HUMAN IL-13 GENE REGULATION AND IMPACT OF POLYMORPHISMS

描述（改编自申请人摘要） 本提案的主题是对影响进行功能分析， 复杂的遗传变异模式对基因表达的影响。 长期 目的是确定调节人IL-13表达的机制， 单核苷酸多态性（SNPs）对IL-13表达的影响， 以及这些多态性导致易感性的机制 过敏和/或哮喘。 我们最近在人IL-13基因中发现了7个SNP， 基因 两个SNP位于IL-13的5'调控区，一个位于IL-13的第三调控区， 内含子，一个位于编码区并导致预测的氨基酸 改变，并且三个在3'非翻译区（UTR）中。 所有SNP，除了 在5'区的，几乎完全连锁不平衡，和 稀有等位基因频率为0.23。 值得注意的是，对于罕见的 变异与血清总IgE增加和喘息密切相关， 年龄6岁，表明遗传变异导致IL-13失调 表情 在此，我们建议： 1.分析IL-13位点的染色质重塑，并评估是否 多态性在控制DNA可接近性中起作用。 我们将使用 用于监测和比较染色质的DNA酶I超敏反应（HS）测定 来自个体的Th 1和Th 2 T细胞克隆中IL-13位点的可及性 不同IL-13基因单倍型的纯合子。 此外，我们将 研究DNA甲基化和组蛋白乙酰化在控制 人IL-13基因的表达。 2.为了分析与核酸酶HS位点相对应的DNA元件， 在IL-13转录调节中的作用，并鉴定任何功能性 与遗传多态性相关的改变。 我们将使用记者 确定含有HS位点的元件的转录作用的测定， 我们将识别出与这些元件结合的转录因子， 确定它们是否调节可及性和/或转录。 在 每一步，野生型和多态性DNA区域将被比较其 功能特性 确定转录后机制在调节IL-13中的作用 基因表达，并确定与多态性相关的任何改变 存在于3 'UTR中。 我们将生成结构来确定是否野生- 3 'UTR影响稳定的报告mRNA的半衰期。 将在3 'UTR中引入缺失和突变以鉴定顺式 mRNA不稳定的相关因素，并测试 单个3 'UTR多态性或其组合。 此外，记者 含有野生型或多态性编码区cDNA的构建体， 异源启动子的控制将用于评估编码 区域含有不稳定元件，其功能可被IL-10改变。 13/+2044。

DESCRIPTION (Adapted from the applicant's abstract) The main theme of this proposal is the functional analysis of the impact that complex patterns of genetic variation have on gene expression. The long-term objective is to determine the mechanisms that regulate human IL-13 expression, the influences of single nucleotide polymorphisms (SNPs) on IL-13 expression, and the mechanisms by which the polymorphisms contribute to susceptibility to allergy and/or asthma. We recently identified seven SNPs in the human IL-13 gene. Two SNPs are in the 5' regulatory region of IL-13, one is in the third intron, one is in the coding region and leads to a predicted amino acid change, and three are in the 3' untranslated region (UTR). All SNPs, except the ones in the 5' region, are in almost complete linkage disequilibrium, and the frequency of the rare alleles is 0.23. Notably, homozygosity for the rare variants is strongly associated to increased total serum IgE and wheezing at age 6, suggesting that genetic variation results in dysregulation of IL-13 expression. Here we propose: 1. To analyze chromatin remodeling at the IL-13 locus and to assess whether the polymorphisms play a role in controlling DNA accessability. We will use DNAse I hypersensitivity (HS) assays to monitor and compare chromatin accessibility at the IL-13 locus in Th1 and Th2 T cell clones from individuals homozygous for different IL-13 gene haplotypes. Furthermore we will investigate the role of DNA methylation and histone acetylation in the control of human IL-13 gene expression. 2. To analyze the DNA elements corresponding to nuclease HS sites for their role in the regulation of IL-13 transcription and to identify any functional alterations associated with genetic polymorphisms. We will use reporter assays to define the transcriptional role of the elements containing HS sites, and we will identify the transcription factors that bind those elements, establishing whether they modulate accessibility and/or transcription. At each step, wild-type and polymorphic DNA regions will be compared for their functional properties. To establish the role of post-transcriptional mechanisms in regulating IL-13 gene expression and to identify any alterations related to polymorphisms present in the 3'UTR. We will generate constructs to determine whether wild- type 3'UTR affects the half-life of an otherwise stable reporter mRNA. Deletions and mutations will be introduced in the 3'UTR to identify cis elements involved in mRNA destabilization and to test the influence of individual 3'UTR polymorphisms or combinations thereof. In addition, reporter constructs containing wild-type or polymorphic coding region cDNA under the control of a heterologous promoter will be used to assess whether the coding region contains a destabilizing element, whose function may be altered by IL- 13/+2044.