Genomic survey of cis-regulatory element function by high-throughput transgenesis

描述(由申请人提供)： 我们的目标是开发非洲爪哇作为一个强大的，相关的和快速的脊椎动物模型系统，用于分析基因特异的保守非编码元件(CNES)所赋予的调节功能。这一提议背后的假设是，许多发育过程，包括胚胎模式和器官发生，在脊椎动物中是高度保守的，而且调节这些过程的遗传机制也可能是保守的。热带非洲爪哇的草稿基因组序列为脊椎动物基因调控的比较分析提供了强大的资源，因为从青蛙到哺乳动物的进化距离使人们能够很容易地识别CNE，在许多情况下，CNE是附近基因的调控元件。基因表达的复杂的长程顺式调控的发现，特别是发育控制基因的发现，是比较基因组学的主要生物学副产品。这一合作项目旨在增加我们对正常器官发生和人类遗传疾病的了解，将使用非洲爪哇的高效和快速转基因系统来筛选系统中可能的调控元件，该系统允许对基因活动进行许多操作并对生物功能进行补充分析。这一方法学的发展具有非常重要的意义，因为它将增强生物学家快速识别和检查基因中的调控元件的能力，甚至是涉及细胞和发育生物学的许多方面的大组基因。该项目通过开发和整合基因组和转基因方法，加强非洲爪哇作为模式生物的效用，为整个非洲爪哇社区服务。我们建议将这种方法应用于眼睛形成和功能的问题，这是格兰杰实验室和合作的Van Heyningen小组的研究主题，尽管是从不同的、互补的角度。格兰杰实验室和其他几个实验室一直在识别与胚胎眼睛形成有关的调控基因。快速识别和研究参与眼睛形成的基因的调控元件的能力已经开始提供，并将继续揭示关于控制眼睛形成的基因网络的重要新见解。一个非常不同的视角是，这些研究可以为理解人类遗传病提供洞察力，就像Van Heyningen实验室所研究的那样。与公共卫生相关：越来越多的证据表明，基因调控元件对正确的基因功能至关重要，当改变时，会导致疾病表型，其中一些调控元件距离编码序列很远。然而，目前还没有系统的方法来识别控制基因表达的增强子元件，因此可能受到突变影响的假定增强子的数据库仅限于一小群偶然发现的元件。这里描述的方法允许人们开发一个大型的数据库，其中包括调控元件、它们在基因表达中的作用以及与它们的突变相关的表型，这将是识别人类患者遗传损伤的一个非常有价值的资源。越来越多的证据表明，基因调控元件，其中一些距离编码序列相当远，对于正确的基因功能是必不可少的，当改变时，会导致疾病表型。然而，目前还没有系统的方法来识别控制基因表达的增强子元件，因此可能受到突变影响的假定增强子的数据库仅限于一小群偶然发现的元件。这里描述的方法允许人们开发一个大型的数据库，其中包括调控元件、它们在基因表达中的作用以及与它们的突变相关的表型，这将是识别人类患者遗传损伤的一个非常有价值的资源。

DESCRIPTION (provided by applicant): Our aim is to develop Xenopus tropicalis as a robust, relevant and rapid vertebrate model system for the analysis of the regulatory functions conferred by gene-specific conserved non-coding elements (CNEs). The hypothesis underlying this proposal is that many developmental processes, including embryonic patterning and organogenesis, are highly conserved among vertebrates, and that the genetic machinery regulating these processes is also likely to be conserved. The Xenopus tropicalis draft genome sequence provides a powerful resource for comparative analysis of vertebrate gene regulation, because the evolutionary distance from frog to mammals permits one to readily identify CNEs, which in many cases act as regulatory elements for nearby genes. Discovery of complex long-range cis-regulation of gene expression, particularly for developmental control genes, is a major biological spin-off of comparative genomics. This collaborative project, aimed at increasing our understanding of both normal organogenesis and human genetic diseases, will use the highly efficient and rapid transgenesis system in Xenopus to screen putative regulatory elements in a system that allows many manipulations of gene activity and complementary assays of biological function. Development of this methodology is highly significant because it will enhance the ability of biologists to rapidly identify and examine regulatory elements in genes, even large sets of genes, involved in many facets of cell and developmental biology. This project serves the Xenopus community at large by developing and integrating genomic and transgenic approaches that strengthen the utility of Xenopus as a model organism. We propose to apply this approach to the problem of eye formation and function, the subject of study in both the Grainger laboratory and the collaborating van Heyningen group, albeit from differing, complementary perspectives. The Grainger lab, and several others, have been involved in identification of regulatory genes involved in embryonic eye formation. The ability to rapidly identify and study the regulatory elements of genes involved in eye formation has begun to provide, and will continue to reveal significant new insights regarding the gene networks controlling eye formation. A very different perspective is the insight that these studies can provide for understanding human genetic diseases, as studied in the van Heyningen laboratory. PUBLIC HEALTH RELEVANCE: Increasingly there is evidence that gene regulatory elements, some quite distant from the coding sequence, are essential for correct gene function, and, when altered, cause disease phenotypes. There has been no systematic way to identify enhancer elements controlling gene expression, however, and therefore the database of putative enhancers that might be affected by mutations is limited to a small group of serendipitously discovered elements. The approaches described here permit one to develop a large database of regulatory elements, their roles in gene expression, and the phenotypes associated with mutations in them, which will be a highly valuable resource for identifying genetic lesions in human patients. Increasingly there is evidence that gene regulatory elements, some quite distant from the coding sequence, are essential for correct gene function, and, when altered, cause disease phenotypes. There has been no systematic way to identify enhancer elements controlling gene expression, however, and therefore the database of putative enhancers that might be affected by mutations is limited to a small group of serendipitously discovered elements. The approaches described here permit one to develop a large database of regulatory elements, their roles in gene expression, and the phenotypes associated with mutations in them, which will be a highly valuable resource for identifying genetic lesions in human patients.