well as pathways which are currently not studied in the context we find them enriched in. All the data generated in this proposal, both computational and functional, will be made available to the community through a dedicated web browser (http://zebrafish.stanford.edu/) as well as integration into ZFIN, Ensembl, and the UCSC genome browser. Combined, our work will advance zebrafish as the major animal model for annotating and characterizing the noncoding portion of the vertebrate genome.