CONTROL OF SWITCH RECOMBINATION BY CD40 LIGAND

CD40 配体对开关重组的控制

• 批准号: 2672358
• 负责人: MICHAEL T BERTON
• 金额: $ 10.13万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672358
• 关键词: B lymphocyte; CD40 molecule; DNA binding protein; DNA footprinting; RNase protection assay; biological signal transduction; gel mobility shift assay; gene rearrangement; gene targeting; genetic mapping; genetic regulatory element; genetic transcription; helper T lymphocyte; immunoglobulin genes; interleukin 4; interleukin 5; ligands; nuclear runoff assay; polymerase chain reaction; site directed mutagenesis; transfection

DESCRIPTION (Adapted from Investigator's abstract): The long-term objective of this proposal is to elucidate the mechanisms underlying helper T (Th) cell-mediated regulation and targeting of isotype switching in murine B-cells. Cytokines such as IL-4 are believed to target switch recombination to specific isotypes by inducing germline Ig gene transcription, possibly making the switch regions accessible to a putative switch recombinase. T-cell contact-dependent signals required for isotype switching are provided by the CD40 ligand (CD40L)-CD40 interaction, but nothing is known about how these signals regulate isotype switching. The investigation has shown that recombinant CD40L induces expression of the germline gamma-1 and epsilon Ig genes in an IL-4 independent manner, and that IL-4 and CD40L synergize to promote maximal germline transcript expression. These results and results from other laboratories suggest that signals delivered via the CD40L-CD40 interaction may regulate isotype switching, at least in part, at the level of germline Ig gene transcription. The central goals of this proposal are to characterize in detail the mechanisms underlying CD40-mediated regulation of germline gamma-1 Ig gene transcription and to determine the role of CD40-mediated signals in activating the gamma-1 switch region for recombination in vivo. A PCR-based assay will be used to measure switch recombination of Sgamma-1 and to determine if and when CD40L and cytokines are required for activating the DNA recombination event. The effects of CD40-mediated signals on germline gamma-1 transcript expression will be characterized in detail by RNAase protection, nuclear run-on transcription assays and mRNA stability studies. CD40-responsive, cis-acting elements will be identified and mapped by transient transfection of germline gamma-1 promoter-luciferase reporter gene constructs. CD40-responsive DNA-binding proteins responsible for regulation of germline gamma-1 Ig gene transcription will be identified by EMSA, binding sites will be defined by DNA footprinting, and function assessed by site-specific mutagenesis and transfection of germline gamma-1 promoter-reporter gene constructs. Novel DNA-binding proteins will be isolated biochemically and/or by cDNA cloning and characterized in detail. Finally, the in vivo role of CD40-responsive germline gamma-1 promoter-binding proteins in regulating endogenous germline gamma-1 transcription and switch recombination to Sgamma-1 will be determined using a combination of protein and gene knock-out strategies.

描述（根据调查员的摘要改编）：长期目标 该提议的是阐明辅助辅助者t（th）的机制 细胞介导的调节和靶向鼠中的同种型切换 B细胞。 据信诸如IL-4之类的细胞因子靶向重组 通过诱导种系Ig基因转录来对特定的同种型，可能 使开关区域可用于推定的开关重组酶。 提供同型切换所需的T细胞触点依赖性信号 通过CD40配体（CD40L）-CD40相互作用，但对如何如何了解 这些信号调节同型切换。 调查表明 重组CD40L诱导种系γ-1和Epsilon Ig的表达 基因以IL-4独立方式，IL-4和CD40L协同为 促进最大种系转录本。 这些结果和结果 来自其他实验室的表明，通过CD40L-CD40传递的信号 交互可能至少部分地调节同种型切换在该级别 生殖线Ig基因转录。 该提议的核心目标是 详细描述CD40介导的调节的机制 种系γ-1 Ig基因转录的作用，并确定 CD40介导的信号激活伽马1开关区域 体内重组。 基于PCR的测定将用于测量开关 Sgamma-1的重组并确定CD40L和何时何时 激活DNA重组事件所必需。 效果 种系γ-1转录本上的CD40介导的信号将是 通过RNAase保护，核跑步转录详细描述 测定和mRNA稳定性研究。 CD40响应性，顺式作用元素 将通过瞬时转染生殖线γ-1鉴定和映射 启动子 - 卢西酶报告基因构建体。 CD40响应性DNA结合 负责调节种系γ-1 Ig基因的蛋白质 转录将由EMSA确定，绑定位点将由 DNA足迹和通过位点特异性诱变评估的功能和功能 种系γ-1启动子重生基因构建体的转染。 小说 DNA结合蛋白将通过生化和/或通过cDNA克隆分离 并详细描述。 最后，CD40响应的体内作用 种系γ-1启动子结合蛋白在调节内源性种系中 伽马1转录和开关重组对Sgamma-1将是 使用蛋白质和基因敲除策略的组合确定。