R-loop-induced DNA damage during immunoglobulin class switch recombination

免疫球蛋白类别转换重组过程中 R 环诱导的 DNA 损伤

• 批准号: 10294876
• 负责人: Jacqueline Barlow
• 金额: $ 3.23万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-05 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294876
• 关键词: Antibodies; Automobile Driving; Award; B-Lymphocytes; Cells; Chromatin; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA Sequence Rearrangement; Defect; Development; Event; Excision; Gene Rearrangement; Genetic Processes; Genomic Instability; Genomics; Heavy-Chain Immunoglobulins; Human; Hybrids; IGH@ gene cluster; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Immunoglobulins; Lymphocyte; Lymphomagenesis; Malignant Neoplasms; Malignant lymphoid neoplasm; Metabolism; Molecular; Mus; Mutation; Nonhomologous DNA End Joining; Oncogenic; Pathway interactions; Play; Production; Proteins; RNA; Recurrence; Ribonucleases; Role; Running; SETX gene; Site; Structure; Transcriptional Activation; Work; chromosome fusion; genome-wide; genomic locus; helicase; insight; novel; nuclease; parent grant; public health relevance; recruit; repaired; tumorigenesis

Project Abstract/Summary for the parent grant Class switch recombination (CSR) is a genetic process where a B cell switches antibody isotype production through site-specific intra-chromosomal DNA rearrangement stimulated by the formation of DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain (IgH) locus. Recurrent oncogenic translocations involving IgH distinguish many human lymphoid malignancies; these translocations originate from mis-repaired DNA DSBs generated during normal lymphocyte development. DSBs are normally repaired by the non-homologous end-joining (NHEJ) and alternative-end joining (alt-EJ) DNA repair pathways. During CSR, DSB formation is highly regulated involving a complex interplay of transcriptional activation, protein recruitment and chromatin reorganization. Understanding the factors regulating DSB formation and repair has a high impact on lymphomagenesis. R loops are three stranded RNA:DNA hybrid structures formed at IgH during CSR. While R loops are implicated in promoting DSB formation at IgH, their role in class switch recombination remains undefined. To determine how persistent R loops impede DNA repair during CSR, and the role R loop metabolism plays in suppressing genome instability at IgH. we bred mice lacking two proteins involved in R loop removal: the helicase Senataxin (Setx-/-) which unwinds R loops; and Rnaseh2b is defective for the RNase H2 nuclease that specifically digests the RNA component of R loops (Rrnaseh2bf/fCD19cre). We find that B cells from Setx-/-Rnaseh2bf/f mice are proficient at class switch recombination, and contain high levels of unrepaired breaks and chromosome fusions at IgH. We hypothesize that persistent R loops block efficient DNA repair by non-homologous end joining at the immunoglobulin heavy chain locus during class switch recombination, leading to persistent, unrepaired breaks. We will functionally dissect the consequences of aberrant R loop formation on DNA repair and chromosome fusions arising during CSR in Setx-/-, Rnaseh2bf/f, and Setx-/- Rnaseh2bf/f cells (Aim 1). To define the impact persistent R loops have on NHEJ, we will characterize DNA repair protein recruitment in Setx-/-, Rrnaseh2bf/f, and Setx-/- Rrnaseh2bf/f cells (Aim 2). We will also identify genomic loci involved in IgH translocations using high-throughput genome-wide translocation sequencing (HTGTS-Seq). Finally, we will define the molecular pathways driving the frequent chromosome fusions observed in Setx-/- Rnaseh2bf/f cells (Aim 3). Our work will define how persistent R loops interfere with class switch recombination, leading to unrepaired breaks, and will uncover the molecular mechanisms promoting chromosome fusions at IgH. We are in the second year of this award which will run until 7/31/2024.

家长资助项目摘要/摘要 类别转换重组 (CSR) 是 B 细胞转换抗体同种型产生的遗传过程 通过 DNA 双链形成刺激的位点特异性染色体内 DNA 重排 免疫球蛋白重链 (IgH) 位点处的断裂 (DSB)。复发性致癌易位涉及 IgH 可区分多种人类淋巴恶性肿瘤；这些易位源自错误修复的DNA DSB 在正常淋巴细胞发育过程中产生。 DSB 通常由非同源修复 末端连接 (NHEJ) 和替代末端连接 (alt-EJ) DNA 修复途径。在 CSR 期间，DSB 的形成是 受到高度调控，涉及转录激活、蛋白质募集和染色质的复杂相互作用 重组。了解调节 DSB 形成和修复的因素对 淋巴瘤发生。 R 环是 CSR 过程中在 IgH 处形成的三链 RNA:DNA 杂合结构。而R 环涉及促进 IgH 处 DSB 的形成，但它们在类别转换重组中的作用仍然存在 不明确的。确定持久性 R 环如何阻碍 CSR 过程中的 DNA 修复，以及 R 环的作用 代谢在抑制 IgH 基因组不稳定性方面发挥着作用。我们培育出缺乏两种与 R 相关的蛋白质的小鼠 环去除：解旋酶 Senataxin (Setx-/-)，它解开 R 环； Rnaseh2b 有缺陷 RNase H2 核酸酶，特异性消化 R 环的 RNA 成分 (Rrnaseh2bf/fCD19cre)。 我们发现来自 Setx-/-Rnaseh2bf/f 小鼠的 B 细胞精通类别转换重组，并且含有高 IgH 处未修复的断裂和染色体融合水平。我们假设持久 R 循环会阻塞 在免疫球蛋白重链位点上通过非同源末端连接进行有效的 DNA 修复 类开关重组，导致持久的、无法修复的中断。我们将从功能上剖析 异常 R 环形成对 CSR 过程中 DNA 修复和染色体融合的影响 Setx-/-、Rnaseh2bf/f 和 Setx-/- Rnaseh2bf/f 细胞（目标 1）。定义持久 R 循环对 NHEJ，我们将表征 Setx-/-、Rrnaseh2bf/f 和 Setx-/- Rrnaseh2bf/f 细胞中 DNA 修复蛋白的招募 （目标 2）。我们还将利用高通量全基因组技术来鉴定参与 IgH 易位的基因组位点 易位测序（HTGTS-Seq）。最后，我们将定义驱动频繁发生的分子途径 在 Setx-/- Rnaseh2bf/f 细胞中观察到染色体融合（目标 3）。我们的工作将定义持久 R 循环的方式 干扰类别转换重组，导致无法修复的断裂，并将揭示分子 IgH 促进染色体融合的机制。我们已进入该奖项的第二年，该奖项将运行 直至 2024 年 7 月 31 日。