Structural and mechanistic studies of V(D)J recombination

V(D)J重组是从分散的V、D和J基因片段阵列中组装抗原受体基因的定点DNA重排。重组是由淋巴特异性的RAG1和RAG2重组酶启动的，它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，并且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，免疫球蛋白基因只在B细胞(不是T细胞)中完全重排，T细胞受体基因在T细胞中组装，但不在B细胞中组装。RAG1和RAG2在非淋巴样细胞中的过度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，必须调节这些基因座对重组酶的可及性。大量证据表明，染色质结构的调节参与了V(D)J重组的调节。 在我们的项目中有两个组件。第一个是研究RAG2蛋白中的PHD结构域，以及它在RAG重组酶调控染色体可及性中的作用，从而调控组织和发育特异性的V(D)J重组。二是RAG1/RAG2重组酶及其与V(D)J重组(RSS)信号DNA复合体的结构。在本财政年度，我们在这两个领域都取得了进展。我们的合作者，麻省总医院的厄廷格博士和斯坦福大学的Gozani博士，已经证明RAG2的PHD结构域识别带有赖氨酸-4三甲基化的组蛋白H3尾巴(H3K4me3)。测定了含有甲基化残基变异的RAG2-PHD与H3K4多肽的6种晶体结构，并测定了RAG2-PHD与甲基化H3多肽的结合常数。我们的结构和生化研究揭示了保守的PHD结构域如何识别不同修饰的H3尾巴的新方面，并导致了V(D)J重组调控机制的提出。这些结果总结在两个手稿中。目前正在对这两本书进行审查，准备出版。 我们与Martin Gellerts博士合作，寻求RAG1/RAG2重组酶的结构解决方案，并在今年终于成功地确定了小鼠RAG1/RAG2重组酶复合体的晶体结构。 参考资料： 金文生，拉普库斯基，M.，杨伟和盖勒特.(2015)V(D)J重组酶RAG1-RAG2的晶体结构，自然，518,507-511. 郭龙迪，G.J.，Gellert，M.和Yang，W.(2010)。在V(D)J重组中，表观遗传信号克服了RAG1和RAG2介导的DNA切割的自动抑制。美国国家航空航天局，第107号，22487-92号。 杨威(2011)核酸酶：结构、功能和机制的多样性。Q Rev BiPhys。44，1-93。 郭、拉蒙-麦克斯等人，Gellert，M.&Yang，W.(2009)。V(D)J重组的初始阶段：RAG1/2和RSS DNA在切割后复合体中的组织。摩尔。电话号码是35,217-227。

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.