CHARACTERIZATION OF AN ALTERNATIVE IL4 GENE TRANSCIPT EXPRESSED IN MAST CELLS

我们之前已经在第二个中描述了DNA调控元件 小鼠IL-4基因的内含子。这个元素展示了肥大细胞- 瞬时转染法检测特异性增强子活性。分析 这种元素的成分揭示了细胞的特异性 活动归因于增强子内的关键部位，这些部位充当 肥大细胞中表达的转录因子的结合位点，但没有 T细胞。尽管该元件与异源启动子一起工作良好 在报告基因检测中，它与5“IL-4只有边际活性 启动子，表明它的生理作用不参与促进 5“IL-4启动子介导的转录。最近变得明显 无TATA启动子元件(一致的INR序列)被定位 就在先前定义的增强子元件的下游。INR- 含有启动子的元件位于之前的下游 定义的增强器元素。含有INR的启动子元件为 活性，并似乎驱动一种独特的、截短的IL-4的转录 文字记录。这份文字记录包含了启动 在外显子II和IV中剪接的INR序列的立即3‘。 对这种“截短”的信使核糖核酸表达谱的研究表明 在未受刺激的肥大细胞中表达水平相对较高 脾文化。这份文字记录的表达方式与此相反 与全长转录本相关的：细胞刺激 结果导致这种mRNA物种的消失，并增加了 全长IL-4元件。根据这些数据，我们假设 截短的IL-4mRNA参与IL-4产生的调节 和/或活性，其转录受内含子控制 增强剂。该提案描述了旨在定义 文字稿的功能及其表达细节的探讨 和监管。

We have previously characterized a DNA regulatory element in the second intron of the murine IL-4 gene. This element exhibits mast cell- specific enhancer activity in transient transfection assays. Analysis of the components of this element revealed that the cell specific activity is due to critical sites within the enhancer that serve as binding sites for transcription factors expressed in mast cells but no T cells. Although this element works well with a heterologous promoter in reporter gene assays, it has only marginal activity with the 5"IL-4 promoter, suggesting its physiological role is not involved in enhancing 5"IL-4 promoter-mediated transcription. It has recently become apparent that a TATA-less promoter element (a consensus Inr sequence) is located just downstream of the previously defined enhancer elements. The Inr- containing promoter element is located just downstream of the previously defined enhancer elements. The Inr-containing promoter element is active and appears to drive transcription of a unique, truncated IL-4 transcript. This transcript contains sequences that initiate immediately 3' of the Inr sequence that are spliced in exons II and IV. Studies of the expression profile of this "truncated" mRNA indicate it is expressed at relatively high levels in unstimulated mast cells and splenic cultures. This expression of this transcript is inversely correlated with that of the full length transcript: cell stimulation results in the disappearance of this mRNA species and an increase in the full length IL-4 element. Based on these data, we hypothesize that the truncated IL-4 mRNA is involved in the regulation of IL-4 production and/or activity, and its transcription is controlled by the intronic enhancer. This proposal describes experiments aimed at defining the function of the transcript and exploring the details of its expression and regulation.