Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox

描述（由申请人提供）：斑马鱼凭借其不断增长的工具库，允许产生转基因、基因敲除和敲除，以及突变体资源，加上其高通量和成本效益，正迅速成为药物筛选和基因相关研究的主要动物模型。然而，与其他脊椎动物基因组一样，斑马鱼基因组的大部分（97%）是由非基因序列组成的，其功能的必要性在很大程度上仍然未知。基因调控是这些区域的一个重要功能，它指示基因在何时何地开启或关闭。然而，与基因不同的是，我们知道它们的基因组位置，它们的代码，以及它们内部核苷酸变化的后果，在基因调控序列中，我们没有这些知识。这些知识是非常重要的，各种各样的临床和分子数据支持这些序列是发展，进化，多样性和疾病的重要驱动力。在这个建议中，我们将结合联合收割机先进的计算工具与高通量斑马鱼功能的研究，注释这个非编码的地形。使用和改进多个脊椎动物基因组比对，我们已经产生了前所未有的166，693个斑马鱼保守非编码元件（CNE），其中至少8，805个区域在人类基因组中具有直接直系同源物。使用斑马鱼转基因增强子测定对这些序列的一部分进行初步研究，发现这些序列中的41%在受精后24至48小时起增强子的作用。利用这种转基因检测，我们的目标是每年筛选200个序列的增强子活性。这些序列将选自我们的大型CNE集，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预测的序列，以及社区要求的序列。这种表征不仅允许这些序列的功能注释，而且还将产生一个新的和非常重要的基因调控元件工具包，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。此外，我们还将使用注释的调控景观来发现具有潜在重要发育功能的新基因。这将通过分析表达模式和功能性后果进行，由于敲除位于丰富的调控区的特征较少的基因，这是重要的发育基因调控因子存在的共同标志。额外的计算技术将用于发现在新的组织环境中受到严格调控的基因，以及目前在我们发现它们富集的背景下没有研究的途径。该提案中生成的所有数据，包括计算数据和功能数据，都将通过专用的网络浏览器（http：//zebrafish.stanford.edu/）以及集成到ZFIN、Enclave和UCSC基因组浏览器中提供给社区。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非编码部分的主要动物模型。公共卫生相关性：斑马鱼基因组调控序列的计算和功能注释虽然基因只占我们DNA的不到3%，但在剩下的97%中还有许多非常重要的序列，如基因调控元件，它们指示基因何时何地打开或关闭。这些基因调控元件的突变对人类疾病有很大的影响，但它们的位置和编码仍对大多数人未知。在本提案中，我们将利用斑马鱼模式生物的独特特性，将先进的计算工具与快速功能性斑马鱼测定结合起来，以注释这些序列并更好地了解脊椎动物基因调控密码，这将是极其重要的。我们理解许多人类疾病的遗传原因。

DESCRIPTION (provided by applicant): 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, Ensemble, 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. PUBLIC HEALTH RELEVANCE: Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox While genes make up less than 3% of our DNA, within the remaining 97% lie other numerous extremely important sequences such as gene regulatory elements, that instruct the genes when and where to turn on or off. Mutations in these gene regulatory elements can have a great impact on human disease, yet their location and code still remains on the majority unknown. In this proposal we will take advantage of the unique properties of the zebrafish model organism to couple advanced computational tools with rapid functional zebrafish assays to annotate these sequences and obtain a better understanding of the vertebrate gene regulatory code, which will be of extreme importance to our comprehension of the genetic cause for numerous human diseases.