Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

Department of Chemistry University of Virginia Charlottesville VA USA

Department of Immunobiology Yale University School of Medicine New Haven CT USA

Department of Pathology University of Virginia Charlottesville VA USA

Institute of Biomedicine University of Turku Turku Finland

Institute of Molecular Genetics Czech Academy of Sciences Videnska 1083 CZ 14220 Prague 4 Czech Republic

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The SV40 virus enhancer functions as a somatic hypermutation-targeting element with potential tumorigenic activity

Ig Enhancers Increase RNA Polymerase II Stalling at Somatic Hypermutation Target Sequences