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.

项目摘要/概要 类别转换重组（CSR）是一种B细胞转换抗体同种型产生的遗传过程 通过由DNA双链形成刺激的位点特异性染色体内DNA重排 在免疫球蛋白重链（IgH）基因座处的断裂（DSB）。复发性致癌易位， IgH可区分许多人类淋巴系统恶性肿瘤;这些易位起源于错误修复的DNA 在正常淋巴细胞发育过程中产生DSB。DSB通常由非同源的 末端连接（NHEJ）和交替末端连接（alt-EJ）DNA修复途径。在CSR期间， 涉及转录激活、蛋白质募集和染色质的复杂相互作用的高度调节 重组。了解调节DSB形成和修复的因素对 淋巴瘤形成R环是在CSR过程中在IgH处形成的三链RNA：DNA杂交结构。而r 环参与促进IgH的DSB形成，它们在类别转换重组中的作用仍然存在 未定义。为了确定持续的R环如何阻碍CSR过程中的DNA修复，以及R环在CSR中的作用， 代谢在抑制IgH的基因组不稳定性中起作用。我们培育了缺乏两种与R相关的蛋白质的小鼠 环去除：解旋酶Senataxin（Setx-/-），其解旋R环;和Rnaseh2b是有缺陷的， RNA酶H2核酸酶，特异性地阻断R环的RNA组分（Rrnaseh2bf/fCD19cre）。 我们发现来自Setx-/-Rnaseh 2bf/f小鼠的B细胞精通类别转换重组，并且含有高水平的 未修复断裂和IgH染色体融合水平。我们假设持续的R环阻断了 通过免疫球蛋白重链基因座处的非同源末端连接的有效DNA修复， 类开关重组，导致持久的，不可修复的中断。我们将从功能上剖析 异常R环形成对DNA修复和染色体融合的影响， Setx-/-、Rnaseh 2bf/f和Setx-/-Rnaseh 2bf/f单元格（目标1）。要定义持久R循环对 NHEJ，我们将表征Setx-/-、Rrnaseh 2bf/f和Setx-/-Rrnaseh 2bf/f细胞中的DNA修复蛋白募集 (Aim 2）。我们还将使用高通量全基因组免疫荧光技术鉴定参与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的 在Setx-/-Rnaseh 2bf/f细胞中观察到染色体融合（Aim 3）。我们的工作将定义持久R循环 干扰类别转换重组，导致未修复的断裂，并将揭示分子 促进IgH处染色体融合的机制。我们是在第二年的这个奖项将运行 直到2024年7月31日。