Dissecting the composition, dynamics, and regulation of immunoglobulin class switch recombination

Abstract

Class switch recombination (CSR) is a somatic recombination reaction occurring in mature B lymphocytes, that involves programmed double-strand break (DSB) formation and repair at immunoglobulin heavy chain locus (Igh). CSR is essential for adaptive immunity since it diversifies the effector functions of antibody responses. Many key players, including components of the DNA damage response and DSB repair, transcriptional regulators, long non-coding RNAs and chromatin remodelers, have been reported to contribute to the different phases of this complex process. However, how all these DNA-RNA-protein interactions are established and evolve to support the repair dynamics of CSR remains elusive. In order to bridge this knowledge gap, I have established a primary B cell model system that couples locus-specific chromatin purification and proximity labelling to high-resolution mass-spectrometry to identify the factors that selectively bind within the Igh locus to support DSB processing and repair during CSR. 53BP1 is a DNA damage response factor that protects DSB ends from nucleolytic processing to promote their repair via the nonhomologous end-joining (NHEJ) pathway. Because of this activity, 53BP1 is not only crucial for CSR, but also facilitates toxic mis-repair (toxic-NHEJ) of DSBs in BRCA1-deficient cells. Phosphorylation of 53BP1 on its N-terminus by the DSB repair kinase ataxia telangiectasia mutated (ATM) is essential for DNA end-protection, and, as a consequence, for both these physiological (CSR) and pathological 53BP1 (toxic-NHEJ) repair events. Through a SILAC-based mass-spectrometry approach, we identified acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) as a potential interactor of 53BP1 under unphosphorylated conditions. I tested the hypothesis that ANP32B might interact with 53BP1 and negatively regulate it during DSB repair, which on further assessment I confirmed is dispensable for repair-associated activity. Rap1-interacting factor 1 (RIF1) is a multi-functional protein that, together with 53BP1, acts as a pro-NHEJ protein because of its ability to mediate DSB end-protection. As a consequence, RIF1 promotes both CSR in mature B cells and toxic-NHEJ reactions on BRCA1-deficient backgrounds. However, the post-translational regulation of RIF1 DNA end-protection function has not yet been elucidated. To this end, I implemented label-free mass-spectrometry in primary B lymphocytes to define potential phosphorylation events supporting RIF1 DNA end-protection activity. I identified a serine residue (S2138) that is phosphorylated upon DSB induction; however, this post-translational modification was dispensable for regulating RIF1 function during DSB repair and CSR. Altogether, CSR repair components, including 53BP1 and RIF1, define a crucial balance between immunity and lymphomagenesis in B cells. Therefore, identifying novel factors and post-translational modifications involved during CSR break repair will expand our understanding of the mechanisms preserving genome stability during antibody diversification in mature B cells.