Structural and mechanistic studies of V(D)J recombination

V(D)J重组是从分散的V、D和J基因片段中组装抗原受体基因的位点特异性DNA重排。重组是由淋巴细胞特异性RAG1和RAG2重组酶启动的，RAG1和RAG2重组酶识别和切割重组信号序列。V(D)J重组受到严格调控，以优先的时间顺序发生，仅在特定的细胞类型和发育阶段发生。免疫球蛋白（Ig）重链重排先于轻链重排，Ig重链D - J连接先于V - DJ重组。此外，Ig基因仅在B细胞（而不是T细胞）中完全重排，T细胞受体基因在T细胞而不是B细胞中组装。非淋巴细胞中RAG1和RAG2的过表达足以诱导人工染色体外底物的重组，但不支持内源性基因座的V(D)J重组。因此，这些基因座对重组酶的可及性必须受到调控。大量证据表明，染色质结构的调控参与了V(D)J重组的调控。

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, this year we published a comprehensive paper on structures along the recombination signal recognition, pathway and DNA cleavage reaction. Six of these structures were determined by X-ray crystallography and one by cryoEM. These are the highest resolution and most revealing structures of RAG1/2 protein-DNA complexes in the world. 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.