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.
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