MECHANISMS FOR EXPRESSION OF IFN-GAMA IN T-CELLS

T 细胞中 IFN-GAMA 的表达机制

• 批准号: 2057181
• 负责人: LAURIA A PENIX
• 金额: $ 9.06万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2057181
• 关键词: DNA binding protein; DNA footprinting; T lymphocyte; affinity chromatography; crosslink; gel mobility shift assay; gene expression; genetic regulatory element; human tissue; interferon gamma; leukocyte activation /transformation; methylation; tissue /cell culture; transcription factor; transfection; ultraviolet radiation

Laurie Penix (hereafter referred to as I) completed residency training in Pediatrics two and one-half years ago, and since that time has been a fellow in Pediatric Infectious Diseases at the University of Washington. I have worked in the laboratory of Dr. Christopher B. Wilson, seeking to explore the basis for the increased susceptibility of the human neonate to infections with intracellular pathogens. Although considerable progress has been made in acquiring the skills necessary to establish an independent laboratory career, an additional period of training supported by the proposed CIA is critical. I believe that Dr. Wilson's laboratory, and the environment provided by its relationship to other investigators in the Department of Immunology and the overall scientific community at the University of Washington, will foster my development into an independent investigator during the tenure of this Clinical Investigator Award. Previous data from our laboratory and others, indicated that a profound deficit in the production of interferon-gamma (IFN-gamma) by T cells from neonates, was a contributing factor to this increased susceptibility. This defect was relatively selective in that the production of IL-2 did not differ between adult and neonatal T cells. this reflected the lack of memory T cells in neonates, since putative naive and memory T cells produce comparable amounts of IL-2, whereas naive T cells (comprising 50- 65% of adult but > 95% of neonatal T cells) produce 10-fold less IFN- gamma. The differences between memory and naive T cells were due to diminished initiation of transcription of the IFN-gamma gene. I joined the laboratory and began to investigate the transcriptional regulation of the IFN-gamma gene, with the long term goal of identifying cis- elements and factors binding thereto, which account for the markedly different expression of this gene in naive T cells. My preliminary data strongly suggest that elements, which are necessary and sufficient for T cell- and activation-specific expression of lacZ reporter constructs are present within 111 bp of the IFN-gamma transcription start site; included therein are elements which bind the transcription factor GATA-3. I will examine the hypothesis that differences in expression/activation of proteins binding to the key regulatory elements in the -111 to +61 region of the IFN-gamma gene, contribute to the differential expression of this gene in naive and memory T cells. This will be done by two complementary approaches (Aims 1 and 2), pursued in parallel. to define the minimal cis-elements in the IFN-gamma promoter that confer T cell specific expression following activation, I will use transient transfection assays in Jurkat T cells. To identify the trans-acting factors interacting with the key cis-elements, I will use electrophoretic mobility shift assays, UV cross-linking, methylation interference and partial affinity purification. Once these elements and factors are delineated, in Aim 3 I will explore their role in the differential expression of IFN-gamma. This will be done by comparing trans-acting factors present in extracts from adult memory T cells to those present in extracts from naive T cells from neonates and adults. This information will enhance our understanding of the molecular basis of T cell development and may provide insight into potential therapeutic approaches to enhance cell-mediated immunity in neonates.

劳里·佩尼克斯（Laurie Penix）（以下称为我）完成了居住培训 两年前在儿科中，从那时起 大学的小儿传染病研究员 华盛顿。 我曾在克里斯托弗·B（Christopher B. B.）的实验室工作。 威尔逊，试图探索提高易感性的基础 人类新生儿对细胞内病原体感染。 虽然 在获取必要的技能方面取得了很大进步 建立独立的实验室职业，额外的时期 提议的中央情报局支持的培训至关重要。 我相信博士 威尔逊的实验室及其与之的关系所提供的环境 免疫学系的其他调查人员和整体 华盛顿大学的科学界将培养我 在此期间，开发成独立研究者 临床研究者奖。 我们实验室和其他实验室的先前数据表明，一个深刻的数据 T细胞中T细胞生产干扰素 - 伽马（IFN-GAMMA）的赤字 新生儿是增加这种敏感性的促成因素。 这种缺陷相对选择性，因为IL-2的产生DID 成人和新生T细胞之间没有差异。 这反映了缺乏 由于推定的天真和记忆T细胞，新生儿的记忆T细胞 产生可比量的IL-2，而天真的T细胞（包括50- 65％的成人但> 95％的新生儿T细胞）产生的IFN-少10倍 伽玛。 记忆和天真T细胞之间的差异是由于 IFN-GAMMA基因转录的开始减少。 我加入了 实验室并开始调查转录调节 IFN-gamma基因的长期目标是确定顺式 该元素和因素具有约束力，这显着解释了 该基因在幼稚T细胞中的不同表达。 我的初步数据 强烈暗示要且足够的要素 LACZ报告基因构建体的T细胞和激活特异性表达 存在于IFN-GAMMA转录起始位点的111 bp之内； 其中包括的是结合转录因子GATA-3的元素。 我将研究表达/激活差异的假设 与-111至+61中的关键调节元件结合的蛋白质 IFN-GAMMA基因的区域有助于差异表达 该基因在幼稚和记忆T细胞中。 这将由两个 互补方法（目标1和2），并并行追求。 定义 赋予T细胞的IFN-GAMMA启动子中的最小顺式元素 激活后的特定表达式，我将使用瞬态 在Jurkat T细胞中的转染测定。 识别跨性别 与关键顺式元素相互作用的因素，我将使用电泳 移动性转移测定，紫外线交联，甲基化干扰和 部分亲和力净化。 一旦这些要素和因素是 划分了，在目标3中，我将探索它们在差异中的作用 IFN-gamma的表达。 这将通过比较反式作用来完成 从成人记忆T细胞提取到存在的因素 在来自新生儿和成人的幼稚T细胞的提取物中。 这 信息将增强我们对t分子基础的理解 细胞开发，并可能深入了解潜在的治疗 增强细胞介导的新生儿免疫力的方法。