Molecular Mechanisms of Class Switch Recombination

类别转换重组的分子机制

• 批准号: 8197214
• 负责人: Frederick W. Alt
• 金额: $ 42.63万
• 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/8197214
• 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.

这一应用提出了对免疫球蛋白重链(IgH)类开关机制的研究 重组(CSR)和体细胞超突变(SHM)。我们已经证明，活化诱导胞苷 脱氨酶(AID)是单链DNA(SsDNA)特异性胞苷脱氨酶，是CSR的启动子。 采用了一系列新的生化和遗传学方法来阐明艾滋病的发病机制 在转录产生的单链DNA上下文中获得转录双链(DS)DNA序列 结构和/或某些艾滋病修饰或辅助因子。我们还表明，企业社会责任可以聘用一般 艾滋病引发的DNA双链断裂(DSB)的突触过程，即一般的DNA修复因子 在企业社会责任中发挥作用，两条不同的末端连接途径融合S区域中断完成企业社会责任。我们的 目前的提案是在三个具体目标的背景下以这些意见为基础的。我们的第一个目标是建议使用 生物化学和遗传学方法，以阐明艾滋病功能和调节的基本机制。在这 在这方面，我们开发了从正常B细胞中纯化AID的方法，并进行了体外转录依赖检测 DsDNA DNA的辅助脱氨基和评估体内AID功能的遗传学方法阐明 生化方面的。我们的第二个目标是解决DNA序列影响AID活性和 它的结果。对于这些研究，我们开发了有针对性的突变分析来取代内源性IGH类 编码IgH可变区的开关(S)区和外显子，以及测试序列，这将使我们能够确定 底物顺序如何影响CSR和SHM中AID的活性和其他相关因素。团结在一起， AIMS 1和AIMS 2的互补生化和遗传分析为阐明 IgH、CSR和SHM启动和调控的因素和机制。第三个提议的目标是 阐明AID诱导的DSB完成CSR的修复过程。对于这些研究，我们再次 开发了大量的试剂和新的方法，包括细胞遗传学方法来跟踪CSR 染色体相关断裂，研究长程突触相关因素的新遗传学方法 DSB和遗传模型来阐明完成IGH CSR的DSB修复途径。我们建议的研究 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有关。就像CSR 这是产生IgE所必需的，这项工作也将与理解过敏性疾病的发病机制有关 还有哮喘。最后，这项工作与B细胞恶性肿瘤有关，因为它们通常涉及染色体 通过异常CSR将易位癌基因连接到IGH S区域的易位。