Targeting of somatic hypermutation in the genome

靶向基因组中的体细胞超突变

• 批准号: 10161714
• 负责人: David G. Schatz
• 金额: $ 41.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10161714
• 关键词: Activator Appliances; Affect; Affinity; Antibodies; Antibody Affinity; Antibody Formation; Area; B lymphoid malignancy; B-Cell Activation; B-Cell Lymphomas; B-Cell Neoplasm; B-Lymphocytes; Binding; Biochemical; Biological; Biological Assay; Cancer Etiology; Cell Cycle; Cells; Cellular biology; Chromatin; Chromosomal translocation; Chromosome abnormality; Cytidine; Cytosine; DNA; DNA Polymerase II; DNA Repair; DNA Repair Pathway; DNA Sequence; DNA-Directed RNA Polymerase; Data; Development; Elements; Enhancers; Environment; Enzyme Activation; Epigenetic Process; Exons; G1 Phase; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Genome; Genomic Instability; Genomic approach; Human; Human Genome; Immune response; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunoglobulins; Infection; Interruption; Malignant Neoplasms; Maps; Mediating; Methods; Modeling; Molecular; Molecular Genetics; Mutate; Mutation; Nuclear; Pathway interactions; Pattern; Point Mutation; Predisposition; Process; Production; Proteins; Publishing; Reaction; Recruitment Activity; Regulation; Reporter; Research; Resistance; Risk; Seminal; Series; Single-Stranded DNA; Site; Tertiary Protein Structure; Testing; Uracil; Vaccination; Work; activation-induced cytidine deaminase; cost; experimental study; genetic approach; genome-wide; genome-wide analysis; in vivo; innovation; insight; integration site; leukemia/lymphoma; novel; novel strategies; reconstitution; repaired; vaccine efficacy; vaccine response; vector

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.

体细胞超突变（SHM）在免疫球蛋白（IG）基因中产生点突变，并允许免疫球蛋白（Ig）基因的突变。 产生高亲和力抗体。这种反应对于防止感染和预防癌症是重要的。 疫苗的功效。SHM是由活化诱导脱氨酶（AID）启动的， 在RNA聚合酶II（Pol II）转录的情况下，单链DNA中的胞苷。而援助 和SHM优先作用于IG基因，它们也影响许多非IG基因座，并且所产生的遗传学效应是由它们所引起的。 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 基因组中的靶向作用还不太清楚。我们实验的中心目标 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 互补的生物化学、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基因的蛋白因子， 确定其作用机制。我们已经鉴定出了 将AID/SHM与IG基因结合，并将其称为DIVAC（多样化激活剂）。批判者的身份 结合DIVAC的蛋白因子和它们介导SHM靶向的机制尚不清楚。 我们将使用生物化学方法鉴定DIVAC结合因子，并使用基因工程方法检测其功能。 靶向和强大的SHM报告基因分析。我们将系统地确定DNA序列， 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。我们还将确定不同的表观遗传，转录和分子 高度突变的靶基因的特征，以测试模型，由我们的初步数据支持， DIVAC通过引起突变靶区域中Pol II的停滞而起作用，从而产生最佳的突变靶区域。 为艾滋病的行动提供物质基础。 目标2.绘制正常和DNA修复中人类基因组的AID/SHM易感区域- 缺陷细胞使用新型慢病毒SHM报告载体和高通量定位前病毒 整合位点，我们将确定：i）人类基因组中对以下易感或耐药的区域： SHM; 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 将产生人类基因组的AID/SHM“脆弱性”图谱，可能具有重要意义 用于理解B细胞肿瘤中的基因组不稳定性。 总之，我们提出的研究具有双重意义，无论是抗体基因的基本机制， 多样化和癌症的原因。