IgH Enhancer Function in an Ectothermic Vertebrate

变温脊椎动物中的 IgH 增强子功能

• 批准号: 6520381
• 负责人: GREGORY W WARR
• 金额: $ 23.85万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520381
• 关键词: B lymphocyte; biochemical evolution; catfish; gene expression; genetic enhancer element; immunogenetics; immunoglobulin genes; mutant; site directed mutagenesis; surface plasmon resonance; transcription factor; transfection

APPLICANT'S DESCRIPTION: Although transcriptional enhancers are essential for the expression of the immunoglobulin heavy chain (IgH) locus, they are also needed for the additional functions of V(D)J joining, V-region hypermutation and immunoglobulin (Ig) class switch recombination. This research is directed towards understanding the function of the enhancer that drives expression of the IgH locus in the channel catfish, a teleost fish and therefore lacking Ig class switching. Preliminary results suggest that the catfish IgH enhancer functions through a novel interaction of transcription factors bound to multiple interacting octamer and muE5 sites. The role of these sites in the function of the enhancer will be dissected by site-directed mutagenesis, and the function of the mutated enhancer will be assessed in transient expression assays upon transfection into catfish B-cells. Catfish muE5-binding factor(s) will be isolated and characterized. The interactions between Oct transcription factors, muE5binding factors and the DNA motifs that they recognize will be measured directly using the BiaCore 2000 instrument that utilizes surface plasmon resonance detection. Such studies will define whether or not cooperative binding reactions occur between the Oct and muE5-binding transcription factors. Gene expression driven by the interaction of octamer-binding (Oct) transcription factors with those binding to muE5 sites (typically bHLH or zinc-finger transcription factors) has not been reported in mammalian systems. Thus, these studies should shed light on novel' transcription factor interactions in Ig enhancers, and will also provide insight into how enhancer function has influenced the evolution of the IgH locus.

申请人的描述：尽管转录增强子对于 免疫球蛋白重链 (IgH) 位点的表达，它们也是 V(D)J 连接、V 区超突变的附加功能所需 和免疫球蛋白（Ig）类开关重组。这项研究是针对 旨在了解驱动表达的增强子的功能 斑点叉尾鮰（一种硬骨鱼）中的 IgH 基因座，因此缺乏 Ig 班级切换。初步结果表明鲶鱼 IgH 增强剂 通过转录因子的新颖相互作用发挥作用 多个相互作用的八聚体和 muE5 位点。这些网站的作用 增强子的功能将通过定点诱变进行剖析，并且 将在瞬时表达中评估突变增强子的功能 转染鲶鱼 B 细胞后进行测定。鲶鱼 muE5 结合因子 将被隔离和表征。 Oct转录之间的相互作用 因子、muE5结合因子和它们识别的DNA基序将被 使用利用表面的 BiaCore 2000 仪器直接测量 等离子共振检测。此类研究将确定是否 Oct 和 muE5 结合之间发生协同结合反应 转录因子。基因表达由相互作用驱动 八聚体结合 (Oct) 转录因子以及与 muE5 位点结合的转录因子 （通常是 bHLH 或锌指转录因子）尚未见报道 哺乳动物系统。因此，这些研究应该揭示小说的意义 Ig 增强子中的转录因子相互作用，并且还将提供 深入了解增强子功能如何影响 IgH 的进化 轨迹。