Molecular Basis of Immunoglobulin Heavy Chain Switch

免疫球蛋白重链开关的分子基础

基本信息

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
项目类别：
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

项目摘要

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（申请人提供）：抗体（免疫球蛋白，Ig）类别转换导致B淋巴细胞从产生IgM转变为产生IgG、IgA或IgE，从而提高抗体清除体内病原体和细菌毒素的能力。类别转换是通过染色体内 DNA 重组事件发生的，必须仔细控制该重组事件，以避免与其他染色体发生异常重组（易位）。然而，癌基因和 IgH 基因座之间确实会发生易位，这可能导致 B 细胞淋巴瘤。在类别转换期间，激活诱导的胞苷脱氨酶 (AID) 会在 Ig 重链基因座 (IgH) 的转换 (S) 区域启动 DNA 双链断裂 (DSB) 的形成，这是类别转换所必需的。我的第一个目标是确定 AID 如何使 Ig S 区域中的 dC 脱氨，形成 dU，从而产生 DSB。我们已经证明，AID 诱导的 dC 脱氨基作用通过碱基切除修复途径导致 DNA 单链断裂 (SSB)。这些 SSB 如何转换为 DSB 尚不清楚。我们已经报道了另一种 DNA 修复途径，错配修复 (MMR) 对于这一步很重要，我们将研究它的作用。我们将通过确定 S 区中 AID 诱导的 dU 的频率和位点来研究 SSB 如何转换为 DSB，AID 靶标的频率和位置如何影响转换频率，以及 AID 本身是否可能将 MMR 蛋白招募到 S 区。在目标 2 中，我们将在本次资助的当前期限内跟进我们的发现，即 AID 可以在激活的 B 细胞中 IgH 基因座以外的位点激发 DSB。我们将确定是什么使这些其他位点成为 AID 的靶点，这些 DSB 是否会导致染色体断裂、缺失和易位，以及 MMR 和已知参与 CSR 的其他 DNA 修复蛋白（例如 ATM、H2AX 和 53BP1）是否是 AID 的靶点。参与制造或阻止这些 DSB。公共健康相关性：大多数人类成熟 B 细胞淋巴瘤的特征是染色体相互易位，通常涉及免疫球蛋白 (Ig) 基因。长期以来的假设是，这些易位是由错误的抗体类别转换产生的，这是一个对于有效的体液免疫反应至关重要的正常过程，直到最近才得到证实。抗体类别转换是一个过程，需要将 DNA 断裂引入 Ig 基因、称为转换区域的特殊区域，然后将其中一个 DNA 断裂末端与同一条染色体上同一 Ig 基因座中的另一个 DNA 断裂末端连接起来。 DNA 断裂的诱导机制以及如何调节该过程以防止 DNA 断裂连接到另一条染色体，这可能导致癌基因激活，从而导致 B 细胞淋巴瘤尚不清楚。这里提出的实验将确定 AID 酶的活性如何被调节以及它如何选择其靶标，AID 酶启动类别转换和染色体易位。我们已经证明，细胞中的一种特定的 DNA 修复途径（称为错配修复）对于修复 DNA 合成过程中的错误至关重要，是类别转换过程中大多数 DNA 断裂形成所必需的。我们寻求进一步了解其在将 AID 引起的损伤转化为 DNA 断裂中的作用。错配修复是消除突变和微卫星不稳定性的重要修复系统，从而防止细胞转化和恶性肿瘤。该途径增加 DNA 断裂的假设似乎违反直觉，但直接源于我们对该途径在类别转换过程中的作用的理解。小鼠成熟 B 细胞浆细胞瘤中 c-myc 癌基因和 Ig 基因座之间的染色体易位需要 AID。此外，许多源自生发中心 B 细胞的人类 B 细胞淋巴瘤显示出涉及 Ig 转换区域的染色体易位，表明涉及类别转换过程。由于与强 IgH 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，AID 靶向 Ig 基因座以外的其他基因座是否会导致这些位点形成 DNA 断裂尚不清楚。通过使用全基因组搜索方法，我们在基因组的其他几个位点发现了 AID 依赖性 DNA 断裂。我们将检查这些其他位点是否参与 Ig 基因座的染色体易位。我们将研究是什么使这些其他位点成为 AID 和 DNA 断裂形成的目标。因此，我们的研究还将揭示国际开发署如何选择目标这一重要问题。