Targeting of somatic hypermutation in the genome

体细胞高度突变(SHM)产生免疫球蛋白(Ig)基因的点突变，并允许 产生高亲和力抗体。这种反应对于预防感染和 疫苗的效力。SHM是由激活诱导脱氨酶(AID)启动的，AID负责脱氨基 核糖核酸聚合酶II(POL II)转录背景下单链DNA中的胞苷。当艾滋病患者 和SHM优先作用于Ig基因，它们也影响许多非Ig基因座，由此产生的遗传 不稳定性导致了一系列B细胞恶性肿瘤的发展。管理AID/SHM的规则 在基因组中的靶向还没有被很好地理解。我们提议的实验的中心目标 是为了确定导致AID/SHM优先靶向Ig基因的机制 并建立规则来管理它们错误地定位于基因组的其他区域。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： 目的1.确定介导SHM优先靶向Ig基因的蛋白质因子和 确定它们的作用机制。我们已经确定了负责靶向的DNA序列 从AID/SHM到Ig基因，称为Divac(多样化激活剂)。批评者的身份 结合Divac的蛋白质因子和它们介导SHM靶向的机制(S)尚不清楚。 我们将用生化方法鉴定Divac结合因子，并用基因测试它们的功能 靶向和强大的SHM记者分析。我们将系统地确定DNA序列和 SHM靶向所需的蛋白质结构域，并使用该信息重建正确靶向的SHM 在非淋巴样细胞中。我们还将确定不同的表观遗传学、转录和分子 一个高度突变的靶基因的特征，以检验我们的初步数据支持的模型， Divac通过使突变靶区中的POL II停滞而起作用，从而产生最佳 爱滋病作用的底物。 目的2.在正常和DNA修复中定位人类基因组的AID/SHM易感区域 缺乏细胞。利用新型慢病毒SHM报告载体和高通量定位前病毒 整合位点，我们将确定：i)人类基因组中易感或抗 在基因组中，AID的作用与高保真的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影响的人类基因组的AID/SHM“易损性”图谱 以了解B细胞肿瘤的基因组不稳定性。 总之，我们提出的研究具有双重意义，既对于抗体基因的基本机制 多样化和致癌原因。

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.