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

基本信息

批准号：
10673639
负责人：
Jacqueline Barlow
金额：
$ 31.4万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/10673639
关键词：
Antibodies Automobile Driving B-Lymphocytes Biology Cell physiology Cells Cellular biology Chromatin Chromosome Fragile Sites Collaborations Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA Sequence Rearrangement Defect Development Dissection Enzymes Event Excision Frequencies G22P1 gene Gene Rearrangement Generations Genetic Processes Genetic Transcription Genomic Instability Genomics Goals Heavy-Chain Immunoglobulins Human Hybrids IGH@ gene cluster Immunoglobulin Class Switching Immunoglobulin Switch Recombination Immunoglobulins Ligase Location Lymphocyte Lymphomagenesis Malignant Neoplasms Malignant lymphoid neoplasm Maps Mediating Metabolism Molecular Monitor Mus Mutation Nonhomologous DNA End Joining Oncogenic Pathway interactions Pharmaceutical Preparations Play Poly(ADP-ribose) Polymerase Inhibitor Production Proteins RAD52 gene RNA Recurrence Research Personnel Ribonucleases Ribonucleotides Role Site Structure Techniques Testing Therapeutic Transcriptional Activation Work cancer cell cancer therapy chromosome fusion defined contribution experience genome-wide genomic locus helicase homologous recombination innovation insertion/deletion mutation insight model organism mouse model mutant novel nuclease public health relevance recruit repaired replication stress synergism tumorigenesis

项目摘要

Project Summary/Abstract 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. 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. We find that mice defective for R loop removal are proficient at class switch recombination, however B cells contain unrepaired breaks and chromosome fusions at IgH. Recurrent oncogenic translocations involving IgH distinguish many human lymphoid malignancies. These translocations originate from mis-repaired DNA double stand breaks (DSBs) generated during normal lymphocyte development. Our goal is 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 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. To test this hypothesis, two mouse models will be employed: the SETX mutant lacks the Senataxin (SETX) helicase that unwinds R loops;; and Rnaseh2b is defective for the RNase H2 nuclease that specifically digests the RNA component of R loops (RNH2B). We will functionally dissect the consequences of aberrant R loop formation on DNA repair and chromosome fusions arising during CSR in SETX-/-, RNH2Bf/f, and SETX-/- RNH2Bf/f cells (Aim 1). To define the impact persistent R loops have on NHEJ, we will characterize DNA repair protein recruitment in SETX-/-, RNH2Bf/f, and SETX-/- RNH2Bf/f cells (Aim 2). We will also identify genomic loci involved in IgH translocations using high-throughput genome-wide translocation sequencing (HTGTS-Seq), in collaboration with Dr. Feyredoun Hormozdiari. Finally, we will define the molecular pathways driving the frequent chromosome fusions observed in SETX-/- RNH2Bf/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. Enzymes regulating R loop metabolism will also provide an attractive target for developing novel cancer treatment.

项目摘要/摘要类开关重组（CSR）是一个遗传过程，其中B细胞开关抗体同种型通过构造刺激的染色体内DNA重排的染色体内DNA重排生产免疫球蛋白重链（IGH）基因座的DNA双链断裂（DSB） DSB是通常由非理论最终连接（NHEJ）和替代末端连接（alt-ej）修复 DNA修复途径。在CSR期间，DSB形成受到高度调节，涉及一个复杂的相互作用转录激活，蛋白质募集和染色质重组。了解因素调节DSB的形成和修复对淋巴作用的影响很高。 R循环是三个滞留的RNA：CSR期间IGH形成的DNA杂交结构。而r循环涉及在IGH时促进DSB形成，在类开关重组中的作用仍然不确定。我们发现无缺的R循环去除的小鼠在类开关重组中精通熟练，但是B细胞在IGH时包含未修复的断裂和染色体融合。复发性致癌易位涉及许多人类淋巴恶性肿瘤。这些易位起源于在正常淋巴细胞发育过程中产生的错误修复的DNA双支架断裂（DSB）。我们的目标是确定持续的r循环如何阻碍CSR期间的DNA修复，以及r循环代谢在抑制IGH的基因组不稳定性方面发挥作用。我们假设持续的R循环在免疫球蛋白重链上连接的非理论末端的块有效的DNA修复课堂开关重组期间的基因座，导致持续，未修复的休息。测试这个假设将采用两种小鼠模型：SETX突变体围巾senataxin（SETX）放松R循环的解旋酶; RNaseH2b对于RNase H2核酸酶是有缺陷的消化R环的RNA分量（RNH2B）。我们将在功能上剖析在setx-/ - ，在CSR期间出现的DNA修复和染色体融合的异常R环形式 RNH2BF/F和SETX - / - RNH2BF/F细胞（AIM 1）。为了定义持续的R循环对NHEJ的影响，我们将在SETX - / - ，RNH2BF/F和SETX - / - RNH2BF/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参与IGH易位的基因组网站全基因组易位测序（HTGTS-SEQ）与Feyredouun博士合作 Hormozdiari。最后，我们将定义驱动频繁染色体融合的分子途径在SETX - / - RNH2BF/F细胞中观察到（AIM 3）。我们的工作将定义持续的R循环如何干扰类开关重组，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。调节R环代谢的酶也将为开发新的癌症治疗提供了一个吸引人的目标。