CONTROL OF SWITCH RECOMBINATION BY CD40 LIGAND

描述(改编自《调查者摘要》)：长期目标 这一建议的目的是阐明辅助T(Th)的机制 小鼠同型转换的细胞调控和靶向性研究 B细胞。IL-4等细胞因子被认为是开关重组的靶点 通过诱导生殖系Ig基因转录，可能与特定的同型有关 使开关区域可被假定的开关重组酶访问。 提供了同种类型切换所需的T-cell触点相关信号 通过CD40配体(CD40L)-CD40的相互作用，但对其如何作用尚不清楚 这些信号调节同型转换。调查表明， 重组CD40L诱导生殖系γ-1和epsilon Ig的表达 基因以IL-4非依赖的方式，以及IL-4和CD40L协同作用 促进生殖系转录本的最大表达。这些结果和结果 来自其他实验室的研究表明，通过CD40L-CD40传递的信号 相互作用可以至少部分地在水平上调节同型转换 生殖系Ig基因转录。这项提议的中心目标是 详细描述CD40介导的调控机制 对生殖系γ-1免疫球蛋白基因转录的影响及确定作用 CD40介导的信号在激活γ-1开关区的作用 体内重组。一种基于聚合酶链式反应的分析方法将被用于测量开关 重组SGamma-1并确定CD40L和细胞因子是否以及何时 是激活DNA重组事件所必需的。的影响 CD40介导的信号对生殖系伽马-1转录表达的影响 以RNAase保护、核连续转录为详细特征 检测和信使核糖核酸稳定性研究。CD40反应元件，顺式作用元件 将通过瞬时转染生殖系伽马-1进行鉴定和定位 启动子-荧光素酶报告基因构建。CD40反应的DNA结合 负责调节生殖系γ-1Ig基因的蛋白质 转录将由EMSA鉴定，结合位点将由 DNA足迹和功能通过定点突变和 生殖系γ-1启动子-报告基因的转染构建。小说 DNA结合蛋白将通过生物化学和/或cdna克隆来分离。 并对其进行了详细的表征。最后，CD40应答在体内的作用 生殖系γ-1启动子结合蛋白在调节内源性生殖系中的作用 伽马-1转录和开关重组到SGamma-1将是 使用蛋白质和基因敲除策略的组合来确定。

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.