MOLECULAR CHARACTERIZATION OF PERFORIN GENE REGULATION

穿孔素基因调控的分子特征

• 批准号: 2096916
• 负责人: Mathias Georg Lichtenheld
• 金额: $ 10.8万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2096916
• 关键词: DNA footprinting; T cell receptor; biological signal transduction; cytolysins; cytotoxic T lymphocyte; gel mobility shift assay; genetic regulation; genetic regulatory element; genetic transcription; laboratory mouse; leukocyte activation /transformation; natural killer cells; northern blottings; nuclear runoff assay; nucleic acid structure; pore forming protein; tissue /cell culture; transcription factor; transfection

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse virus-infected cells and possibly emerging tumor cells but they also contribute to transplant rejection and perhaps to autoimmune tissue destruction. One of the lytic molecules of CTL and NK cells is a pore-forming protein known as perforin. Perforin gene expression is restricted to CTL and NK cells. While NK cells express perforin constitutively, CTL express perforin upon their activation. Our objective is to characterize relevant molecular mechanisms 1) that determine the CTL and NK-cell restricted expression of perforin; 2) that determine the induction of perforin in CTL upon their activation; and we propose 3) to identify the transcription factors involved. Within the first objective, we will characterize a cell-type specific cis-acting element in the 5' flanking region of the perforin gene (specific aim 1. 1), determine the role of the proximal perforin promoter (specific aim 1.2) and analyze and determine the role of the chromatin structure of the perforin gene locus (specific aim 1.3). To achieve these aims we will analyze fragments of the perforin 5' flanking and promoter region for their control over the "CAT" reporter gene after transient transfection into different cell types and will compare DNase I hypersensitivity sites of the perforin gene locus between different cell types. Within the second objective, we will determine whether perforin mRNA induction in CTL occurs primarily at the transcriptional level or the posttranscriptional level (specific aim 2.1). We anticipate that the regulation event takes place at the transcriptional level based on our preliminary data and therefore propose to identify regulatory elements involved (specific aim 2.2). To achieve these aims we will analyze, by Northern blot analysis and nuclear run-off assay, perforin gene induction or upregulation in cloned CTL in response to IL-2, T-cell receptor signals or second-messenger pathway activating agents. We will identify the cis-acting responsive regions by reporter gene analysis as outlined above. Within the third objective, we propose to identify putative transcription factors acting on relevant regulatory elements defined in the previous aims (specific aim 3). We will apply in vitro DNase I footprinting and will analyze a particular DNA binding site by gel shift analysis.

细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞溶解 病毒感染的细胞和可能出现的肿瘤细胞，但它们也 导致移植排斥反应， 杀伤性 CTL和NK细胞的溶解分子之一是 一种叫做穿孔素的成孔蛋白。 穿孔蛋白基因表达是 仅限于CTL和NK细胞。 而NK细胞表达穿孔素 组成型地，CTL在其活化时表达穿孔素。 我们 目的是表征相关的分子机制1）， 确定穿孔素的CTL和NK细胞限制性表达; 2） 确定CTL激活后穿孔素的诱导; 建议3）鉴定所涉及的转录因子。 内 第一个目标，我们将表征细胞类型特异性顺式作用 穿孔素基因的5'侧翼区中的元件（特异性目的1. 1）、确定近端穿孔素启动子的作用（特异性靶向 1.2)并分析和确定染色质结构的作用， 穿孔素基因位点（特异性目的1.3）。为了实现这些目标，我们将 分析穿孔素5'侧翼和启动子区域的片段， 它们在瞬时转染后对“CAT”报告基因的控制 并将比较DNA酶I超敏位点 不同细胞类型之间的穿孔素基因位点。 内 第二个目标，我们将确定是否穿孔素mRNA诱导， CTL主要发生在转录水平或转录水平。 转录后水平（具体目标2.1）。我们预计 调控事件发生在转录水平上，基于我们的 初步数据，因此建议确定监管要素 参与（具体目标2.2）。为了实现这些目标，我们将分析， 北方印迹分析和核溢流分析，穿孔素基因诱导 或在克隆的CTL中响应于IL-2、T细胞受体 信号或第二信使途径激活剂。 我们将确定 如所概述的，通过报告基因分析顺式作用应答区 以上 在第三个目标中，我们建议确定假定的 作用于相关调控元件的转录因子 目标3（具体目标3）。 我们将在体外应用DNase I 足迹，并将分析一个特定的DNA结合位点的凝胶位移 分析.