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.

父母赠款的项目摘要/摘要 类开关重组（CSR）是一个遗传过程，B细胞开关抗体同种型产生 通过位点特异性的染色体内DNA重排，由DNA双链的形成刺激 免疫球蛋白重链（IGH）基因座的断裂（DSB）。涉及的复发性致癌易位 IGH区分许多人类淋巴恶性肿瘤；这些易位源于错误修复的DNA 在正常淋巴细胞发育过程中产生的DSB。通常通过非同学修复DSB 最终连接（NHEJ）和替代端连接（Alt-EJ）DNA修复途径。在CSR期间，DSB的形成为 高度调节，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形成和维修的因素对 淋巴作用。 R环是三链RNA：CSR期间IGH形成的DNA杂交结构。而r 循环涉及促进IGH处的DSB形成，它们在类开关重组中的作用仍然存在 不明确的。为了确定持续的R环如何阻碍CSR期间的DNA修复，并且作用R环路 代谢在抑制IGH的基因组不稳定性方面发挥作用。我们育种小鼠缺乏涉及R的两种蛋白质 循环去除：解旋蛋白酶senataxin（setx - / - ）放松r循环； RNaseH2b对于 RNase H2核酸酶特异性消化了R loop的RNA成分（RRNASEH2BF/FCD19CRE）。 我们发现来自setX - / - rnaseh2bf/f小鼠的B细胞熟练地重组，并且包含高 IGH时未修复的断裂和染色体融合的水平。我们假设持续的R循环块 在免疫球蛋白重链基因座上通过非同源末端连接的有效DNA修复 类开关重组，导致持久，未修复的休息。我们将在功能上剖析 异常R环形成对DNA修复和染色体融合在CSR中产生的后果 setX - / - ，rnaseh2bf/f和setx - / - rnaseh2bf/f单元格（AIM 1）。定义持续的R循环对影响的影响 nhej，我们将在setx - / - ，rrnaseh2bf/f和setx - / - rrnaseh2bf/f细胞中表征DNA修复蛋白募集 （目标2）。我们还将确定使用高通量基因组的基因组基因座参与IGH易位 易位测序（HTGTS-SEQ）。最后，我们将定义驱动频繁的分子途径 在SETX - / - RNASEH2BF/F细胞中观察到的染色体融合（AIM 3）。我们的工作将定义持续的R循环 干扰类开关重组，导致未修复的断裂，并将发现分子 促进IGH染色体融合的机制。我们将获得该奖项的第二年 直到7/31/2024。