Molecular Basis of Immunoglobulin Heavy Chain Switch

免疫球蛋白重链开关的分子基础

• 批准号: 8090512
• 负责人: Janet M. Stavnezer
• 金额: $ 1.95万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-28 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8090512
• 关键词: APEX1 gene; Address; Affect; Antibodies; B-Cell Lymphomas; B-Lymphocytes; Bacterial Toxins; Base Excision Repairs; Binding; Cell Cycle; Cells; Chromosomal Breaks; Chromosomal translocation; Chromosomes; Complex; DNA; DNA Double Strand Break; DNA Repair Gene; DNA Repair Pathway; DNA Single Strand Break; DNA biosynthesis; DNA repair protein; DNA-(apurinic or apyrimidinic site) lyase; Deamination; Dependence; Enhancers; Enzyme Activation; Event; Frequencies; G1 Phase; Genes; Genetic Recombination; Genome; Grant; Heavy-Chain Immunoglobulins; Human; IgE; Immune response; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulin Switch Recombination; Immunoglobulins; Lead; Lesion; Ligation; Light; MYC gene; Malignant Neoplasms; Maps; Mature B-Lymphocyte; Mediating; Methods; Microsatellite Instability; Mismatch Repair; Modeling; Molecular; Mus; Mutation; Oncogene Activation; Oncogenes; Pathway interactions; Plasmacytoma; Positioning Attribute; Process; Proteins; Publishing; Recruitment Activity; Relative (related person); Reporting; Role; Site; Structure of germinal center of lymph node; System; Time; activation-induced cytidine deaminase; base; c-myc Genes; chromatin immunoprecipitation; follow-up; genome-wide; improved; metaplastic cell transformation; pathogen; prevent; public health relevance; repair enzyme; repaired; research study; uracil-DNA glycosylase

DESCRIPTION (provided by applicant): Antibody (immunoglobulin, Ig) class switch causes B lymphocytes to switch from producing IgM to producing IgG, IgA or IgE, which improves the ability of the antibody to remove pathogens and bacterial toxins from the body. Class switching occurs by an intrachromosomal DNA recombination event that must be carefully controlled in order to avoid aberrant recombination with other chromosomes (translocations). However, translocations do occur between oncogenes and the IgH locus, and this can lead to B cell lymphomas. During class switching, activation-induced cytidine deaminase (AID) initiates the formation of DNA double strand breaks (DSBs) at switch (S) regions in the Ig heavy chain gene locus (IgH), which are necessary for class switching. My first Aim is to determine how deamination of dC's in Ig S regions by AID, forming dU's, results in DSBs. We have shown that AID-induced deamination of dC leads to DNA single-strand breaks (SSBs) via the base excision repair pathway. How these SSBs are then converted to DSBs is less clear. We have reported that another DNA repair pathway, mismatch repair (MMR) is important for this step, and we will investigate its role. We will investigate how SSBs are converted to DSBs by determining the frequency and sites of AID- induced dU's in S regions, how the frequency and positions of AID targets affects frequency of switching, and whether MMR proteins might be recruited to S regions by AID itself. In Aim 2 we will follow up on our finding during the current term of this grant that AID can instigate DSBs at sites other than the IgH locus in activated B cells. We will determine what makes these other sites targets for AID, and if these DSBs lead to chromosome breaks, deletions and translocations, and whether MMR and other DNA repair proteins known to be involved in CSR, for example, ATM, H2AX, and 53BP1 are involved in making or preventing these DSBs. PUBLIC HEALTH RELEVANCE: The majority of human mature B-cell lymphomas are characterized by reciprocal chromosomal translocations that often involve the immunoglobulin (Ig) genes. The longstanding assumption that these translocations are generated by erroneous antibody class switching, a normal process that is essential for an effective humoral immune response, was verified only recently. Antibody class switching is a process that requires the introduction of DNA breaks into the Ig genes, into special regions called switch regions, and then joining one of these DNA break ends with another DNA break end in the same Ig locus, on the same chromosome. The mechanism of induction of DNA breaks and also how this process is regulated to prevent joining the DNA breaks to another chromosome, which can result in activation of oncogenes and thus B cell lymphomas not understood. The experiments proposed here will determine how the activity of the enzyme AID, which initiates class switching and also chromosomal translocations, is regulated and how it chooses its targets. We have shown that a specific DNA repair pathway in cells, called mismatch repair, which is essential for repairing mistakes during DNA synthesis, is required for formation of most of the DNA breaks during class switching. We seek to further understand its role in converting AID-induced lesions to DNA breaks. Mismatch repair is an essential repair system for eliminating mutations and microsatellite instability, thereby protecting against cellular transformation and malignancy. The hypothesis that this pathway increases DNA breaks seems counterintuitive, but arises directly from our understanding of the role of this pathway during class switching. AID is required for chromosomal translocations between the c-myc oncogene and the Ig locus in mature B-cell plasmacytomas in mouse. Also, many human B-cell lymphomas that originate from germinal center B cells show chromosomal translocations involving the Ig switch regions, suggesting the involvement of class switch processes. IgH translocations often greatly increase oncogene expression due to juxtaposition to the strong IgH enhancers, leading to cellular transformation and malignancy. Whether AID targeting to other loci besides the Ig loci leads to formation of DNA breaks at these sites is, however, unknown. By the use of a genome- wide search method, we have found AID-dependent DNA breaks at several other sites in the genome. We will examine whether these other sites are involved in chromosomal translocations with the Ig locus. We will investigate what makes these other sites targets for AID, and for DNA break formation. Thus our studies will also shed light on the important question of how AID chooses its targets.

