Epigenetic control of V(D)J recombination

In the course of lymphoid development, V(D)J recombination is subject to stringent locus-specific and temporal regulation. These constraints are ultimately responsible for several features peculiar to lymphoid development, including the lineage specificity of antigen receptor assembly, allelic exclusion and receptor editing. Regulation of V(D)J recombination involves interactions between the V(D)J recombinase---a heteromeric complex consisting of the recombination activating gene (RAG) proteins RAG-1 and RAG-2 subunits---and macromolecular assemblies extrinsic to the recombinase. This thesis will characterize those features of the recombinase itself---and in particular the RAG-2 subunit---that interact with extrinsic factors to establish patterns of temporal control and locus specificity in developing lymphocytes.;I will show that RAG-2 binds specifically to histone H3 and that this binding is absolutely dependent on di- or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction requires a non-canonical plant homeodomain (PHD) that has previously been described within the non-core region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-JH-C locus exhibits a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger (W453A) abolishes binding to H3K4me3 and greatly impairs recombination of extrachromosomal and endogenous immunoglobulin gene segments.;I will proceed to present evidence that hypermethylated H3K4 may not simply act as a docking site for the recombinase but rather may play a more active role as an allosteric trigger of RAG catalysis. To this end, I have identified an inhibitory domain within the non-core region of RAG-2. Mutations within this region restore activity of the RAG-2 (W453A) mutant to a level similar to that of core RAG-2. Moreover, a single mutation within the putative inhibitory domain yields a hyperactive form of RAG-2 whose activity exceeds that of wild type in extrachromosomal assays for recombination.;Results presented in this thesis provide a bridge between one chemical mark of active chromatin and the V(D)J recombinase machinery. An understanding of how histone modification is linked mechanistically to V(D)J recombination may provide a starting point from which to address the larger problem of allelic exclusion.