DNA损伤结合蛋白DDB1及其底物识别蛋白DCAF2复合体在B淋巴细胞发育过程中的功能机制研究

Regarded for their capacity to produce antibody responding to antigens, B lymphocytes was critical for maintain physiological homeostasis. Amount of DNA replication and VDJ recombination at pro-B stage happened during the development of B lymphocytes will generate DNA damage widely that put the severe stress on the genomic stability. The negative effect of accumulated DNA damage on the proliferation of B lymphocytes results in the decrease of B lymphocytes, leading to a weakened immune system. However, the precise mechanistic roles of regulators of DNA damage repair and cell cycle in B lymphocytes development remain poorly understood. How DNA damage response regulates the development of B lymphocyte is not known. We have preliminary data indicating the specific deletion of Damage Specific DNA Binding Protein 1 (DDB1) leads to DNA damage increased and has a severe effect on the cell cycle of proliferative pro-B cell that lead to abnormal development of B lymphocyte. At the same time, we found specific deletion DCAF2 responsible to substrate recognition for CRL4DDB1 show us similar phenotype to DDB1 knock-out mice. To explore the molecular mechanism of DDB1-DCAF2 complex in DNA damage response and proliferation during B lymphocyte development, we will determine the relationship of DDB1 and DCAF2 in the pro-B cell and how they cooperate with each other to regulate the downs-stream key proteins important for normal B lymphocytes cell cycle and DNA damage response.

B淋巴细胞是机体产生抗体的细胞群，对机体识别并清除外源病原菌具有重要意义。B淋巴细胞发育和分化要经历大量DNA复制和重组等过程。这些过程都极其容易产生DNA损伤，尤其是pro-B阶段的VDJ重排，从而对维持基因稳定构成威胁。而过量积累DNA损伤会影响B淋巴细胞正常增殖从而减少B淋巴细胞的数目，导致机体免疫力降低。然而B淋巴细胞的发育过程中DNA损伤修复机制以及增殖调控机制都有待进一步阐明。本研究组发现DDB1(DNA损伤结合蛋白)缺失会导致祖B细胞（pro-B）DNA损伤增多，细胞周期被严重阻碍，进而影响早期B淋巴细胞在骨髓中的增殖和发育。另一方面，其底物识别蛋白DCAF2敲除小鼠也表现出相似表型。本课题组拟从细胞和小鼠水平研究DDB1和DCAF2两者的联系，及其对下游重要DNA损伤修复相关蛋白的功能调控，探讨B淋巴细胞发育过程中调控细胞DNA损伤应答和细胞增殖的具体分子机制。

小鼠骨髓中的HSC不断发育为B细胞，为外周提供成熟的B细胞，参与体液免疫，维持机体稳态。Prepro-B细胞被认为是最出现的B细胞前体，随之发育为pro-B细胞，在pro-B细胞阶段会发生免疫球蛋白重链的V-DJ重排，形成μ链。此时细胞还没有成熟的轻链，有两个替代型轻链λ链和VpreB，μ链和这两个替代型轻链组装成pre-BCR。此时B细胞发育进入pre-B细胞阶段，在pre-BCR信号刺激下，pre-B细胞进行增殖，同时进行轻链的重排，形成成熟的BCR，在经历阳性选择后发育为immature B细胞和mature B细胞。.DDB1，是在DNA损伤结合蛋白1的简称，与DDB2形成损伤修复复合体，参与UV引起的DNA损伤修复，后续研究发现DDB1作为一个接头蛋白，参与CRL4 E3泛素连接酶的形成。DDB1和CUL4相关因子（DDB1-CUL4-associated factor，DCAF）结合，DCAF分子具有识别特异性底物的能力，CRL4通过DDB1结合不同的DCAF，可以在特定的时空降解特定的靶蛋白，调控细胞各项活动。. 为了特异性研究DDB1在B细胞中的功能，我们将Ddb1fl/fl小鼠和Mb1Cre小鼠杂交，得到在prepro-B后期敲除DDB1的小鼠。表型分析发现，DDB1缺失会导致pro-B细胞大量减少，并且pro-B细胞的减少不是由于存活能力减弱凋亡增加，也不是因为免疫球蛋白重链重排失败，而是由于细胞大量阻滞在G2/M期，不能成功进行细胞分裂。进一步的质谱分析，发现DDB1通过DCAF2参与调控pro-B细胞发育的,Dcaf2fl/flMb1Cre小鼠呈现出跟Ddb1fl/flMb1Cre小鼠相似的表型。而已知由DCAF2识别并介导降解的G2/M期检查点蛋白CHK1和P21在DDB1或DCAF2缺陷的pro-B细胞中都有大量积累。综上实验结果，我们认为在B细胞发育过程中，DDB1通过与DCAF2相互作用，识别并降解CHK1和P21，保证pro-B细胞周期的顺利进行。这为了解泛素化调控在B细胞发育过程中的作用增添了新的一笔。