Structural Annotation of the human Genome

描述（由申请人提供）：基因表达的调节对于正常发育至关重要，该过程中的缺陷可能导致疾病。更好地了解有助于调节疾病基因的遗传元件可以揭示疾病的原因，并可能刺激新疗法的开发。对基因组的大规模分析，如ENCODE项目发起的分析，已经开始揭示许多调控元件的位置。然而，在大多数情况下，不知道哪些元件调节任何给定的基因。由于调控元件和基因沿染色体沿着的线性顺序往往不能反映它们之间的功能关系，因此理解调控元件和基因之间的关系变得复杂。例如，调控元件可能会影响远端基因，但不会影响紧邻它们的基因。因此，基因和调控元件之间的功能连接必须通过实验来确定。该提议基于这样的假设，即调控元件通过形成染色质环与其靶基因物理关联。我们建议通过绘制ENCODE联盟选择的1%基因组中基因和调控元件之间的环状相互作用来验证这一假设。我们将使用一种独特的方法，染色体构象捕获（3C）方法检测染色质环。在过去的2年中，我们开发了一种新的高通量3C应用程序，称为5C（3C-碳拷贝），它采用微阵列或定量测序检测染色质环。我们将通过分析β-珠蛋白基因座进一步优化5C（目的1）。我们将使用5C来识别基因和调控元件之间的染色质环，这些基因和调控元件贯穿人类基因组的充分研究的ENCODE区域（目的2）。对于每个基因，我们将确定远距离元件，如与其启动子相互作用的增强子。我们将通过FISH验证循环相互作用（目的3）。我们将使用瞬时转染并通过将成环数据与ENCODE联盟获得的其他数据（如组蛋白修饰、DNasel超敏反应和靶基因表达）整合来测试成环元件的功能（目的3）。我们将通过UCSC基因组浏览器提供循环数据。这些研究将绘制基因和调控元件之间的连接网络，并将揭示长期基因调控机制的新见解。

DESCRIPTION (provided by applicant): Regulation of gene expression is crucial for normal development and defects in this process can result in disease. A better understanding of the genetic elements that contribute to regulation of disease genes can reveal causes of disease and may spur development of novel treatments. Large-scale analyses of genomes, such as those initiated by the ENCODE project, have started to reveal the positions of many regulatory elements. However, in most cases it is not known which elements regulate any given gene. Understanding the relationships between regulatory elements and genes is complicated by the fact that the linear order of elements and genes along chromosomes often does not reflect functional relationships between them. For instance, regulatory elements may affect distal genes but not those located immediately next to them. Therefore, functional connections between genes and regulatory elements must be experimentally determined. This proposal is based on the hypothesis that regulatory elements physically associate with their target gene through formation of chromatin loops. We propose to test this hypothesis by mapping of looping interactions between genes and regulatory elements throughout the well-studied 1 % of the genome selected by the ENCODE consortium. We will detect chromatin loops using a unique approach, Chromosome Conformation Capture (3C) methodology. During the last 2 years we have developed a new high-throughput 3C application, called 5C (for 3C-carbon-copy) which employs microarrays or quantitative sequencing for detection of chromatin loops. We will further optimize 5C by analysis of the beta-globin locus (aim 1). We will employ 5C to identify chromatin loops between genes and regulatory elements throughout the well-studied ENCODE regions of the human genome (aim 2). For each gene we will identify distant elements such as enhancers that interact with its promoter. We will validate looping interactions by FISH (aim 3). We will test the function of looping elements using transient transfections and by integrating looping data with other data obtained by the ENCODE consortium such as histone modifications, DNasel hypersensitivity and target gene expression (aim 3). We will make looping data available through the UCSC genome browser. These studies will map the network of connections between genes and regulatory elements and will reveal new insights into the mechanisms that underlie long-range gene regulation.