Identification of Factors Involved in the DNA Repair Phase of B Cell Class Switch Recombination

B cells undergo class switch recombination (CSR) in order to modify their antibody isotypes during an immune response. Different antibody isotypes convey unique immune functions, predominate in different tissue environments, and are responsible for clearance of a distinct array of pathogens. CSR requires the timely repair of DNA double-stranded breaks (DSBs) generated at the immunoglobulin locus that emerge from self-inflicted genetic lesions introduced by the enzyme activation-induced cytidine deaminase (AID). Researchers have taken advantage of the programmed DSBs generated during CSR to make significant conceptual insights into the mechanisms of DSB sensing, the DNA damage response (DDR), and the resolution of DSBs through various recombination or end joining pathways. Nonetheless, our comprehension of the DSB repair phase of CSR remains incomplete, with significant knowledge gaps in understanding which factors are recruited to DSBs, how they assemble in the context of various post-translational modifications, and how their dynamic functions influence the choice between different downstream repair pathways. We completed a genome-wide RNA interference screen in the murine B cell line CH12F3-2 with the goal of identifying novel factors involved in the repair phase of CSR. With this system, we selected three candidates, Kin, Eny2, and Usp22, as factors for further investigation. We confirm that Kin is critical for optimal CSR and is required for the functionality of multiple DSB repair pathways. Moreover, we demonstrate that Eny2 and Usp22, both members of the H2B deubiquitination module of the SAGA complex, maintain CSR by regulating the expression of the ÎłH2AX histone mark, a key intermediate in the DNA damage response. These findings uncover new ground for our growing understanding of cell-ubiquitous DSB repair, with implications in cancer, immunity, and aging.Ph.D