Regulatory mechanisms in V(D)J recombination

During lymphoid development, a diverse array of immunoglobulin and T cell receptor genes are assembled in a series of site-specific recombination reactions termed V(D)J recombination. This dissertation investigates several mechanisms involved in the regulation of V(D)J recombination. To better understand how RAG transposition is suppressed in vivo, I defined the steps of the transposition reaction pathway. I show that both V(D)J cleavage and release of flanking coding DNA occur before the RAG proteins bind target DNA and commit to the transposition pathway, suggesting that coding DNA may aid in preventing the transpositional resolution of V(D)J recombination intermediates. I also demonstrate that the C-terminal portion of RAG2 inhibits transposition of uncleaved substrates and that this block in transposition is enforced at the step of target capture, further supporting the notion that coding end release is a key step in the regulation of RAG transposition. In order to better understand how V(D)J recombination is developmentally regulated, I collaborated with Or Gozani (Stanford) and Wei Yang (NIH) to examine whether RAG2 binds modified histories. We find that a plant homeodomain (PHD) finger present in the C-terminal portion of RAG2 specifically recognizes histone H3 that is concurrently trimethylated at lysine 4 and symmetrically dimethylated at arginine 2. This interaction is functionally significant because mutations that abrogate RAG2's recognition of methylated H3 severely impair V(D)J recombination in vivo. Likewise, reducing the level of H3K4me3 also leads to a decrease in V(D)J recombination in vivo. A conserved tryptophan residue (W453) that is essential for RAG2's recognition of methylated H3 is mutated in patients with immunodeficiency syndromes. Finally, in the absence of a modified histone peptide, a cis-peptide occupies the substrate-binding site, suggesting a potential autoregulatory mechanism for RAG2. Taken together, this work identifies a novel function for histone methylation in DNA recombination. Furthermore, this is the first example of a single domain synergistically recognizing two adjacent histone modifications, arguing for increased diversity and complexity in the read-out of combinatorial histone modifications. Finally, this work provides the first evidence suggesting that disrupting the read-out of histone modifications can cause an inherited human disease.