Computational & Functional Annotation of the Zebrafish Genome Regulatory Toolbox

计算型

• 批准号: 7943127
• 负责人: Nadav Ahituv
• 金额: $ 38.91万
• 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/7943127
• 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; 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; public health relevance; 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）：斑马鱼的工具库日益增长，允许产生转基因，基因敲低和淘汰赛，以及突变资源，以及其高通量和成本效率，迅速成为药物筛查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，大多数斑马鱼基因组（97％）由非基因序列组成，其功能必要性在很大程度上尚不清楚。这些区域中明显嵌入的一个重要功能是基因调节，指导基因在何时何地打开或关闭。但是，与我们知道其基因组位置的基因不同，它们的代码和核苷酸内部的后果在基因调节序列中不存在。这些知识至关重要，拥有多种临床和分子数据，这些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。在此提案中，我们将将高级计算工具与高通量斑马鱼功能研究相结合，以注释这一非编码地形。使用和完善多个脊椎动物基因组比对，我们产生了前所未有的166,693个斑马鱼保守的非编码元件（CNE），其中至少8,805个区域在人类基因组中具有直接矫形器。使用斑马鱼转基因增强剂测定法对这些序列的一部分进行初步研究，发现这些序列的41％在受精后24至48小时在增强子起作用。利用这种转基因测定，我们旨在每年筛选200个序列以增强活性。这些序列将从我们的大型CNE集合中选择，其增强器活性和组织时间特异性的序列将使用复杂的计算工具以及社区请求的序列预测。这种表征不仅将允许这些序列的功能注释，而且还将生成一个新颖且极为重要的基因调节元素工具包，该工具包可以在精确位置和精确的发育时间点上驱动任何感兴趣的基因的表达。此外，我们还将使用带注释的调节景观来发现具有潜在重要发育功能的新基因。这将通过分析由于含有丰富的调节区域的特征性较低的基因而导致的表达模式和功能后果来进行，这是重要发育基因调节剂存在的常见迹象。 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基因组浏览器。结合在一起，我们的工作将促进斑马鱼作为注释和表征脊椎动物基因组的非编码部分的主要动物模型。公共卫生相关性：斑马鱼基因组调节工具箱的计算和功能注释，而基因占我们DNA的不到3％，其余的97％在其他众多极为重要的序列（例如基因调节元件）中，这些序列（例如基因调节元素）指导基因何时何时打开或关闭。这些基因调节元件中的突变可能会对人类疾病产生很大的影响，但是它们的位置和代码仍然对大多数未知。在该提案中，我们将利用斑马鱼模型生物的独特特性，使其与快速功能性斑马鱼测定法相结合，以注释这些序列，并更好地了解脊椎动物基因调节法规，这对我们对我们对多种人类疾病的遗传原因的理解非常重要。