Molecular Mechanisms of Class Switch Recombination

描述(申请人提供)：本申请提出对免疫球蛋白重链(IGH)类开关重组(CSR)和体细胞超突变(SHM)机制的研究。我们已经证明，CSR的启动子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，我们利用一系列新的生化和遗传学方法来阐明AID在转录产生的ssDNA结构和/或某些AID修饰或辅助因子的背景下获得转录双链(DS)DNA序列的机制。我们还表明，CSR可能采用一般的过程来突触艾滋病引发的DNA双链断裂(DSB)，一般的DNA修复因子在CSR中发挥作用，两条不同的末端连接途径融合S区域的断裂而完成CSR。我们目前的建议建立在这些观察结果的基础上，具体目标有三个。我们的第一个目标是利用生物化学和遗传学的方法来阐明艾滋病的基本功能和调控机制。在这方面，我们开发了从正常B细胞中纯化AID的方法，体外转录依赖AID脱氨基的双链DNA检测方法，以及从生化角度评估体内AID功能的遗传学方法。我们的第二个目标是解决DNA序列影响AID活性及其结果的机制。在这些研究中，我们开发了有针对性的突变分析方法，用测试序列取代内源IgH类开关(S)区域和编码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CSR和SHM中AID和其他相关因素的活性。总之，AIMS 1和AIMS 2的互补生化和遗传分析为阐明与IgH、CSR和SHM的启动和调控有关的因素和机制提供了强有力的途径。第三个被提议的目的是阐明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，包括跟踪CSR相关染色体断裂的细胞遗传学方法，研究DSB长距离突触所涉及的因素的新的遗传学方法，以及解释完成IGH CSR的DSB修复途径的遗传模型。我们提出的研究将为通过IgH CSR产生抗体的机制提供新的见解，因此，对于理解免疫缺陷、疫苗免疫学和自身免疫性疾病具有重要意义。由于CSR是产生IgE所必需的，这项工作也将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细胞恶性肿瘤有关，因为它们通常涉及染色体易位，通过异常的CSR将易位的癌基因连接到IGH S区域。公共卫生相关性：我们拟议的研究将继续为不同类型的抗体通过称为免疫球蛋白重链类开关重组(CSR)的基因重排过程产生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机制对于全面了解过敏性疾病和哮喘也很重要，因为增加一类特定抗体的产生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某些免疫系统癌症的基础因素，例如淋巴瘤，它通过异常的CSR激活致癌基因。

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.