Molecular Mechanisms of Class Switch Recombination

类别转换重组的分子机制

• 批准号: 8386894
• 负责人: Frederick W. Alt
• 金额: $ 40.08万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386894
• 关键词: Address; Alanine; Allergic Disease; Antibodies; Antibody Formation; Autoimmune Diseases; B lymphoid malignancy; B-Lymphocytes; Biochemical Genetics; Biological Assay; Cancer Etiology; Cells; Chromosomal translocation; Chromosome Pairing; Chromosomes; Complex; Cytidine Deaminase; Cytogenetics; DNA; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA Structure; Deamination; Disease; Double Strand Break Repair; Exons; Extrinsic asthma; Frequencies; G22P1 gene; Gene Rearrangement; Genes; Genetic; Genetic Models; Genetic Transcription; Heavy-Chain Immunoglobulins; Higher Order Chromatin Structure; IGH@ gene cluster; IgE; Immune system; Immunoglobulin Class Switching; Immunoglobulin Joining Region; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunologic Deficiency Syndromes; Immunology; Indium; Link; Lymphoma; Malignant Neoplasms; Mediating; Methods; Modification; Molecular; Molecular Conformation; Mus; Mutant Strains Mice; Mutation; Nonhomologous DNA End Joining; Oncogenes; Outcome; Pathogenesis; Pathway interactions; Phosphorylation; Process; Production; Proteins; Reagent; Regulation; Reporter; Role; Series; Serine; Single-Stranded DNA; Site; Specificity; Testing; Transgenic Organisms; Vaccines; Work; Yeasts; Zebrafish; activation-induced cytidine deaminase; chromatin immunoprecipitation; ds-DNA; endonuclease; in vitro Assay; in vivo; insight; mutant; novel; novel strategies; protein complex; repaired; replication factor A; response

This application proposes studies of the mechanisms of immunoglobulin heavy chain (IgH) class switch recombination (CSR) and Somatic Hypermutation (SHM). We have shown that Activation Induced Cytidine Deaminase (AID), the initiator of CSR, is a single strand DNA (ssDNA) specific cytidine deaminase and we employed a series of novel biochemical and genetic approaches to elucidate mechanisms by which AID gains access to transcribed double strand (ds)DNA sequences in the context transcription-generated ssDNA structures and/or certain AID modifications or co-factors. We also showed that CSR may employ general processes for synapsis of AID-initiated DNA double strand breaks (DSBs), that general DNA repair factors function in CSR, and that two distinct end-joining pathways fuse S region breaks to complete CSR. Our current proposal builds on these observations in the context of three specific Aims. Our first aim proposes use of biochemical and genetic approaches to elucidate basic mechanisms of AID function and regulation. In this regard, we developed methods to purify AID from normal B cells, in vitro assays for transcription-dependent AID deamination of dsDNA DNA, and genetic approaches to evaluate in vivo AID functions elucidated biochemically. Our second aim addresses mechanisms by which DNA sequences influence AID activity and its outcome. For these studies, we developed targeted mutation assays to replace endogenous IgH class switch (S) regions and exons encoding IgH variable regions with test sequences that will allow us to determine how substrate sequences influence activities of AID and other relevant factors in CSR and SHM. Together, the complementary biochemical and genetic assays of Aims 1 and 2 offer a powerful approach for elucidating factors and mechanisms involved in initiation and regulation of IgH CSR and SHM. A third proposed aim is to elucidate processes involved in the repair of AID induced DSBs to complete CSR. For these studies, we again have developed a large array of reagents and novel approaches, including cytogenetic methods to follow CSR related breaks in chromosomes, novel genetic approaches to study factors involved in long range synapsis of DSBs, and genetic models to elucidate DSB repair pathways that complete IgH CSR. Our proposed studies should provide novel insights into the mechanism of antibody production via IgH CSR and, therefore, be relevant to understanding immunodeficiencies, vaccine immunology, and autoimmune diseases. As CSR is required for IgE production, the work will also be relevant to understanding pathogenesis of allergic diseases and asthma. Finally, the work is relevant to B cell malignancies as they often involve chromosomal translocations that link translocated oncogenes to IgH S regions via aberrant CSR.

本申请提出了免疫球蛋白重链 (IgH) 类别转换机制的研究 重组（CSR）和体细胞超突变（SHM）。我们已经证明激活诱导胞苷 脱氨酶 (AID) 是 CSR 的引发剂，是一种单链 DNA (ssDNA) 特异性胞苷脱氨酶，我们 采用了一系列新颖的生化和遗传学方法来阐明 AID 获益的机制 在转录生成的 ssDNA 中获取转录的双链 (ds)DNA 序列 结构和/或某些 AID 修饰或辅因子。我们还表明，企业社会责任可以采用一般 AID 引发的 DNA 双链断裂 (DSB) 的突触过程，即一般 DNA 修复因子 在 CSR 中发挥作用，并且两个不同的末端连接途径融合 S 区断裂以完成 CSR。我们的 当前的提案以三个具体目标的背景下的这些观察为基础。我们的首要目标是建议使用 生物化学和遗传学方法来阐明 AID 功能和调节的基本机制。在这个 为此，我们开发了从正常 B 细胞中纯化 AID 的方法，并进行转录依赖性体外测定 阐明双链 DNA 的 AID 脱氨基作用以及评估体内 AID 功能的遗传方法 生物化学上。我们的第二个目标是解决 DNA 序列影响 AID 活性的机制 它的结果。对于这些研究，我们开发了靶向突变检测来替代内源性 IgH 类 使用测试序列转换编码 IgH 可变区的 (S) 区域和外显子，这将使我们能够确定 底物序列如何影响 AID 的活性以及 CSR 和 SHM 中的其他相关因素。在一起， 目标 1 和目标 2 的互补生化和基因检测提供了一种强有力的方法来阐明 参与 IgH CSR 和 SHM 启动和调节的因素和机制。拟议的第三个目标是 阐明参与 AID 诱导 DSB 修复以完成 CSR 的过程。对于这些研究，我们再次 开发了大量试剂和新方法，包括遵循 CSR 的细胞遗传学方法 染色体的相关断裂，研究参与长程突触的因素的新遗传学方法 DSB 和遗传模型阐明完成 IgH CSR 的 DSB 修复途径。我们提出的研究 应该为通过 IgH CSR 产生抗体的机制提供新的见解，因此， 与了解免疫缺陷、疫苗免疫学和自身免疫性疾病相关。正如企业社会责任是 IgE 产生所需，这项工作也将与了解过敏性疾病的发病机制相关 和哮喘。最后，这项工作与 B 细胞恶性肿瘤相关，因为它们通常涉及染色体 通过异常 CSR 将易位癌基因与 IgH S 区域连接起来的易位。