Immunoglobulin class switch DNA recombination

免疫球蛋白类别转换 DNA 重组

• 批准号: 8513563
• 负责人: Paolo Casali
• 金额: $ 52.26万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513563
• 关键词: 14-3-3 Proteins; Adaptor Signaling Protein; Address; Advanced Development; Affinity; Allergic; Antibodies; Antibody Formation; Ascaridil; Autoantibodies; Autoantigens; Autoimmunity; B-Lymphocytes; Binding; Biological; C-terminal; Catalytic Domain; Cell surface; Cells; ChIP-seq; Characteristics; Chromatin; Cyclic AMP-Dependent Protein Kinases; DNA; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA lesion; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Data; Deoxycytidine; Dependency; Development; Elements; Enzymes; Estrogens; Family; Fluorescence Resonance Energy Transfer; Funding; Generations; Genes; Genetic Recombination; Genetic Transcription; Grant; HIV; Histones; Homologous Protein; IgE; IgG1; IgG3; Immune system; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Impairment; Indium; Infection; Interleukin-4; Knock-out; Lead; Lesion; Mediating; Molecular; Mus; Mutant Strains Mice; Mutation; Pathway interactions; Pattern; Peptides; Play; Polymerase; Predisposition; Process; RNA Sequences; Receptors, Antigen, B-Cell; Recruitment Activity; Regulation; Resolution; Role; Salmonella; Serum; Stimulus; Stream; Syndrome; T-Lymphocyte; TNFRSF5 gene; TNFSF5 gene; Testing; Therapeutic; Toll-like receptors; Trans-Activators; Vaccines; activation-induced cytidine deaminase; base; combinatorial; cytokine; effective therapy; fighting; high throughput screening; influenzavirus; innovation; microbial; novel; pathogen; promoter; repair enzyme; research study; scaffold; small molecule; small molecule libraries; therapeutic development; tumor

DESCRIPTION (provided by applicant): We want to understand the molecular mechanisms of Ig class switch DNA recombination (CSR). CSR entails generation of double-strand DNA breaks (DSBs) and DSB resolution, as mediated by AID, Ung and other DNA-binding proteins/repair enzymes. In this grant current funding period (years 06-10), we have made important discoveries, including the: (i) identification of HoxC4 as trans-activator of AID gene promoter and demonstration of HoxC4 promoter activation by estrogen; (ii) characterization of 5'-AGCT-3' as the major con- served repeats in all IgH switch (S) regions and specific targets of 14-3-3 proteins; (iii) demonstration that 14-3- 3 function as DNA/protein adaptors that bind S regions/AID and PKA for CSR; (iv) identification of a post- cleavage role of AID in CSR; and (v) demonstration of an important role of the Rev1 DNA polymerase in CSR. Here, we want to identify the role of "open chromatin" in recruiting/stabilizing 14-3-3, AID, PKA, Rev1 and Ung to/on S regions, characterize the scaffolding functions of these factors in CSR and identify mechanisms of CSR inhibition. We hypothesize that 14-3-3, which have a high affinity for 5'-AGCT-3' repeats in all S regions, target only those that will undergo recombination due to the open chromatin state (reflected by germline transcription and combinatorial patterns of histone PTMs - our preliminary findings) of these regions, where they are stabilized by histone PTMs (Aim 1). Prompted by our preliminary findings that Rev1 recruits/stabilizes Ung to/on S regions, we contend that in addition to 14-3-3, which are bona fide "scaffolding" adaptors, AID, PKA, Rev1 and Ung possess scaffolding functions that reciprocally contribute to their recruitment/stabilization to/on S regions, and disruption of such scaffolding functions by naturally occurring or synthetic molecules aborts CSR (Aim 2). We will test our hypotheses by: (Aim 1.1) assessing the open chromatin state of the recombining S regions, including germline transcription and combinatorial patterns of histone PTM H3S10ph, H3K9/K14ac H3K4me3, H2BK120ub and H2BS14ph; (Aim 1.2) analyzing the selective binding of 14-3-3, AID, PKA, Rev1 and Ung to the recombining S regions, and its dependency on histone PTMs; (Aim 1.3) addressing the role of H3K9acS10ph, H3S10phK14ac and/or H2BK120ub PTMs in recruitment/stabilization of 14-3-3, Rev1 and AID to/on the recombining S regions; (Aim 2.1) defining 14-3-3, AID, PKA, Rev1 and/or Ung molecular interactions and their scaffolding functions that are central to CSR; (Aim 2.2) further understanding 14-3-3, AID, PKA, Rev1 and/or Ung scaffolding functions and interfering with such scaffolding functions to abort CSR by using naturally occurring Vpr/short Vpr peptides; (Aim 2.3) identifying, by high-throughput screening (HTS), synthetic small molecules that interfere with 14-3-3, AID, PKA, Rev1 and/or Ung interactions to abort CSR. Our experiments are timely and based on convincing preliminary data. They use innovative approaches, such as BiFC, qChIP, ChIP-seq, Seq-ChIP, RNA-seq, FRET, Vpr peptides, HTS and new KO/mutant mice, and they will significantly advance the development of therapeutics that block unwanted CSR, e.g., to IgG autoantibodies or atopic IgE.

