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

描述（由申请人提供）： 我们的目的是开发一个强大的，相关的和快速的脊椎动物模型系统，用于分析基因特异性保守非编码元件（CNEs）所赋予的调控功能。支持这一提议的假设是，许多发育过程，包括胚胎形成和器官发生，在脊椎动物中是高度保守的，并且调节这些过程的遗传机制也可能是保守的。热带爪蟾基因组序列草图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允许人们容易地识别CNE，在许多情况下，CNE作为附近基因的调控元件。发现基因表达的复杂的长距离顺式调控，特别是发育控制基因，是比较基因组学的一个主要生物副产品。这个合作项目，旨在增加我们对正常器官发生和人类遗传疾病的理解，将使用非洲爪蟾的高效和快速的转基因系统来筛选系统中假定的调控元件，该系统允许许多基因活性的操作和生物功能的互补测定。这种方法的发展是非常重要的，因为它将提高生物学家的能力，以快速识别和检查基因中的调控元件，甚至是大量的基因，涉及细胞和发育生物学的许多方面。该项目通过开发和整合基因组和转基因方法，加强非洲爪蟾作为模式生物的实用性，为非洲爪蟾社区提供服务。我们建议将这种方法应用于眼睛的形成和功能的问题，在格兰杰实验室和合作的货车Heyningen组的研究主题，虽然从不同的，互补的角度。格兰杰实验室和其他几个实验室已经参与了鉴定胚胎眼睛形成中的调控基因。快速识别和研究眼睛形成相关基因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。一个非常不同的观点是，这些研究可以为理解人类遗传疾病提供见解，正如货车Heyningen实验室所研究的那样。公共卫生相关性：越来越多的证据表明，基因调控元件，一些相当远离编码序列，是正确的基因功能所必需的，当改变时，会导致疾病的表型。然而，还没有系统的方法来鉴定控制基因表达的增强子元件，因此可能受突变影响的推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开发一个大型的调控元件数据库，它们在基因表达中的作用，以及与它们中的突变相关的表型，这将是一个非常有价值的资源，用于识别人类患者的遗传病变。越来越多的证据表明，基因调控元件，一些相当远离编码序列，是正确的基因功能所必需的，当改变时，会导致疾病的表型。然而，还没有系统的方法来鉴定控制基因表达的增强子元件，因此可能受突变影响的推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开发一个大型的调控元件数据库，它们在基因表达中的作用，以及与它们中的突变相关的表型，这将是一个非常有价值的资源，用于识别人类患者的遗传病变。

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.