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 (S{micro}) 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 S{micro} region. Successful recovery of BRD2 from the S{micro} 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.