RAG and AID biology

RAG 和 AID 生物学

• 批准号: 8746502
• 负责人: rafael c casellas
• 金额: $ 330.33万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746502
• 关键词: Activated B-Lymphocyte; Affinity; Amplifiers; Animals; Antibodies; Antibody Affinity; Apoptosis; Autoimmunity; Automobile Driving; B-Cell Lymphomas; B-Lymphocytes; Bacteria; Binding; Binding Proteins; Bioinformatics; Biology; Burkitt Lymphoma; Cell Cycle; Cell Cycle Stage; Cell Differentiation process; Cell surface; Cells; Chromosomal translocation; Complex; DNA; DNA Damage; Deposition; Development; Disease; ERCC3 gene; Ensure; Enzyme Activation; Enzymes; Equipment and supply inventories; Eukaryota; Exhibits; Fc Receptor; G1 Phase; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Genes; Genetic Processes; Genetic Recombination; Genetic Transcription; Genome; Genomics; Goals; Growth; Human; Hyperplasia; Immediate-Early Genes; Immune response; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Infection; Invaded; Journals; Laboratories; Lead; Lesion; Literature; Lymphocyte; Malignant - descriptor; Manuscripts; Mediating; Messenger RNA; Metabolism; Mitosis; Molecular; Molecular Biology; Monitor; Multienzyme Complexes; Multiple Myeloma; Mus; Nature; Oncogene Deregulation; Oncogenes; Pathway interactions; Peripheral; Physiological; Point Mutation; Polymerase; Process; Proteins; Publications; Publishing; RNA Polymerase II; Reaction; Reporting; Resected; Rest; Role; S Phase; Scientist; Site; Specificity; System; Techniques; Transcription Initiation Site; Transgenic Organisms; V(D)J Recombination; Virus; activation-induced cytidine deaminase; base; cell transformation; chromatin modification; embryonic stem cell; genome-wide; helicase; homologous recombination; in vivo; interest; intestinal villi; melting; mouse model; nuclease; pathogen; promoter; receptor; repaired; response; tool; transcription factor TFIIH; tumor; tumorigenesis

B lymphocytes recognize and destroy viruses and bacteria though special receptors on their cell surface called antibodies. The affinity and specificity of these receptors for pathogens depends to a great extent on three genetic processes: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles the 5 end of the antibody gene by combining related DNA segments. The recombination is catalyzed by the RAG1 and RAG2 enzymes. Somatic hypermutation on the other hand introduces random point mutations to increase the binding affinity of the antibody for the pathogen in question. Lastly, CSR introduces further changes to facilitate the elimination of the invading pathogen. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced Cytidine Deaminase (AID). The importance of RAGs and AID in the immune response is highlighted in humans and animals deficient for these enzymes, which are highly susceptible to infection and exhibit gut flora-dependent hyperplasia of intestinal villi. Complex diseases such as autoimmunity have long been associated with RAG and AID-dependent activity and both enzyme complexes are promiscuous, in that they can also damage non-immunoglobulin genes, including oncogenes (tumor-inducing genes). This off-targeting activity can lead to DNA mutations and oncogene deregulation, resulting in malignant transformation. Predominant among these irregularities are chromosomal translocations, which drive the formation of B cell lymphomas (e.g. Burkitt lymphomas and multiple myeloma) in humans. Thus, unraveling how RAG and AID activities are regulated under normal conditions and deregulated during tumorigenesis is key. This fiscal year we have furthered our understanding of AID biology and B cell transformation in several ways: i) we have resolved the nature of RPA recruitment to AID-mediated DNA breaks. As mentioned above, AID promotes chromosomal translocations by inducing DNA breaks at immunoglobulin genes and oncogenes in the G1 phase of the cell cycle. RPA is a ssDNA-binding protein that associates with damaged DNA in the S phase and facilitates the assembly of factors involved in homologous repair such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage. Because of the discrepancy in cell cycle stages, scientists have suggested that RPA might have a role in immunoglobulin gene recombination outside its homologous repair one. In a manuscript published in the January issue of Cell Reports we have demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1. However, recruitment in S-G2/M is extensive and associated with Rad51 accumulation as expected if RPA functions mainly in homologous recombination. Thus, most RPA recruitment are antibody genes represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. ii) AID is expressed in activated B lymphocytes. This process is initiated by a global increase in mRNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring ssDNA genome-wide, we have recently shown in the journal Cell that the genome of nave cells is poised for rapid activation. In G0, 90% of promoters from genes to be expressed in cycling lymphocytes are loaded with polymerases but unmelted and thus they support only basal transcription. Furthermore, we have found that the transition from abortive to productive elongation is kinetically limiting causing polymerases to accumulate nearer transcription start sites. Resting lymphocytes also limit expression of the TFIIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our publication identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens. iii) The Myc protein, which is deregulated by chromosomal translocations mediated by AID, has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and cell differentiation. No principle yet unifies Myc action due partly to an incomplete inventory of Myc's targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, we collaborated with David Levens laboratory from NCI and created a mouse model that helps visualize Myc expression in vivo. In a publication in Cell we used these mice to analyze the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications in activated B cells and ES cells. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action thereore explains the vast majority of Myc biology observed in literature and provides a rationale as to how this protein transforms B lymphocytes.

