Allosteric Regulation of V(D)J Recombination By Active Chromatin

V(D)J recombination, the process by which antigen receptor genes are assembled, is implicated in the pathogenesis of lymphoid malignancies. Rearrangement is initiated by the RAG recombinase, a heterotetrameric complex consisting of two subunits each of RAG-1 and RAG-2, which cleaves participating DNA segments at conserved signal sequences. The susceptibility of gene segments to undergo recombination is positively correlated with active chromatin marks, such as tri-methylation of histone H3 at lysine 4 (H3K4me3). RAG-2 interacts directly with H3K4me3 through a plant homeodomain (PHD), and this interaction is required for efficient V(D)J recombination in vivo. Genetic and biochemical evidence suggest that H3K4me3 stimulates substrate binding and DNA cleavage by RAG through an allosteric mechanism. This predicted that H3K4me3 binding induces conformational changes in RAG-2 that are transmitted to RAG-1. To determine whether the conformation of the RAG complex is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols using pulse-alkylation mass spectrometry. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within the DNA-binding domains and in the ZnH2 domain, which comprises part of the scaffold for the catalytic center. Our results demonstrate that engagement of the RAG-2 PHD induces dynamic conformational changes in the RAG-1 catalytic subunit, consistent with a model of allosteric control of RAG by active chromatin.