CONTROL OF SWITCH RECOMBINATION BY CD40 LIGAND

描述（改编自研究者摘要）：长期目的 这一建议的目的是阐明辅助性T细胞（Th）的机制， 小鼠同种型转换的细胞介导调控和靶向 B细胞 细胞因子如IL-4被认为靶向开关重组 通过诱导生殖系IG基因转录，可能 使得开关区域可接近推定的开关重组酶。 提供同种型转换所需的T细胞接触依赖性信号 通过CD 40配体（CD 40 L）-CD 40相互作用，但不知道如何 这些信号调节同种型转换。 调查显示， 重组CD 40 L诱导种系γ-1和ε IG的表达 IL-4和CD 40 L协同作用， 促进最大生殖系转录本表达。 这些结果和成果 来自其他实验室的研究表明，通过CD 40 L-CD 40传递的信号 相互作用可以调节同种型转换，至少部分地，在水平上， 生殖系IG基因转录。 该提案的核心目标是 详细描述CD 40介导的调节机制， 生殖系γ-1 IG基因转录的影响，并确定 CD 40介导的激活γ-1开关区的信号， 体内重组。 将使用基于PCR的试验测量转换 重组的Sgamma-1，并确定是否和何时CD 40 L和细胞因子 是激活DNA重组事件所必需的。 的影响 CD 40介导的信号对生殖系γ-1转录本表达的影响将被研究。 其特征在于RNA酶保护，核连续转录 分析和mRNA稳定性研究。 CD 40应答性顺式作用元件 将通过瞬时转染生殖系γ-1 启动子-荧光素酶报告基因构建体。 CD 40应答性DNA结合 负责调节生殖系γ-1 IG基因的蛋白质类 转录将通过EMSA鉴定，结合位点将通过 DNA足迹法和通过位点特异性诱变评估的功能， 生殖系γ-1启动子-报告基因构建体的转染。 小说 DNA结合蛋白将通过生物化学和/或cDNA克隆进行分离 并详细描述了其特征。 最后，研究了CD 40-应答性T细胞在体内的作用。 调节内源性生殖系的生殖系γ-1启动子结合蛋白 γ-1转录和转换重组为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.