描述（由申请人提供）：抗体（免疫球蛋白，IG）类开关导致B淋巴细胞从产生IgM转变为产生IgG，IgA或IgE，从而提高了抗体去除体内的病原体和细菌毒素的能力。类别切换是通过肉体内DNA重组事件进行的，必须仔细控制，以避免与其他染色体（易位）异常重组。然而，确实发生了易发性和IGH基因座之间的易位，这可能导致B细胞淋巴瘤。在课堂交换过程中，激活诱导的胞苷脱氨酶（AID）启动了IG重链基因基因座（IGH）中开关区域的DNA双链断裂（DSB）的形成，这对于类开关是必需的。我的第一个目的是确定DC在Ig S区域的脱氨酸，形成DU的结果，结果是DSB的。我们已经表明，辅助诱导的直流脱氨基通过碱基切除修复途径导致DNA单链断裂（SSB）。然后，如何将这些SSB转换为DSB并不清楚。我们报告说，另一种DNA修复途径，不匹配修复（MMR）对于此步骤很重要，我们将研究其作用。我们将通过确定S区域的AID诱导DU的频率和位点，如何将SSB转换为DSB，AID目标的频率和位置如何影响开关的频率以及是否可以通过AID本身招募MMR蛋白。在AIM 2中，我们将在本赠款的当前术语中进行跟进，即援助可以在激活B细胞中的IGH基因座以外的其他位点启动DSB。我们将确定是什么使这些其他站点成为AID的目标，以及这些DSB是否导致染色体断裂，缺失和易位，以及MMR和其他已知参与CSR的DNA修复蛋白是否参与CSR，例如ATM，H2AX和53BP1参与制造或预防这些DSB。 公共卫生相关性：大多数人类成熟的B细胞淋巴瘤的特征是相互染色体易位，通常涉及免疫球蛋白（IG）基因。长期以来，这些易位是通过错误的抗体类切换产生的，这是一种正常过程，这对于有效的体液免疫反应至关重要。抗体类切换是一个过程，需要将DNA断裂到IG基因，称为开关区域的特殊区域，然后将这些DNA断裂之一连接到同一Ig基因座的另一个DNA断裂端，在同一染色体上。 DNA断裂诱导的机制以及如何调节该过程以防止连接DNA与另一个染色体的分解，这可能导致癌基因的激活，因此无法理解B细胞淋巴瘤。此处提出的实验将决定如何调节酶AID的活性以及启动类转换以及染色体易位的活性以及如何选择其靶标。我们已经表明，细胞中的特定DNA修复途径称为不匹配修复，这对于在DNA合成过程中修复错误至关重要，这对于在课堂切换过程中形成大多数DNA断裂是必需的。我们寻求进一步了解其在将辅助诱导的病变转化为DNA断裂中的作用。不匹配修复是消除突变和微卫星不稳定性的必不可少的修复系统，从而防止细胞转化和恶性肿瘤。该途径增加DNA断裂的假说似乎是违反直觉的，但直接源于我们对该途径在课堂切换过程中的作用的理解。 小鼠成熟的B细胞浆细胞瘤中C-Myc癌基因与Ig基因座之间的染色体易位需要辅助。同样，许多来自生发中心B细胞的人类B细胞淋巴瘤都显示出涉及IG开关区域的染色体易位，这表明类开关过程的参与。由于与强IGH的增强子并置，IGH易位通常会大大增加癌基因的表达，从而导致细胞转化和恶性肿瘤。然而，除了IG基因座以外，针对其他基因座的辅助是否导致在这些位点形成DNA断裂是未知的。通过使用基因组搜索方法，我们发现了基因组中其他几个部位的辅助依赖性DNA断裂。我们将检查其他这些位点是否与IG基因座有关染色体易位。我们将调查是什么使这些其他地点目标是辅助的，并导致DNA断裂形成。因此，我们的研究还将阐明援助如何选择目标的重要问题。