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类开关重组（CSR）和IG变量区域（VDJ）外显子体细胞超突变（SHM），重点关注阐明激活引起胞苷脱氨酸酶（AID）的机制，CSR和SHM的发起，CSR和SHM的启动。基于各种证据，我们假设特定序列基序和独特的转录特征针对特定基因组部位的目标有助于。基于支持初步数据，我们建议通过强大的新方法检验这一假设。我们建议阐明CSR期间DSB生成和定向连接的机制。 IGH开关区域是辅助发射的DSB的目标，在两个不同的S区域之间连接以实现CSR，并由具有异常特征的重复序列组成。我们开发了一个功能强大的新V（D）J乘客等位基因测定系统，该系统使我们能够表明S区域在CSR激活的B细胞以及GC B细胞，GC B细胞以及在GC B细胞中，在GC B细胞中，在第一次执行Core s Shm模式的SHM SHM模式的SHM SHM模式的SHM SHM模式时，S SHM和DSB都会经历SHM和DSB。我们建议使用乘客等位基因系统对小鼠和人类区域基序进行系统分析，以及其他物种的基序来识别靶向靶向基序的辅助，并阐明如何对S区域DSB的特定方向连接来促进生产性生产性，Deletional CSR。 V（d）J外显子通过与激活CSR激活的B细胞中的S区域不同的机制在SHM中的辅助活性靶向。在V（d）J外显子中，互补性确定区域（CDR）是主要援助目标，即使它们并没有富含已知辅助热点基序。我们建议使用乘客等位基因系统剖析CDR中的潜在辅助靶向元素，并检验以下假设：抗HIV中高水平的SHM，缺失和插入通过抗体亲和力成熟过程中的辅助热点基序促进了抗HIV广泛中和抗体的可变区域。我们还将检验以下假设：GC B细胞中VH（d）JH外显子的收敛转录靶向SHM的帮助，并且IGH 3'调节区域的元素调节了这种转录。许多人类B细胞淋巴瘤是GC起源的，并且怀疑有助于发出的促进性致癌易位。现在，我们建议通过我们新开发的高吞吐量易位测序方法来鉴定小鼠GC B细胞中辅助依赖性的DSB/易位，并通过GRO-SEQ（另一种高吞吐量方法）表征其转录特征。我们还将测试我们的乘客等位基因系统中先前定义的和新鉴定的辅助非Ig基因靶标（“非目标”），以阐明靶向靶标和转录在使其辅助目标中的作用。最后，我们将扩展我们的乘客等位基因分析以测试易位的人B细胞癌基因，以测试它们是否确实是优惠的辅助靶标，并阐明其内部潜在的靶标基序及其转录的作用。