鉴定活化 B 细胞中 IgH 开关序列处与 R 环相关的 RNA-DNA 杂交体。

Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells.

机构信息

Department of Microbiology and Immunology, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Department of Pharmacy, School of Pharmacy, Jeonbuk National University, Jeonju, South Korea.

出版信息

Methods Mol Biol. 2022;2528:55-66. doi: 10.1007/978-1-0716-2477-7_5.

DOI:10.1007/978-1-0716-2477-7_5

PMID:35704185

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9261291/

Abstract

During transcription and replication, R-loops that contain RNA-DNA hybrids are generated across numerous genomic loci and contribute to many biological events. Using S9.6, a monoclonal antibody against RNA-DNA hybrids, accelerated the study of R-loop biology. An outpouring of recent studies has implicated various contributions of R-loop in physiological cellular functions. Earlier studies using nondenaturing sodium bisulfite probing also supported existence of DNA-RNA hybrids formation in mammalian cells. In activated B cells, RNA-DNA hybrids formation at IgH gene locus of B cells is crucial for class switch recombination that ensure the proper effector function of the antibody. Here, we describe the identification of R-loops associated with the IgH locus using RNA-DNA hybrid immunoprecipitation sequencing and nondenaturing sodium bisulfite probing. This will be helpful for future studies of R-loops status on whole genome as well as on IgH locus in B cells.

摘要

在转录和复制过程中，含有 RNA-DNA 杂交体的 R 环会在许多基因组位置产生，并促成许多生物学事件。使用针对 RNA-DNA 杂交体的单克隆抗体 S9.6，加速了 R 环生物学的研究。最近的大量研究表明，R 环在生理细胞功能中具有多种作用。早期使用非变性亚硫酸氢钠探测的研究也支持哺乳动物细胞中 DNA-RNA 杂交体形成的存在。在活化的 B 细胞中，B 细胞中 IgH 基因座的 RNA-DNA 杂交体的形成对于保证抗体的适当效应功能的类别转换重组至关重要。在这里，我们使用 RNA-DNA 杂交体免疫沉淀测序和非变性亚硫酸氢钠探测描述了与 IgH 基因座相关的 R 环的鉴定。这将有助于未来对整个基因组以及 B 细胞中 IgH 基因座上的 R 环状态进行研究。