描述(申请人提供)：我们想了解免疫球蛋白类开关DNA重组(CSR)的分子机制。CSR需要在AID、Ung和其他DNA结合蛋白/修复酶的介导下产生双链DNA断裂(DSB)和DSB分解。在本次资助期间(06-10年)，我们取得了一些重要发现，包括：(1)鉴定出HoxC4是AID基因启动子的反式激活因子，并证实了雌激素对HoxC4启动子的激活作用；(2)鉴定了5‘-AGCT-3’作为所有IgH开关(S)区域和14-3-3蛋白的特定靶点的主要保守重复序列；(3)证实了14-3-3-3作为DNA/蛋白质接头结合S区域/AID和PKA用于CSR；(4)鉴定了AID在CSR中的切割后作用；以及(V)Rev1 DNA聚合酶在CSR中的重要作用。在这里，我们想要确定“开放染色质”在招募/稳定14-3-3、AID、PKA、Rev1和Ung到S区域中的作用，表征这些因子在企业社会责任中的支架作用，并确定企业社会责任抑制的机制。我们假设，在所有S区域都对5‘-AGCT-3’重复具有高亲和力的14-3-3只针对那些由于这些区域的开放染色质状态(反映在胚系转录和组蛋白PTM的组合模式-我们的初步发现)而发生重组的区域，其中它们被组蛋白PTMS稳定(目标1)。根据我们的初步发现，Rev1在S区域招募/稳定NGG，我们认为，除了14-3-3是真正的“支架”接头外，Aid、PKA、Rev1和Ung还具有支架功能，这些功能相互促进它们在S区域招募/稳定，并且这种支架功能被自然发生的或合成的分子破坏而中止CSR(目标2)。我们将通过(目标1.1)评估重组的S区域的开放染色质状态，包括组蛋白PTM H3S10ph、H3K9/K14ac H3K4me3、H 2BK120ub和H 2BS14ph的胚系转录和组合模式；(目标1.2)分析14-3-3、Aid、PKA、Rev1和Ung与重组的S区域的选择性结合及其对组蛋白PTM的依赖；(目标1.3)研究H3K9acS10ph、H3S10phK14ac和/或H 2BK120ub的PTMS在14-3-3、Rev1和AID to/对重组的S区域的招募/稳定中的作用；(目标2.1)定义对CSR至关重要的14-3-3、AID、PKA、Rev1和/或Ung分子相互作用及其支架功能；(目标2.2)进一步了解14-3-3、AID、PKA、Rev1和/或Ung的支架功能，并干扰这些支架功能，通过使用天然存在的VPR/VPR短肽来终止CSR；(目标2.3)通过高通量筛选(HTS)鉴定干扰14-3-3、AID、PKA、Rev1和/或Ung相互作用的合成小分子以终止CSR。我们的实验是及时的，并且基于令人信服的初步数据。他们使用创新的方法，如BIFC、qChIP、CHIP-SEQ、SEQ-CHIP、RNA-SEQ、FRET、VPR肽、HTS和新的KO/突变小鼠，他们将极大地推动阻断不想要的CSR的治疗方法的发展，例如，针对免疫球蛋白自身抗体或特应性IgE。