Molecular Mechanisms of Class Switch Recombination

类别转换重组的分子机制

• 批准号: 7577240
• 负责人: Frederick W. Alt
• 金额: $ 42.33万
• 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/7577240
• 关键词: Address; Alanine; Allergic; 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）：本申请提出了有关免疫球蛋白重链（IGH）类开关重组（CSR）和体细胞超偏（SHM）机制的研究。 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修改或联合因素。我们还表明，企业社会责任可能采用一般过程来实现辅助发射的DNA双链断裂（DSB）的突触，其中一般的DNA修复因子在CSR中起作用，并且两个不同的最终连接途径融合了融合的区域断裂以完成CSR。我们当前的建议是基于这些观察结果的三个特定目标。我们的第一个目的建议使用生化和遗传方法来阐明援助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​​中纯化辅助的方法，在体外测定DSDNA DNA的转录依赖性辅助脱氨酸以及评估体内辅助功能的遗传方法。我们的第二个目的解决了DNA序列影响辅助活动及其结果的机制。在这些研究中，我们开发了靶向突变测定，以替代用测试序列编码IGH可变区域的内源性IGH类开关和外显子，这将使我们能够确定底物序列如何影响CSR和SHM中AID和其他相关因素的活动。目的1和2的互补生化和遗传测定法为阐明IGH和SHM的启动和调节涉及的因素和机制提供了强大的方法。提出的第三个目标是阐明涉及援助诱导的DSB的过程以完成CSR。在这些研究中，我们再次开发了大量的试剂和新方法，包括遵循CSR相关的染色体中与CSR相关断裂的细胞遗传学方法，研究与DSBS远程突触的研究因素的新型遗传方法以及阐明CSR完整IGH CSR的DSB修复途径的遗传模型。我们提出的研究应通过IGH CSR提供有关抗体生产机制的新见解，因此与了解免疫缺陷，疫苗免疫学和自身免疫性疾病有关。由于企业社会责任是IgE生产所必需的，因此该工作也将与了解过敏性疾病和哮喘的发病机理有关。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易位，这些染色体易位通过异常CSR连接了肿瘤基因与IGHS区域。公共卫生相关性：我们提出的研究将继续提供有关通过称为免疫球蛋白重链链类转换重组（CSR）的基因重排过程产生不同类型抗体的机制的新见解。阐明CSR机制对于理解免疫缺陷和自身免疫性疾病具有很大的意义。阐明企业社会责任机制对于完全理解过敏性疾病和哮喘也很重要，因为增加的一类抗体的产生是这些疾病发病机理的重要组成部分。最后，这项工作将有助于阐明免疫系统某些癌症基础的因素，例如淋巴瘤，这些因素通过异常CSR激活癌症，从而激活癌症。