Structural and mechanistic studies of V(D)J recombination

V（D）J重组是一种位点特异性的DNA重排，它将抗原受体基因从分散的V、D和J基因片段阵列中组装起来。淋巴细胞特异性RAG 1和RAG 2重组酶可识别并切割重组信号序列，从而启动重组。V（D）J重组受到严格的调控，以优选的时间顺序发生，并且仅在特定的细胞类型和发育阶段中发生。免疫球蛋白（IG）重链重排先于轻链重排，IG重链D至J连接先于V至DJ重组。此外，IG基因仅在B细胞（而不是T细胞）中完全重排，并且T细胞受体基因在T细胞中组装，但在B细胞中不组装。RAG 1和RAG 2在非淋巴细胞中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，必须调节这些基因座对重组酶的可及性。大量证据表明，V（D）J重组的调控涉及染色质结构的调节。 我们的项目有两个组成部分。首先是研究RAG 2蛋白中的PHD结构域及其在调节RAG重组酶的染色体可及性中的作用，从而研究组织和发育特异性V（D）J重组。第二部分是RAG 1/RAG 2重组酶的结构及其与V（D）J重组（RSS）信号DNA的复合物。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Oettinger博士和斯坦福大学的Gozani博士，已经证明RAG 2的PHD结构域识别具有赖氨酸-4三甲基化的组蛋白H3尾（H3 K4 me 3）。我们已经确定了RAG 2-PHD与H3 K4肽复合的六种晶体结构，其中含有甲基化残基的变化，并测量了RAG 2-PHD和甲基化H3肽的结合常数。我们的结构和生物化学研究揭示了保守的PHD结构域如何识别不同修饰的H3尾的新方面，并提出了V（D）J重组的调控机制。结果总结在两个手稿。这两本书都在审查中。 与Martin Gellman博士的实验室合作，我们一直在寻求RAG 1/RAG 2重组酶的结构解决方案，并最终在今年成功确定了小鼠RAG 1/RAG 2重组酶复合物的晶体结构。 参考文献： 格伦迪，G. J.，Gellert，M. &杨，W.（2010年）。由RAG 1和RAG 2介导的DNA切割的自抑制被V（D）J重组中的表观遗传信号克服。PNAS，107，22487-92. 杨，W.（2011）核酸酶：结构，功能和机制的多样性。Q Rev Biophys. 44，1-93。 格伦迪，G. J.，Ramn-Maiques，S.，例如，Gellert，M. &杨，W.（2009年）。V（D）J重组的初始阶段：RAG 1/2和RSS DNA在切割后复合物中的组织。摩尔手机，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: 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.