Molecular Mechanisms of Class Switch Recombination

类别转换重组的分子机制

• 批准号: 7743798
• 负责人: Frederick W. Alt
• 金额: $ 42.36万
• 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 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743798
• 关键词: Address; Alanine; Allergic Disease; Antibodies; Antibody Formation; Asthma; 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; 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; Immunoglobulins; 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; public health relevance; repaired; replication factor A; response

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Our proposed studies will continue to provide novel insights into the mechanism by which different types of antibodies are produced through the gene rearrangement process termed immunoglobulin heavy chain class switch recombination (CSR). Elucidation of the CSR mechanism has great relevance for understanding immunodeficiency and autoimmune diseases. Elucidation of the CSR mechanism also has importance for fully understanding allergic diseases and asthma, as increased production of a particular class of antibodies is an important component of the pathogenesis of these diseases. Finally, the work will help elucidate factors that underlie certain cancers of the immune system, such as lymphomas, which activate cancer causing genes through aberrant CSR.

描述（由申请人提供）：本申请提议研究免疫球蛋白重链（IgH）类转换重组（CSR）和体细胞超突变（SHM）的机制。我们已经证明，CSR 的引发剂激活诱导胞苷脱氨酶 (AID) 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 在转录生成的 ssDNA 结构和/或某些 AID 的背景下获得转录双链 (ds)DNA 序列的机制。 修饰或辅因子。我们还表明，CSR 可能采用 AID 引发的 DNA 双链断裂 (DSB) 突触的一般过程，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。我们当前的提案建立在三个具体目标背景下的这些观察结果之上。我们的首要目标是使用生化和遗传学方法来阐明 AID 功能和调节的基本机制。在这方面，我们开发了从正常 B 细胞中纯化 AID 的方法、双链 DNA 转录依赖性 AID 脱氨基的体外测定，以及评估生物化学阐明的体内 AID 功能的遗传方法。我们的第二个目标是解决 DNA 序列影响 AID 活动及其结果的机制。对于这些研究，我们开发了靶向突变测定，用测试序列替换内源性 IgH 类别转换 (S) 区和编码 IgH 可变区的外显子，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。总之，目标 1 和目标 2 的互补生化和遗传检测为阐明 IgH CSR 和 SHM 的启动和调节所涉及的因素和机制提供了一种强大的方法。第三个目标是阐明修复 AID 诱导的 DSB 以完成 CSR 所涉及的过程。对于这些研究，我们再次开发了大量试剂和新方法，包括跟踪 CSR 相关染色体断裂的细胞遗传学方法、研究 DSB 长程突触相关因素的新遗传学方法，以及阐明完成 IgH CSR 的 DSB 修复途径的遗传模型。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，因此与理解免疫缺陷、疫苗免疫学和自身免疫性疾病相关。由于 CSR 是 IgE 产生所必需的，因此这项工作也与了解过敏性疾病和哮喘的发病机制有关。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易位，通过异常的 CSR 将易位的癌基因与 IgHS 区域连接起来。公共健康相关性：我们提出的研究将继续为通过称为免疫球蛋白重链类别转换重组（CSR）的基因重排过程产生不同类型抗体的机制提供新的见解。阐明 CSR 机制对于理解免疫缺陷和自身免疫性疾病具有重要意义。阐明 CSR 机制对于充分了解过敏性疾病和哮喘也很重要，因为特定类别抗体的产生增加是这些疾病发病机制的重要组成部分。最后，这项工作将有助于阐明导致某些免疫系统癌症（例如淋巴瘤）的因素，这些癌症通过异常的 CSR 激活致癌基因。