Complex regulation of somatic hypermutation by cis-acting sequences in the endogenous IgH gene in hybridoma cells

To create high-affinity antibodies, B cells target a high rate of somatic hypermutation (SHM) to the Ig variable-region genes that encode the antigen-binding site. This mutational process requires transcription and is triggered by activation-induced cytidine deaminase (AID), which converts deoxycytidine to deoxyuridine. Mistargeting of AID to non-Ig genes is thought to result in the malignant transformation of B cells, but the mechanism responsible for targeting SHM to certain DNA regions and not to others is largely unknown. Cis-acting elements have been proposed to play a role in directing the hypermutation machinery, but the motifs required for targeting SHM have been difficult to identify because many of the candidate elements, such as promoters or enhancers, are also required for transcription of Ig genes. Here we describe a system in cultured hybridoma cells in which transcription of the endogenous heavy-chain Ig gene continues in the absence of the core intronic enhancer (Eμ) and its flanking matrix attachment regions (MARs). When AID is expressed in these cells, SHM occurred at the WT frequency even when Eμ and the MARs were absent together. Interestingly, SHM occurred at less than the WT frequency when Eμ or the MARs were individually absent. Our results suggest that these intronic regulatory elements can exert a complex influence on SHM that is separable from their role in regulating transcription.