Isolation of region-specific factors assembled on the Immunoglobulin heavy chain locus during antibody class switch recombination

Activation-Induced Cytidine Deaminase (AID) induces DNA double-strand breaks (DSBs) at the switch (S) regions of the Immunoglobulin heavy chain ( Igh ) locus, which are essential for Class Switch Recombination (CSR) and Somatic Hypermutation (SHM), key processes for effective antibody production. While AID activity is critical, its off-target effects, such as DSBs at the Myc locus, can cause chromosomal translocations like Igh–Myc fusions, contributing to B-cell lymphomas. The factors assembled on Igh locus that tether AID-induced DSBs and subsequently the CSR, remain unknown. To address this, we developed a method to isolate CSR-specific factors by inserting a 5X-GAL4-UAS sequence at the switch-mu ( Sµ ) region in CH12 cells. This engineered site enables recruitment of a 3-FLAG-GAL4 DNA-binding protein (3F-GAL4-DBD), allowing specific pulldown of proteins enriched at the Sµ region. Successful recovery of BRD2 from the Sµ region, a known CSR regulator, validated this approach. Characterizing these factors may uncover novel regulators of CSR and highlight mechanisms that balance antibody diversification with genomic integrity in B cells.