Genome-wide discovery of beta cell gene control elements

描述（由申请人提供）： 糖尿病正迅速成为一个主要的公共卫生问题，2000年在北美报告了1420万例病例，全世界有1.51亿例病例，预计到2010年全世界发病率将增加46%。在1型和2型糖尿病中，产生胰岛素的胰腺β细胞在疾病的病理生理学中起着核心作用。因此，更好地了解β细胞生物学对于开发降低与这种疾病相关的发病率和过早死亡率的治疗策略至关重要。 基因组学的出现大大扩展了β细胞的知识基础，发现数千个基因在内分泌胰腺中特异性表达。这些发现提出了这种组织特异性表达如何产生的问题。胰腺转录因子在胰岛素基因表达和胰腺发育中的重要作用已得到充分表征。此外，转录因子基因的突变与糖尿病的遗传形式有关，再次提出了β细胞中基因控制的问题。我们将用一种新的策略来解决这个生物学问题，这种策略允许在全基因组水平上识别基因调控元件。迄今为止尚未实现基因控制元件的这种全面鉴定，并且将使β细胞成为关于基因表达控制的最好理解的细胞类型。 我们提出了一个整合的方法，β细胞特异性基因控制元件的全基因组识别相结合的实验研究与生物信息学和计算分析。从人基因组文库中，将凭借其在转染入β细胞后驱动抗生素抗性基因表达的能力来选择携带β细胞特异性增强子的DNA片段。将通过种间序列比较生成胰腺特异性表达基因的保守非编码区数据库。我们将交叉引用实验确定的增强子与这个数据库的保守区域，分析这些区域的转录因子结合位点，纳入基因表达的公共数据，并表征基因间的共调控机制。总之，我们的研究将显示斑点基因表达是如何在β细胞中实现的，并提供一个框架来整合基因转录和β细胞生物学。

DESCRIPTION (provided by applicant): Diabetes is rapidly becoming a major public health concern, with 14.2 million cases reported in North America for the year 2000, 151 million cases worldwide, and a projected increase of 46% for worldwide incidence by the year 2010. In both Type 1 and Type 2 diabetes, the insulin-producing pancreatic beta cell plays a central role for the pathophysiology of the disease. Therefore, a better understanding of beta cell biology is crucial for the development of therapeutic strategies that reduce morbidity and premature mortality associated with this disease. The advent of genomics has considerably expanded the knowledge base on beta cells with the discovery that several thousand genes are specifically expressed in the endocrine pancreas. These findings bring up the question of how this tissue-specific expression arises. The prominent role of pancreatic transcription factors for insulin gene expression and pancreatic development is well characterized. Furthermore, mutations in transcription factor genes are linked to genetic forms of diabetes, again raising the question of gene control specifically in the beta cell. We will address this biological problem with a new strategy that allows identification of gene regulatory elements on a genome-wide level. Such a comprehensive identification of gene control elements has not been achieved to date and will make the beta cell the best understood cell type with regard to control of gene expression. We propose an integrated approach to genome-wide identification of beta cell-specific gene control elements that combines experimental investigation with bioinformatics and computational analysis. From a human genomic library, DNA fragments carrying beta cell-specific enhancers will be selected by virtue of their ability to drive expression of an antibiotic resistance gene after transfection into beta cells. A database of conserved non-coding regions for genes with specific expression in the pancreas will be generated through interspecies sequence comparisons. We will cross-reference the conserved regions on experimentally identified enhancers with this database, analyze such regions for transcription factor binding sites, incorporate public data on gene expression, and characterize mechanisms of co-regulation among genes. Together, our studies will show how speck gene expression is achieved in beta cells and provide a framework to integrate gene transcription and beta cell biology.