Structural and mechanistic studies of V(D)J recombination

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V(D)J recombination is the site-specific DNA rearrangement that assembles antigen receptor genes from dispersed arrays of V, D and J gene segments. Recombination is initiated by the lymphoid-specific RAG1 and RAG2 recombinase, which recognizes and cleaves the recombination signal sequences. V(D)J recombination is tightly regulated, occurring in a preferred temporal order and only in specific cell types and developmental stages. Immunoglobulin (Ig) heavy chain rearrangement precedes light chain rearrangement and Ig heavy-chain D to J joining precedes V to DJ recombination. In addition, Ig genes are fully rearranged only in B cells (not T cells) and T cell receptor genes are assembled in T but not B cells. Over-expression of RAG1 and RAG2 in non-lymphoid cells is sufficient to induce recombination of an artificial extrachromosomal substrate, but does not support V(D)J recombination of endogenous loci. Therefore, the accessibility of these loci to the recombinase must be regulated. A large body of evidence suggests that the regulation of chromatin structure is involved in the regulation of V(D)J recombination. There are two components in our project. The first is to study the PHD domain in RAG2 protein and its role in regulating chromosome accessibility by RAG recombinase and thus tissue- and development-specific V(D)J recombination. The second is the structures of RAG1/RAG2 recombinase and their complex with signal DNA for V(D)J recombination (RSS). We have made progresses in both areas in this fiscal year. Our collaborators, Dr. Oettinger at Mass General Hospital and Gozani at Stanford University, have shown that the PHD domain of RAG2 recognizes histone H3 tail with lysine-4 trimethylated (H3K4me3). We have determined six crystal structures of RAG2-PHD complexed with H3K4 peptide containing variations of methylated residues and measured binding constants of RAG2-PHD and methylated H3 peptides. Our structural and biochemical studies have revealed new aspects of how the conserved PHD domains can recognize differently modified H3 tail and led to proposals of a regulatory mechanism of V(D)J recombination. The results are summarized in two manuscripts. Both are being reviewed for publication. In collaboration with Dr. Martin Gellerts lab, we have pursued a structural solution of RAG1/RAG2 recombinase and have finally succeeded in determining the crystal structure of mouse RAG1/RAG2 recombinase complex this year. References: Kim, M.S., Lapkouski, M., Yang, W. & Gellert M. (2015) Crystal structure of the V(D)J recombinase RAG1-RAG2, Nature, 518, 507-511. Grundy, G. J., Gellert, M. & Yang, W. (2010). Auto-inhibition of DNA cleavage mediated by RAG1 and RAG2 is overcome by an epigenetic signal in V(D)J recombination. PNAS, 107, 22487-92. Yang , W. (2011) Nucleases: diversity in structure, function and mechanism. Q Rev Biophys. 44, 1-93. Grundy, G. J., Ramn-Maiques, S., et al., Gellert, M. & Yang, W. (2009). Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the post-cleavage complex. Mol. Cell, 35, 217-227.