Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox

计算型

• 批准号: 8119701
• 负责人: Nadav Ahituv
• 金额: $ 38.5万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-29 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119701
• 关键词: Animal Model; Automobile Driving; Binding Sites; Biological; Biological Assay; Candidate Disease Gene; Cataloging; Catalogs; Clinical; Code; Communities; Complex; Comprehension; Computational Technique; Computer Simulation; Coupled; DNA; Data; Development; Developmental Gene; Developmental Process; Disease; Elements; Embryo; Enhancers; Evolution; Fertilization; Functional RNA; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genome; Genomics; Health; Hour; Human; Human Development; Human Genome; In Situ Hybridization; Individual; Internet; Knock-out; Knowledge; Lampreys; Location; Modeling; Molecular; Morphologic artifacts; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Open Reading Frames; Organism; Orthologous Gene; Pathway interactions; Pattern; Phylogenetic Analysis; Plague; Preclinical Drug Evaluation; Property; Regulation; Regulator Genes; Regulatory Element; Reporter Genes; Research; Resources; Scanning; Shark; Specificity; Testing; Time; Tissues; Transgenes; Transgenic Organisms; Validation; Work; Zebrafish; base; computerized tools; cost; drug candidate; gene discovery; genome sequencing; genome-wide; high throughput screening; human disease; improved; in vivo; interest; knock-down; mutant; novel; promoter; research study; small molecule; teleost fish; tool; transcription factor; vertebrate genome; zebrafish genome

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.

描述（由申请人提供）：斑马鱼拥有越来越多的工具库，可以产生转基因，基因敲除和敲除，以及突变资源，加上其高通量和成本效率，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，其功能必要性在很大程度上仍然未知。在这些区域中明显嵌入的一个重要功能是基因调控，指示基因何时何地开启或关闭。然而，不像基因，我们知道它们的基因组位置，它们的密码，以及它们内部核苷酸变化的后果，在基因调控序列中，我们不知道这些。这些知识是极其重要的，各种各样的临床和分子数据支持这些序列是发育、进化、多样性和疾病的重要驱动因素。在本提案中，我们将结合先进的计算工具和高通量斑马鱼功能研究来注释这个非编码地形。利用和完善多个脊椎动物基因组比对，我们已经生成了一组前所未有的166693个斑马鱼保守非编码元件（CNEs），其中至少8805个区域在人类基因组中具有直接同源性。使用斑马鱼转基因增强子试验对这些序列的一部分进行初步研究，发现41%的这些序列在受精后24至48小时起增强子的作用。利用这种转基因试验，我们的目标是每年筛选200个序列的增强子活性。这些序列将从我们的大型CNE集中选择，这些序列的增强子活性和组织时间点特异性将使用复杂的计算工具进行预测，以及社区要求的序列。这种表征不仅允许对这些序列进行功能注释，而且还将生成一个新的和极其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。这将通过分析位于丰富调控区域的低特征基因的表达模式和功能后果来实现，这是重要发育基因调控存在的常见标志。额外的计算技术将用于在新的组织环境中发现严格调控的基因，以及目前尚未在我们发现它们富集的环境中研究的途径。该提案中生成的所有数据，无论是计算数据还是功能数据，都将通过专用的web浏览器（http://zebrafish.stanford.edu/）提供给社区，并集成到ZFIN， Ensemble和UCSC基因组浏览器中。综上所述，我们的工作将推动斑马鱼成为脊椎动物基因组非编码部分注释和表征的主要动物模型。虽然基因只占我们DNA的不到3%，但在剩下的97%中还有其他许多极其重要的序列，如基因调控元件，它们指示基因何时何地开启或关闭。这些基因调控元件的突变可以对人类疾病产生重大影响，但它们的位置和编码在大多数情况下仍然未知。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斑马鱼检测相结合，对这些序列进行注释，并更好地了解脊椎动物基因调控代码，这将对我们理解许多人类疾病的遗传原因至关重要。