AID Targeting Mechanisms for IgH Switch Recombination and Somatic Hypermutation

IgH 开关重组和体细胞超突变的 AID 靶向机制

• 批准号: 8697880
• 负责人: Frederick W. Alt
• 金额: $ 43.85万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697880
• 关键词: Affinity; Alleles; Antibody Affinity; B-Cell Lymphomas; B-Lymphocytes; Base Composition; Biological Assay; Characteristics; Chickens; Complementarity Determining Regions; Coupled; Data; Ducks; Elements; Exons; Funding; Gene Targeting; Generations; Genetic Transcription; Genome; Genomics; Goals; HIV; Human; Immune response; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Indium; Knock-in Mouse; Maps; Methods; Mus; Nucleic Acid Regulatory Sequences; Oncogenes; Oncogenic; Pattern; Process; Rana; Repetitive Sequence; Role; Running; Sequence Analysis; Site; Structure of germinal center of lymph node; System; Testing; Time; activation-induced cytidine deaminase; base; density; design; insertion/deletion mutation; insight; neutralizing antibody; novel; novel strategies; public health relevance; research study

DESCRIPTION (provided by applicant): We propose to study IgH class switch recombination (CSR) and Ig variable region (VDJ) exon somatic hypermutation (SHM) with a focus on elucidating mechanisms that direct Activation Induced Cytidine Deaminase (AID), the initiator of CSR and SHM, to specific targets. Based on various lines of evidence, we hypothesize that particular sequence motifs and unique transcription features target AID to particular genomic sites. Based on supporting preliminary data, we propose to test this hypothesis with powerful new approaches. We propose to elucidate mechanisms of DSB generation and directional joining during CSR. IgH switch (S) regions are targets of AID-initiated DSBs that are joined between two different S regions to achieve CSR and are composed of repetitive sequences with unusual characteristics. We developed a powerful new V (D) J passenger allele assay system that allowed us to show that S regions undergo both SHMs and DSBs when inserted in place of a V (D) J exon in CSR-activated B cells and in GC B cells and to perform, for the first time, in depth analysis of SHM patterns of core S regions. We propose to use the passenger allele system to perform a systematic analysis of mouse and human S region motifs, as well as those from other species to identify AID targeting motifs and to elucidate how orientation-specific joining of S region DSBs is enforced to promote productive, deletional CSR. V (D) J exons are targeted by AID activity during SHM in germinal center (GC) B cells through different mechanisms than S regions in B cells activated for CSR. Within V(D)J exons, complementarity determining region (CDRs) are prime AID targets even though they are not enriched for known AID hotspot motifs. We propose to use the passenger allele system to dissect potential AID targeting elements in CDRs and test the hypothesis that high levels of SHMs, deletions and insertions in anti-HIV broadly neutralizing antibody variable regions are promoted by acquisition of AID hotspots motifs during the antibody affinity maturation process. We also will test the hypothesis that convergent transcription of VH(D)JH exons in GC B cells targets AID for SHM and that that elements of the IgH 3' regulatory region regulate such transcription. Many human B cell lymphomas are of GC origin and harbor suspected AID-initiated oncogenic translocations. We now propose to identify AID-dependent DSB/translocation off-targets in mouse GC B cells by our newly developed high throughput translocation sequencing method and to characterize their transcriptional signatures by Gro-Seq, another high throughput approach. We also will test activity of previously defined and newly identified AID non-Ig gene targets ("off-targets") in our passenger allele system to elucidate targeting motifs and roles of transcription in making them AID targets. Finally, we will extend our passenger allele analyses to test translocated human B cell oncogenes to test whether they are, indeed, preferential AID targets and, if so, to elucidate potential target motifs within them and roles for transcription.

描述（由申请人提供）：我们建议研究 IgH 类别转换重组 (CSR) 和 Ig 可变区 (VDJ) 外显子体超突变 (SHM)，重点是阐明将激活诱导胞苷脱氨酶 (AID)（CSR 和 SHM 的引发剂）引导至特定靶点的机制。基于各种证据，我们假设特定的序列基序和独特的转录特征将 AID 靶向特定的基因组位点。基于支持初步数据，我们建议用强大的新方法来检验这一假设。我们建议阐明 CSR 期间 DSB 生成和定向加入的机制。 IgH 开关 (S) 区域是 AID 启动的 DSB 的目标，这些 DSB 在两个不同的 S 区域之间连接以实现 CSR，并且由具有不寻常特征的重复序列组成。我们开发了一种强大的新型 V (D) J 过客等位基因检测系统，该系统使我们能够证明，当插入 CSR 激活的 B 细胞和 GC B 细胞中的 V (D) J 外显子时，S 区域会同时经历 SHM 和 DSB，并首次对核心 S 区域的 SHM 模式进行深入分析。我们建议使用过客等位基因系统对小鼠和人类 S 区基序以及其他物种的基序进行系统分析，以识别 AID 靶向基序并阐明如何强制执行 S 区 DSB 的方向特异性连接以促进生产性、删除性 CSR。 V (D) J 外显子在生发中心 (GC) B 细胞的 SHM 期间通过与 CSR 激活的 B 细胞中的 S 区域不同的机制被 AID 活性靶向。在 V(D)J 外显子内，互补决定区 (CDR) 是主要的 AID 靶点，尽管它们并未富集已知的 AID 热点基序。我们建议使用乘客等位基因系统来剖析CDR中潜在的AID靶向元件，并测试这样的假设：在抗体亲和力成熟过程中获得AID热点基序，可以促进抗HIV广泛中和抗体可变区中高水平的SHM、缺失和插入。我们还将测试以下假设：GC B 细胞中 VH(D)JH 外显子的聚合转录以 AID 为目标，实现 SHM，并且 IgH 3' 调节区的元件调节这种转录。许多人类 B 细胞淋巴瘤是 GC 起源的，并含有疑似 AID 引发的致癌易位。我们现在建议通过我们新开发的高通量易位测序方法来鉴定小鼠 GC B 细胞中 AID 依赖性 DSB/易位脱靶，并通过另一种高通量方法 Gro-Seq 来表征其转录特征。我们还将测试我们的过客等位基因系统中先前定义的和新识别的 AID 非 Ig 基因靶标（“脱靶”）的活性，以阐明靶向基序以及转录在使它们成为 AID 靶标中的作用。最后，我们将扩展我们的过客等位基因分析，以测试易位的人类 B 细胞癌基因，以测试它们是否确实是 AID 的优先靶标，如果是，则阐明其中的潜在靶基序和转录作用。