Targeting of somatic hypermutation in the genome

体细胞超突变 (SHM) 会在免疫球蛋白 (Ig) 基因中产生点突变，并允许 高亲和力抗体的生产。该反应对于预防感染和预防感染很重要 疫苗的功效。 SHM 由激活诱导脱氨酶 (AID) 启动，该酶可脱氨 RNA 聚合酶 II (Pol II) 转录过程中单链 DNA 中的胞苷。虽然援助 和SHM优先作用于Ig基因，它们也影响许多非Ig基因座，以及由此产生的遗传 不稳定性会导致一系列 B 细胞恶性肿瘤的发生。管理 AID/SHM 的规则 基因组中的靶向尚不清楚。我们提出的实验的中心目标 确定 AID/SHM 优先靶向 Ig 基因的机制 并建立规则来管理它们对基因组其他区域的错误定位。我们将使用 互补的生化、分子、遗传和基因组方法，以实现以下目标： 目标 1. 确定介导 SHM 优先靶向 Ig 基因的蛋白质因子，并 确定它们的作用机制。我们已经确定了负责靶向的 DNA 序列 AID/SHM 转化为 Ig 基因，并将其称为 DIVAC（多样化激活剂）。关键人物的身份 结合 DIVAC 的蛋白质因子及其介导 SHM 靶向的机制尚不清楚。 我们将使用生化方法来鉴定 DIVAC 结合因子，并使用基因测试其功能 靶向和强大的 SHM 报告基因检测。我们将系统地确定 DNA 序列并 SHM 靶向所需的蛋白质结构域，并使用此信息来重建正确靶向的 SHM 在非淋巴细胞中。我们还将确定独特的表观遗传、转录和分子特征 高度突变的目标基因的特征，以便测试模型，并得到我们初步数据的支持， DIVAC 通过在突变目标区域阻止 Pol II 发挥作用，从而创建最佳的 AID作用的底物。 目标 2. 绘制正常和 DNA 修复中人类基因组的 AID/SHM 易感区域图谱 缺乏细胞。使用新型慢病毒 SHM 报告载体和原病毒的高通量作图 整合位点，我们将确定： i) 人类基因组中易感或耐药的区域 健康管理； ii) 在基因组中，AID 的作用与高保真 DNA 修复相反，以及 iii) 如何 AID/SHM 靶向规则受 DIVAC 结合因子和细胞周期的影响。这些实验 将产生可能具有重要意义的人类基因组 AID/SHM“脆弱性”图 用于了解 B 细胞肿瘤的基因组不稳定性。 总之，我们提出的研究对于抗体基因的基本机制具有双重意义 多样化和癌症成因。

Somatic hypermutation (SHM) generates point mutations in immunoglobulin (Ig) genes and allows for the production of high affinity antibodies. The reaction is important for protection against infection and for the efficacy of vaccines. SHM is initiated by the activation induced deaminase (AID), which deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase II (Pol II). While AID and SHM act preferentially on Ig genes, they also affect numerous non-Ig loci, and the resulting genetic instability contributes to the development of a range of B cell malignancies. The rules that govern AID/SHM targeting in the genome are not well understood. The central objectives of our proposed experiments are to determine the mechanisms responsible for the preferential targeting of AID/SHM to Ig genes and to establish the rules that govern their mis-targeting to other regions of the genome. We will use complementary biochemical, molecular, genetic, and genomic approaches to achieve the following aims: Aim 1. Determine the protein factors that mediate preferential targeting of SHM to Ig genes and determine their mechanism of action. We have identified the DNA sequences responsible for targeting of AID/SHM to Ig genes, and refer to them as DIVAC (diversification activator). The identity of the critical protein factors that bind DIVAC and the mechanism(s) by which they mediate SHM targeting are not known. We will use biochemical methods to identify DIVAC-binding factors and will test their function using gene targeting and powerful SHM reporter assays. We will systematically determine the DNA sequences and protein domains required for SHM targeting and use this information to reconstitute properly targeted SHM in non-lymphoid cells. We will also determine the distinctive epigenetic, transcriptional, and molecular features of a highly mutating target gene so as to test the model, supported by our preliminary data, that DIVAC functions by causing the arrest of Pol II in the mutation target region, thereby creating an optimal substrate for the action of AID. Aim 2. Map the AID/SHM-susceptible regions of the human genome in normal and DNA repair- deficient cells. Using novel lentiviral SHM reporter vectors and high-throughput mapping of proviral integration sites, we will determine: i) the regions of the human genome that are susceptible or resistant to SHM; ii) where in the genome the action of AID is opposed by high-fidelity DNA repair, and iii) how AID/SHM targeting rules are influenced by DIVAC-binding factors and the cell cycle. These experiments will yield AID/SHM "vulnerability" maps of the human genome that are likely to have important implications for understanding genomic instability in B cell tumors. Together, our proposed studies have a dual significance, both for basic mechanisms of antibody gene diversification and for the causes of cancer.