B淋巴细胞通过其细胞表面上称为抗体的特殊受体识别并摧毁病毒和细菌。这些受体对病原体的亲和力和特异性在很大程度上取决于三个遗传过程：V（D）J重组，体细胞超突变和类别转换重组（CSR）。第一种机制通过结合相关的DNA片段组装抗体基因的5端。重组由RAG 1和RAG 2酶催化。另一方面，体细胞超突变引入随机点突变以增加抗体对所讨论的病原体的结合亲和力。最后，CSR引入了进一步的变化，以促进入侵病原体的消除。体细胞超突变和开关重组都是通过B细胞特异性酶：激活诱导的胞苷脱氨酶（AID）进行的。RAG和AID在免疫应答中的重要性在缺乏这些酶的人类和动物中突出，这些酶高度易受感染并表现出肠道菌群依赖性肠绒毛增生。复杂疾病如自身免疫长期以来与RAG和AIDS依赖性活性相关，并且两种酶复合物是混杂的，因为它们也可以损害非免疫球蛋白基因，包括致癌基因（肿瘤诱导基因）。这种脱靶活性可导致DNA突变和癌基因失调，导致恶性转化。在这些不规则性中占主导地位的是染色体易位，其驱动人类B细胞淋巴瘤（例如伯基特淋巴瘤和多发性骨髓瘤）的形成。因此，阐明RAG和AID活性如何在正常条件下调节和在肿瘤发生期间解除调节是关键。本财年，我们通过以下几种方式进一步加深了对AID生物学和B细胞转化的理解： i）我们已经解决了RPA募集到艾滋病介导的DNA断裂的性质。如上所述，AID通过在细胞周期的G1期诱导免疫球蛋白基因和癌基因的DNA断裂来促进染色体易位。RPA是一种ssDNA结合蛋白，在S期与受损DNA结合，并促进参与同源修复的因子（如Rad 51）的组装。值得注意的是，RPA沉积也标志着艾滋病介导的损伤部位。由于细胞周期阶段的差异，科学家们认为RPA可能在其同源修复之外的免疫球蛋白基因重组中发挥作用。在发表于1月号Cell Reports的一篇论文中，我们证明RPA与切除的ssDNA不对称地结合，以响应AID、RAG或其他核酸酶产生的损伤。在G1的援助目标处存放了少量的RPA。然而，如果RPA主要在同源重组中起作用，则S-G2/M中的募集是广泛的，并且与预期的Rad 51积累相关。因此，大多数RPA募集是抗体基因，其代表在细胞周期的S-G2/M期期间通过基于同源性的途径对未修复的断裂的挽救。 ii）AID在活化的B淋巴细胞中表达。这一过程是由mRNA合成的整体增加启动的。然而，在免疫应答期间驱动转录组扩增的机制是未知的。通过监测ssDNA全基因组，我们最近在《细胞》杂志上表明，原始细胞的基因组正处于快速激活的状态。在G 0，90%的启动子从基因表达的循环淋巴细胞加载聚合酶，但未融化，因此他们只支持基础转录。此外，我们已经发现，从流产到生产性延伸的过渡是动力学限制，导致聚合酶积累更接近转录起始位点。静息淋巴细胞也限制TFIIH复合物的表达，包括参与启动子解链和开放复合物延伸的XPB和XPD解旋酶。到目前为止，两个限速步骤已被证明是控制全球基因表达的真核生物：preinitiation复合体组装和聚合酶暂停。我们的出版物确定启动子熔解作为第三个关键的调节步骤，并提出这种机制确保了迅速的淋巴细胞对入侵病原体的反应。 iii）由AID介导的染色体易位解除调节的Myc蛋白已经涉及生理或病理生长、增殖、凋亡、代谢和细胞分化。Myc的行动还没有统一的原则，部分原因是Myc的目标清单不完整。为了观察Myc在时间和生理调节的系统中的Myc靶表达和功能，我们与NCI的大卫莱文斯实验室合作，创建了一种有助于可视化体内Myc表达的小鼠模型。在《细胞》杂志上发表的一篇文章中，我们使用这些小鼠分析了激活的B细胞和ES细胞中Myc、RNA聚合酶II和染色质修饰的转录组和全基因组分布。从这种定量分析中出现了一个非常简单的规则：Myc不是基因活性的开关指定器，而是表达的非线性放大器，普遍作用于活性基因，除了在Myc之前强烈诱导的立即早期基因。Myc作用的这一规律解释了文献中观察到的绝大多数Myc生物学，并为该蛋白质如何转化B淋巴细胞提供了基本原理。