In antigen-stimulated B cells, the immunoglobulin (Ig) heavy chain constant region can be changed through a mechanism called Class Switch Recombination (CSR). This process alters the effector function of the Ig molecule while maintaining the antigen specificity. Defective CSR may result in hyper-IgM syndrome, chromosomal translocations, and autoimmune diseases. Thus, understanding the mechanism of CSR will provide valuable insights into these diseases. This work used a mouse B cell line (CH12F3) to study the mechanism of CSR.CSR is initiated by a B-cell-specific factor called activation-induced cytidine deaminase (AID), which converts cytosines to uracils in a single stranded DNA at switch (S) regions. In order for AID to access a S region, a germline transcription (GLT) through that S region is absolutely required. GLT is driven by cytokine-dependent promoter upstream of a non-coding I exon located upstream of each S region. GLT is also regulated by a large enhancer complex called the 3' regulatory region (3'RR) located at the 3'end of the last C region (Cα in mice). One of the functions of 3'RR is to stimulate and enhance GLT required for AID targeting and CSR. This 28kb region contains four enhancers identified as DNase I Hypersensitive (HS) sites. We show the four HS sites contain all functional elements of the 3’RR for CSR.Repair of AID-generated uracils in S regions leads to double strand break (DSB) formation. Widely accepted hypothesis in the field is that when uracils are generated within close proximity on opposing DNA strands, this can lead to closely generated nicks, which can result in DSBs. We show that AID deaminates evenly across the S region and between the top and bottom strands. In addition, the AID footprints show DSB formation occurs mostly with distal nicks. Several AID mutantations that cause hyper-IgM syndrome have been reported to possess active deaminase activity in biochemical assays but are defective for CSR. Why these mutants cannot support CSR is unknown. We focused on a mutation that results in AID with truncated C-terminus (AID∆C). Our study shows that AID∆C acts as a null allele, which is contrary to human patients. This suggests a possibility of organismal difference in AID regulation or functionality. These studies have improved our understanding of AID-targeting and AID action to S regions during CSR. Additionally, the creation of cell lines in these studies will allow further mutagenesis study of 3’RR and provide an important tool for identifying CSR-specific defect of other AID mutations.
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