SUMMARY V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.

V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.DNA recombination | genomic plasticity | allosteric control | epigenetic modification | immune development A ll forms of DNA processing-replication, transcription, recombination, and repair-use allosteric regulation, often as a basis for molecular discrimination but also to establish a sequence of interactions or to bias the outcome of a reaction. In many instances the allosteric ligand is a specific DNA structure, as in Cre-mediated recombination, in which the Holliday junction intermediate effects allosteric conformational changes that switch active and inactive Cre monomers (1). In other instances the allosteric ligand is a DNA-bound protein array, as in λ integration, which is driven to completion by a flanking DNA-protein array that biases the conformation of λ-integrase (2).V(D)J recombination, the process by which antigen receptor genes are assembled, is also subject to allosteric control, but in this case the allosteric ligand is a specific chromatin mark rather than a DNA structure. V(D)J recombination is initiated by recombinationactivating gene (RAG)-1 and RAG-2, which together cleave DNA at recombination signal sequences (RSSs) flanking the participating gene segments (3). There are two classes of RSS, termed 12-RSS and 23-RSS, in which heptamer and nonamer elements are separated by spacers of 12 bp or 23 bp; physiological DNA cleavage requires the pairing of a 12-RSS with a 23-RSS (3). V(D)J recombination acts in an ordered, locus-specific fashion during lymphoid development. The accessibility of gene segments to V(D)J recombination is positively correlated with transcription at the unrearranged locus and with histone modifications characteristic of active chromatin, including hypermethylation of histone H3 at lysine 4 (H3K4me3) (4-10).RAG-1 and RAG-2 are 1,040 and 527 aa residues long, respectively. The catalytic core and DNA-binding functions are largely contained within RAG-1 (11). RAG-2, which is also ess...

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.