RAG2 involves the Igκ locus demethylation during B cell development

The genes encoding the immunoglobulin kappa light chain are assembled during B cell development by V(D)J recombination. For efficient rearrangement, the Ig kappa locus must undergo a series of epigenetic changes. One such epigenetic mark is DNA methylation. The mechanism that the Ig kappa locus is selectively demethylated at the pre-B cell stage has not previously been characterized. Here, we employed bisulfite DNA-modification assays to analyze the methylation status of the Ig kappa locus in primary pre-B cells from RAG-deficient mice with pre-rearranged Ig kappa knock-in allele. We observed that the Ig kappa locus was hypermethylated in RAG2-deficient pre-B cells but hypomethylated in RAG1-deficient pre-B cells, indicating that wild-type (WT) RAG2 involves the Ig kappa locus demethylation in a RAG1-independent manner prior to rearrangement. We generated a series of RAG2 mutants between residue 350 and 383. We showed that these mutants mediated the Ig kappa rearrangement but failed to regulate the Ig kappa gene demethylation. We further analyzed that these mutants could increase RAG recombinase activity in vivo. We conclude that residues 350-383 region are responsible for endogenous Ig kappa locus demethylation at pre-B cells. We propose that WT RAG2 has an intrinsic function to regulate the Ig kappa locus demethylation.