A combined computational and experimental approach to the evolution and role of the DNA sequence environment in targeting mutations to antibody V regions

项目摘要 我们对突变如何优先针对变量的理解存在根本性的差距 (V)免疫球蛋白（IG）基因座在体细胞超突变（SHM）期间的区域。这种差距的持续存在 限制了我们对激活诱导脱氨酶（AID）致突变机制的理解 在免疫反应和AID在导致B细胞淋巴瘤的错误靶向突变中的作用， 其他癌症。拟议研究的长期目标是了解 免疫力的突变是保护我们免受感染所必需的。作为来自人类的高通量数据， 抗体免疫反应变得可用，它为我们提供了新的机会， 解释SHM潜在机制的假说。我们现在提出进一步的假设 使用应用于附加数据库的计算模型，并使用细胞模型验证这些假设， 动物实验。我们的目标是了解是什么引导SHM跨越许多人类IG重 链V区。我们的中心假设是V区SHM过程高度依赖于DNA 以很大程度上确定性的方式驱动突变的序列签名。这一假设得到了支持 通过我们使用来自一些人V区基因的人体内数据的初步结果， 使用独立的数据库和在人B细胞系中的实验进行验证。基本原理是 基于生物学机制的计算数据的评估，以及适当的生物学 实验，将揭示IGHV区域（IGHV 3-23，4-34，1-18，1-02等）之间的关键差异。的 导致这些V区中的每一个在对医学上重要的抗原的应答中占主导地位。我们 假设将通过追求两个具体目标进行测试：1）确定DNA签名的程度 确定了在疾病中重要的四个人IGHV基因的突变过程 2）检查所有其他人类中AID热点和Polη热点之间的关系 V区基因，从而严格定义了突变靶向标签。这两个目标也需要研究 人V区基因及其在表达人V区基因的人细胞系和小鼠中的修饰 区域，以进一步确认签名并确定体内分子机制。我们的做法是 创新，因为我们提出的计算模型将是机械驱动的聚焦， 关于AID和Polη热点之间的相互作用，从而测试分子机制， 经典的统计模型使用整个V区序列，忽略了潜在的生物学。此外， 我们将利用来自人类V区的新的高通量数据， 进行抗原选择。我们的研究结果将与人IgV库分析高度相关， 目前难以解释的免疫反应将有助于未来的疫苗和治疗性抗体 发展，以及帮助了解人类恶性肿瘤中的突变，其中艾滋病起着关键作用。

Project summary There is a fundamental gap in our understanding of how mutations are preferentially targeted to the variable (V) regions of the Immunoglobulin (Ig) loci during somatic hypermutation (SHM). The persistence of this gap has limited our understanding of the mutagenic mechanisms involving activation-induced deaminase (AID) in the immune response and in the role of AID in mis-targeting mutations leading to B-cell lymphomas and other cancers. The long-term goal of the proposed research is to understand the global targeting of mutations in immunity that are required to protect us from infections. As high-throughput data from human antibody immune responses became available, it provided us with new opportunities to generate hypotheses to explain the underlying mechanisms of SHM. We now propose to generate further hypotheses using computational models applied to additional databases and to validate these hypotheses using cellular and animal experiments. Our objective is to understand what directs SHM across the many human Ig heavy chain V-regions. Our central hypothesis is that the V-region SHM process is highly dependent on a DNA sequence signature(s) that drives mutations in a largely deterministic fashion. This hypothesis is supported by our preliminary results using human in vivo data from a few human V region genes and has begun to be validated using independent databases and experiments in human B cell lines. The rationale is that evaluations of computational data based upon biological mechanisms, together with appropriate biological experiments, will reveal the key differences between IGHV regions (IGHV 3-23, 4-34, 1-18, 1-02, etc.) that lead to the dominance of each of those V regions in the responses to medically important antigens. Our hypothesis will be tested by pursuing two specific aims: 1) identify the extent to which a DNA signature determines the mutation process in four individual human IGHV genes that are important in disease responses; 2) examine the relationship between AID hotspots and Polη hotspots across all the other human V region genes, thus rigorously defining a mutation targeting signature. Both aims will also entail studying human V region genes and modifications of them in human cell lines and in mice expressing a human V region to further confirm the signature and identify molecular mechanisms in vivo. Our approach is innovative because the computational models we are proposing will be mechanistically motivated focusing on the interaction between AID and Polη hotspots, thus testing molecular mechanisms as opposed to classic statistical models using whole V region sequences that ignore the underlying biology. In addition, to focus on mechanisms we will leverage new high-throughput data from human V regions that have not undergone antigen selection. Our results will be highly relevant to human IgV repertoire analyses from immune responses that are currently hard to interpret and will help future vaccine and therapeutic antibody development, as well as help to understand mutations in human malignancies where AID plays a key role.