Molecular Mechanisms of Class Switch Recombination

描述（由申请人提供）：本申请提出了免疫球蛋白重链（IgH）类别转换重组（CSR）和体细胞超突变（SHM）机制的研究。我们已经表明，激活诱导的胞苷脱氨酶（AID），CSR的启动子，是一种单链DNA（ssDNA）特异性胞苷脱氨酶，我们采用了一系列新的生化和遗传学方法来阐明机制，AID获得的背景下转录产生的ssDNA结构和/或某些AID修饰或辅因子的转录双链（ds）DNA序列。我们还表明，CSR可能采用艾滋病引发的DNA双链断裂（DSB）的突触的一般过程，一般的DNA修复因子在CSR中的功能，以及两个不同的末端连接途径融合S区断裂完成CSR。我们目前的建议是在三个具体目标的范围内根据这些意见提出的。我们的第一个目标是利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。在这方面，我们开发的方法，从正常的B细胞，在体外测定转录依赖性的双链DNA的AID脱氨基，和遗传方法，以评估在体内的AID功能阐明生物化学。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。对于这些研究，我们开发了靶向突变测定，以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和CSR和SHM中其他相关因子的活性。总之，目标1和2的互补生化和遗传测定为阐明IgH CSR和SHM的启动和调节所涉及的因素和机制提供了强有力的方法。第三个目标是阐明AID诱导的DSB修复完成CSR的过程。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染色体中CSR相关的断裂，新的遗传学方法来研究DSB长距离突触中涉及的因素，以及阐明完成IgH CSR的DSB修复途径的遗传模型。我们提出的研究应提供新的见解抗体生产的机制，通过IgH CSR，因此，有关了解免疫缺陷，疫苗免疫学，和自身免疫性疾病。由于CSR是IgE产生所必需的，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，这项工作是相关的B细胞恶性肿瘤，因为它们往往涉及染色体易位，连接易位的癌基因IgH S区通过异常的CSR。公共卫生相关性：我们提出的研究将继续提供新的见解的机制，不同类型的抗体是通过基因重排过程中产生的免疫球蛋白重链类转换重组（CSR）。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。CSR机制的阐明对于充分理解过敏性疾病和哮喘也具有重要意义，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免疫系统某些癌症的基础因素，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.