Identification of an inhibitory domain in RAG-2 that gates access of the V(D)J recombinase to chromatin
Ward, Alyssa Maria
MetadataShow full item record
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 characterized features of this PHD-mediated inhibitory domain and identified a second, independent, inhibitory domain. Mutation of this second inhibitory domain allows bypass of the requirement for engagement of H3K4me3 by the RAG-2 PHD. Disruption of this inhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate. We further characterized this domain genetically and determined that the inhibitory function is imposed by acidic residues between residues 352 and 405. Further, we were able to demonstrate that the inhibitory domain mutation is refractory to removal of the entire noncore portion of RAG-2. Therefore, this inhibitory domain acts independently of the PHD. Inactivation of the inhibitory domain confers a gain-of-function recombination phenotype and permits rearrangement at endogenous IgH and Igκ loci in the absence of H3K4me3 binding. In B progenitor cells, localization of wild-type RAG-2 to the IgH locus and actively transcribed loci is abolished by the W453A mutation, indicating that this pattern of chromatin localization is dependent on recognition of H3K4me3. This same mutation also abolishes association of RAG-1 with the IgH locus. Strikingly, disruption of the inhibitory domain permits association of RAG-2 and RAG-1 with the IgH locus even in the absence of H3K4me3 engagement by RAG-2. Thus, the RAG-2 inhibitory domain serves as a binary gate that permits the association of RAG-1 and RAG-2 with chromatin only if H3K4me3 is engaged by the RAG-2 PHD finger.