Regulation of Gene Expression in the Th2 Cytokine Locus

描述（由申请人提供）：在Th2 T细胞效应物的发展过程中，IL-4基因座在染色质结构上经历了显著的变化，促进了基因座内基因的协调表达。这些变化包括但不限于DNA酶1敏感性、组蛋白乙酰化和DNA甲基化的变化。在许多已确定的基因座中，这些变化是协调的，并可能受到顺式作用元件的调节，如基因座控制区（Locus Control Regions， LCR）和绝缘子或边界元件。到目前为止，在IL-4基因座中还没有发现这样的元件。我们已经发现了一个以前未被识别的RAD50基因区域，该区域在Th2细胞中特异性地成为DNase 1超敏（HS），并包含基因座控制区的特性。我们假设这个区域包括基因座控制区的元素，也可能是绝缘体。该项目的主要目标是描述和功能表征该区域内具有LCR和/或绝缘体活性的元素。第一个具体目标是表征区域内HS元件的潜在增强子活性，因为lcr具有很强的谱系特异性增强子活性。我们将使用转基因小鼠系统来实现这一目标。第二个具体目标是评估整个基因座的染色质特征，重点关注乙酰化和DNasel敏感性模式的转变。这些实验旨在识别轨迹内潜在的边界元素。后续的实验将根据这些元件阻断增强子功能和/或防止浓缩染色质侵入的能力来表征它们。

DESCRIPTION (provided by applicant): During development into Th2 T cell effectors, the IL-4 locus undergoes significant changes in chromatin structure that facilitate coordinated expression of the genes within the locus. These changes include, but are not limited to, changes in DNase l sensitivity, histone acetylation, and DNA methylation. In a number of identified loci, these changes are coordinated, and possibly regulated by cis-acting elements, such as Locus Control Regions (LCR) and insulators or boundary elements. No such elements in the IL-4 locus have been identified to date. We have identified a previously unrecognized region of the RAD50 gene, which becomes DNase l hypersensitive (HS) specifically in Th2 cells and contains the properties of a Locus Control Region. We hypothesize that this region comprises elements of a Locus Control Region and possibly an insulator. The principal goal of this project is to delineate and functionally characterize elements within this region that possess LCR and/or insulator activity. The first specific aim is to characterize potential enhancer activity of HS elements within the region, as LCRs possess strong lineage-specific enhancer activity. We will use a transgenic mouse system to fulfill the goals of this aim. The second specific aim is to assess the chromatin character across the entire locus, focusing on transitions in acetylation and DNasel sensitivity patterns. These experiments are designed to identify potential boundary elements within the locus. Subsequent experiments will be performed to characterize these elements in terms of their ability to block enhancer function and/or prevent the encroachment of condensed chromatin. Inappropriate Th2 differentiation has been implicated in a number of pathological states, most notably allergic diseases. The information gleaned from these studies will contribute to our understanding of the process of Th2 differentiation, and will hopefully enable the ultimate goal, which is to intervene in this process, and enhance our ability to avoid or correct these pathological manifestations.