RAG-mediated DNA Damage Responses in Immune Development and Function

项目摘要 淋巴细胞发育被精确控制，以使克隆扩增和表达不同的免疫原性。 免疫球蛋白受体库，其通过DNA双链断裂（DSB）进行， RAG内切核酸酶。这两个二分法，但相互依存的过程，是通过管理， 不同细胞信号的合作，以防止具有DSB的细胞进入细胞周期， 异常修复为易位。在早期B细胞发育过程中，前B细胞受体（pre-BCR），通过 SYK激酶的激活，协调前B细胞的增殖扩增和 免疫球蛋白受体基因。需要前BCR的负调控以确保细胞周期停滞， 限制免疫球蛋白受体基因组装过程中产生的DNA断裂数量。的确， 前BCR信号传导，特别是增加的SYK活性，驱动增殖和白血病转化。 然而，抑制SYK和pre-BCR信号传导的机制尚不清楚，并且仍然是一个关键的空白， 我们对B细胞成熟的理解。我们已经确定了一种新的细胞类型的特定程序激活的信号 来自抑制SYK和抑制前BCR信号传导的RAG DSB。在DNA损伤中的防御- 介导的反馈回路导致前B细胞白血病的起始。令人惊讶的是，这个信号网络不是 由所有DNA损伤触发，而是特异于免疫球蛋白受体基因表达过程中产生的RAG DSB。 组装件.我们的目标是确定来自RAG DSB的信号如何与发育程序整合 以协调B细胞成熟并防止白血病转化。我们建议RAG DSB 抑制SYK以加强细胞周期停滞，从而防止DNA断裂B细胞重新进入细胞 周期这种DNA损伤介导的检查点程序将允许迭代尝试产生成熟的 抗原受体，以促进B细胞分化，同时防止白血病的启动。此外，我们建议， 这些DNA损伤信号通过RAG核酸内切酶的不同结构域被激活， DSB位点的蛋白质来调节信号通路。B细胞中的这种RAG特异性机制区分了 在正常和错误的DSB之间激活适当的细胞反应。利用创新的实验 一种允许在B细胞发育程序的背景下询问DSB信号的方法， 拟议的研究将定义RAG DSB信号如何维持前B细胞检查点，并将解决 区分RAG介导和非RAG介导的DNA损伤的机制。完成这些研究 将描绘出抑制早期B细胞增殖信号的关键途径，建立限制增殖的信号， 白血病发生，并确定RAG核酸内切酶在调节DNA损伤反应中的新功能。

PROJECT SUMMARY Lymphocyte development is precisely controlled to enable clonal expansion and expression of a diverse immunoglobulin receptor repertoire, which proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These two dichotomous, but interdependent processes, are managed through the cooperation of diverse cellular signals to prevent cells with DSBs from entering cell cycle where they could be aberrantly repaired as translocations. During early B cell development, the pre-B cell receptor (pre-BCR), through activation of the SYK kinase, coordinates both the proliferative expansion of pre-B cells and the assembly of immunoglobulin receptor genes. Negative regulation of the pre-BCR is required to ensure cell cycle arrest and limit the number of DNA breaks generated during immunoglobulin receptor gene assembly. Indeed, unopposed pre-BCR signaling, particularly increased SYK activity, drives proliferation and leukemic transformation. However, the mechanisms that repress SYK and pre-BCR signaling are not known and remain a critical gap in our understanding of B cell maturation. We have identified a novel cell-type specific program activated by signals from RAG DSBs that suppresses SYK and inhibits pre-BCR signaling. Deficiencies in this DNA damage- mediated feedback circuit result in initiation of pre-B cell leukemia. Surprisingly, this signaling network is not triggered by all DNA injury but, rather, is specific to RAG DSBs generated during immunoglobulin receptor gene assembly. Our goal is to determine how signals from RAG DSBs integrate with developmental programs to coordinate B cell maturation and prevent leukemic transformation. We propose that RAG DSBs suppress SYK to enforce cell cycle arrest and, thereby prevent B cells with DNA breaks from re-entering cell cycle. This DNA damage-mediated checkpoint program would permit iterative attempts at generation of a mature antigen receptor to promote B cell differentiation while preventing leukemic initiation. Further, we propose that these DNA damage signals are activated through distinct domains of the RAG endonuclease that interact with proteins at sites of DSBs to modulate signaling pathways. This RAG-specific mechanism in B cells discriminates between normal and errant DSBs to activate appropriate cellular responses. Utilizing an innovative experimental approach that allows interrogation of DSB signals within the context of B cell developmental programs, our proposed studies will define how RAG DSB signals maintain pre-B cell checkpoint and will resolve the mechanisms that distinguish RAG-mediated from non-RAG-mediated DNA damage. Completion of these studies will delineate pathways critical for dampening proliferative signals in early B cells, establish signals that restrict leukemogenesis, and define novel functions of the RAG endonuclease in regulating DNA damage responses.