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）基因座的表达，它们也 需要V（D）J连接的额外功能，V区超突变 和免疫球蛋白（IG）类别转换重组。这项研究是针对 为了理解增强子的功能， 斑点叉尾鱼中的IgH位点，一种硬骨鱼，因此缺乏IG 阶级转换初步结果表明，鲶鱼IgH增强剂 通过转录因子的新型相互作用发挥作用， 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来剖析，并且 突变增强子的功能将在瞬时表达中评估 在转染到鲶鱼B细胞中后的测定中。鲶鱼muE5结合因子 将被分离并鉴定。Oct转录与 因子，muE5结合因子和它们识别的DNA基序将被 使用BiaCore 2000仪器直接测量，该仪器利用表面 等离子体共振检测这些研究将确定是否 协同结合反应发生在Oct和muE5结合之间 转录因子基因表达由以下相互作用驱动： 与muE5位点结合的八聚体结合（Oct）转录因子 （通常是bHLH或锌指转录因子）尚未报道， 哺乳动物系统因此，这些研究应该对小说的发展有所启示。 转录因子相互作用的IG增强子，也将提供 深入了解增强子功能如何影响IgH的进化 基因座