重排酶RAG2非核心区PHD结构域的新功能与作用机制

The function of the non-core region of recombination-activation genes RAG1/2 has attracted great concern since a lot of studies revealed that it’s involved in the regulation of V(D)J recombination. PHD domain is the most important domain identified in RAG2’s non-core region, which has been reported to regulate V(D)J recombination via regulating histone binding. Importantly, all the seven amino acids mutations identified in SCID (Severe Combined Immunodeficiency) patients are included within PHD domain, indicating its critical role in immune development. In order to explore the novel function of PHD domain of RAG2’s non-core region, we constructed C478Y mutation mouse, with mutation frequently reported in PHD domain of RAG2’s non-core region of SCID patients. Our primary data showed that this mutation mice lacked T and B cells with predominant reduced V(D)J recombination efficiency. By using this mouse model, this project will further study if C478Y mutation will affect the accuracy of V(D)J recombination and its mechanism regulating V(D)J recombination, including its effects on RAG2 stability, RAG1 function and the recruiting of DNA repairing proteins. Our study will not only discover novel functions of RAG2’s non-core region via elucidating its mechanism regulating V(D)J recombination, but will also provide evidence of the pathological mechanism of SCID.

受体重排酶RAG1/2非核心区的功能研究越来越受到关注，多个研究表明它参与调控V(D)J重排。PHD结构域是重排酶RAG2非核心区已鉴定的最重要的结构域，并被报导通过调控与核小体的结合来调控V(D)J重排。已鉴定出的重症联合免疫缺陷SCID病人的7种RAG2氨基酸突变都被覆盖在RAG2的PHD区域，说明其与免疫发育密切相关。为了深入研究RAG2非核心区PHD结构域的新功能，我们构建了在SCID病人中报导较多的该结构域C478Y点突变小鼠模型。初步研究结果表明，该小鼠T、B细胞缺失，而且V(D)J重排效率显著下降。本项目将以该突变小鼠为主要研究模型，进一步研究C478Y是否会影响V(D)J重排的正确性以及调控机制，包括对RAG2蛋白稳定性、RAG1功能及相关修复蛋白招募的影响等机制。该研究将揭示RAG2非核心区的新功能及调控V(D)J重排的作用机制，并为相关的免疫缺陷疾病发病机制提供新依据。

PHD结构域是重排酶RAG2非核心区已鉴定的最重要的结构域，并被报导参与调控V(D)J重排。已鉴定出的重症联合免疫缺陷SCID病人的7种RAG2氨基酸突变都被覆盖在RAG2的PHD区域，说明其与免疫发育密切相关。为了深入研究RAG2非核心区PHD结构域的新功能，我们构建了在SCID病人中报导较多的该结构域C478Y点突变（RAG2-C478Y）小鼠模型。本项目以该突变小鼠为主要研究模型，旨在揭示RAG2非核心区调控V(D)J重排的机制。我们的研究结果表明，该突变小鼠T、B细胞缺失，而且V(D)J重排效率显著下降。机制研究表明RAG2-C478Y突变导致RAG2蛋白质不稳定。而另一个位于RAG2的非核心区域且与C478相邻的非锌离子结合位点的点突变N474S却不会影响RAG2的稳定性或抑制淋巴细胞的发育，这说明锌指结构对于RAG2的蛋白稳定性和V(D)J重排十分重要。该研究揭示RAG2非核心区的新功能及调控V(D)J重排的作用机制，并为相关的免疫缺陷疾病发病机制提供新依据。