人体可产生多样化的抗体以对抗病原微生物的侵染，该过程主要由胞苷脱氨酶AID决定。申请人在研究AID对靶位点特异性识别的机制时，意外的在质谱数据中发现Dhx9可与AID结合，但具体作用方式、功能及机制不明。利用免疫共沉淀、超高分辨率成像和体外pull-down实验我们发现Dhx9与AID直接相互作用并且在活化的B细胞中共定位。意外的是，在B细胞中敲除Dhx9后，高效价抗体的水平显著上升。与此对应，体外脱氨基实验也证实Dhx9可封闭AID的活性，这些结果提示Dhx9可能是抗体类别转化过程中的关键性负调控因子。在机制上，我们初步发现Dhx9敲除会导致G4和R-loop结构的大量堆积，进而引起DNA损伤和断裂，促进抗体重排。在此基础上，本申请计划深入探究Dhx9与AID结合的生理及病理意义，解析Dhx9调控AID活性和DNA重排的详细分子机制，为免疫缺陷病的诊断和治疗提供新的思路和靶点。

Human body can produce a huge number of different antibodies to execute immune clearance of pathogenic microorganisms. The diversity of antibody repertory was mainly determined by activation-induced cytidine deaminase (AID). During studying the mechanism of AID targeting, we unexpectedly found a candidate AID partner called Dhx9 in the mass spectrometry data. However, the detailed interaction mode, function and mechanism were unclear. Using co-immunoprecipitation, super resolution imaging and RNA pull-down assay, we confirmed that these two proteins not only interact with each other but also perfectly co-localized in the activated B cells. Surprisingly, we found that the higher-titer antibody production was dramatically increased in Dhx9-ablated B cells. In line with this observation, Dhx9 protein is able to block AID activity in an in vitro deamination assay. These data collectively indicate that Dhx9 may function as a negative regulator of class switch recombination. Mechanistically, we found that Dhx9-ablation leads to a large accumulation of both G4 and R-loop structures, which in turn will cause DNA damage and breakage, and thus promote DNA rearrangement. On this basis, this proposal plans to further explore the physiological and pathological meaning of the interaction between Dhx9 and AID, and to decipher the molecular mechanisms of Dhx9 in the negative regulation of AID and DNA rearrangement. We hope this study will finally provide new strategies and targets for the diagnosis and treatment of immunodeficiency diseases.