Journal Article

Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching

Meng, Feilong, Nussenzweig, Michel C., Dong, Junchao, Zhang, Yu, Robbiani, Davide F., Volpi, Sabrina A., Zhang, Tingting, Du, Zhou, Meyers, Robin M., Panchakshari, Rohit A., Ho, Yu-Jui, Manis, John P., Gostissa, Monica, Hu, Jiazhi, Alt, Frederick W.

2015

Overview

High-throughput genome-wide sequencing reveals why class switch recombination in the IgH locus, an essential step in the process of antibody generation, has a directional joining bias towards deletion rather than inversion. Orientation-specific DNA joining The process of antibody generation requires rearrangements in the immunoglobulin heavy chain (IgH) locus to juxtapose single V, D and J gene segments, by excising all the remaining segments. In principle, the process making these deletions could also result in inversions. Frederick Alt and colleagues now use high-throughput genome-wide sequencing to examine the long-standing question of why the process at this locus has a directional bias towards deletion rather than inversion. They find that it involves sequences within the IgH locus itself, double-strand DNA breaks initiated by the AID deaminase, and double-strand break repair factors 53BP1 and ATM. During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons 1 , 2 . In mice, six additional sets of constant region exons (C H s) lie 100–200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons 2 . Long repetitive switch (S) regions precede Cμ and downstream C H s. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream C H (ref. 2 ). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region 2 , 3 ; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors 3 . Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing 4 into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 13/106 / 13/109 / 13/21 / 13/31 / 42/41 / 49/23 / 631/250/1619/40 / 631/337/1427/2122 / Animals / Antibodies / Ataxia Telangiectasia Mutated Proteins - metabolism / B-Lymphocytes - enzymology / B-Lymphocytes - immunology / B-Lymphocytes - metabolism / Chromosomal Proteins, Non-Histone - metabolism / Cytidine Deaminase - metabolism / Deamination / Deoxyribonucleic acid / DNA / DNA Breaks, Double-Stranded / DNA Repair - genetics / DNA-Binding Proteins - metabolism / Genetic aspects / Genetic recombination / Genetic research / Genomes / Humanities and Social Sciences / Immunoglobulin Class Switching - genetics / Immunoglobulin Constant Regions - genetics / Immunoglobulin Heavy Chains - genetics / Immunoglobulins / Lesions / letter / Mice / multidisciplinary / Nucleotides / Science / Sequence Deletion - genetics / Translocation / Tumor Suppressor p53-Binding Protein 1 / VDJ Exons - genetics / Viral antibodies