title: Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching.
abstract: 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. In mice, six additional sets of constant region exons (CHs) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors. 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 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.
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Junchao
authors: J
authors: Dong
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Rohit A
authors: RA
authors: Panchakshari
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Tingting
authors: T
authors: Zhang
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Yu
authors: Y
authors: Zhang
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Jiazhi
authors: J
authors: Hu
authors: Boston Children's Hospital and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, Massachusetts, USA.
authors: Sabrina A
authors: SA
authors: Volpi
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Robin M
authors: RM
authors: Meyers
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Yu-Jui
authors: YJ
authors: Ho
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Zhou
authors: Z
authors: Du
authors: Howard Hughes Medical Institute, Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
authors: Davide F
authors: DF
authors: Robbiani
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Feilong
authors: F
authors: Meng
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Monica
authors: M
authors: Gostissa
authors: Howard Hughes Medical Institute, Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.
authors: Michel C
authors: MC
authors: Nussenzweig
authors: Boston Children's Hospital and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, Massachusetts, USA.
authors: John P
authors: JP
authors: Manis
authors: Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors: Frederick W
authors: FW
authors: Alt
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003564:Cytidine Deaminase / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003641:Deamination; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D051379:Mice; D017384:Sequence Deletion / Q000235:genetics; D000071857:Tumor Suppressor p53-Binding Protein 1; D050441:VDJ Exons / Q000235:genetics; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; C498302:Trp53bp1 protein, mouse; D000071857:Tumor Suppressor p53-Binding Protein 1; D064007:Ataxia Telangiectasia Mutated Proteins; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase