Defining the Mechanism and Function of DNA Double Strand Break Induced Inhibition of V(D)J Recombination

项目摘要：B细胞介导的适应性免疫依赖于DNA双链的程序性诱导 中断(DSB)以创建不同的免疫球蛋白(Ig)基因库。RAG1/RAG2(RAG)内切酶 通过重组免疫球蛋白基因座的可变(V)、多样性(D)和连接(J)基因片段来组装免疫球蛋白基因。 这个过程对获得性免疫和哺乳动物的生存至关重要，但它也带来了风险，因为大多数B血统 癌症包含涉及免疫球蛋白基因和原癌基因的克隆性易位，强调了至关重要的 严格调控V(D)J重组的重要性。我的论文实验室发现抹布或具有基因毒性的双链球菌 通过ATM激酶(细胞的关键调节因子)发出信号，快速抑制Rag1/2基因的转录 DSB响应。ATM-/-小鼠发育B细胞的频率较高，其中Ig等位基因和 有免疫球蛋白易位的成熟B淋巴细胞。此外，小鼠中B细胞特异性ATM的缺失增加了 伴有Ig易位的B细胞性淋巴瘤的发病率。这些数据与DSB诱导的抑制一致 Rag1/2对于防止Ig易位至关重要；然而，鉴于ATM在DSB中的多功能作用 这些表型不能直接归因于DSB诱导的Rag1/2表达的抑制。 遗传毒性DSB诱导依赖atm的NFκB必需调节物(NEMO)蛋白磷酸化 激活核因子κB转录因子。我发现发育中的NEMO/-B细胞削弱了对Rag1/2的抑制 对DSB的反应，在Ig基因座上表现为RAG DSB增加。我的数据与之前的ATM-/-研究一致， 但与ATM-/-细胞不同，Nemo-/-细胞保持正常的DSB修复和检查点/凋亡激活。因此， Nemo-/-模型让我有机会更直接地检验我的中心假设，即DSB诱导 NEMO/NFκB介导的Rag1/2转录抑制免疫球蛋白转位和生成的淋巴组织 癌症。我建议阐明NEMO依赖的Rag1/2抑制的机制。本提案的目标1 将检验这样一种假设，即NFκB因子直接结合RAG1/2 ERAG增强子来介导DSB诱导的 通过抑制转录延长来抑制Rag1/2。这可能揭示了核因子κB的新机制- 介导的转录抑制，这在很大程度上是不确定的。我将确定双链球菌诱导的核因子κB结合 Rag1/2抑制中的位点及其功能作用。此外，我将定义Rag1/2的转录状态 在没有或存在DSB的情况下，确定DSB抑制Rag1/2的机制水平。目标2 将测试DSB诱导的Rag1/2抑制限制RAG DSB从而抑制RAG DSB的假设 致癌免疫球蛋白易位。具有B细胞特异性Nemo缺失的小鼠将被用于i)定量RAG DSB 前B和前B细胞中的免疫球蛋白等位基因，II)量化非恶性B细胞中的Ig易位，以及III)评估 具有致癌Ig易位的B系癌症的易感性。这些研究的圆满完成 应阐明DSB诱导RAG抑制的机制，为NFκB介导的研究提供新的见解 转录抑制，并可能揭示支撑B系癌症的病理机制。

Project Summary: B cell-mediated adaptive immunity relies on the programmed induction of DNA double strand breaks (DSBs) to create diverse immunoglobin (Ig) gene repertoires. The RAG1/RAG2 (RAG) endonuclease assembles Ig genes through recombination of variable (V), diversity (D), and joining (J) gene segments of Ig loci. This process is vital for adaptive immunity and mammalian survival yet, it also confers risk, as most B lineage cancers contain clonal translocations involving an Ig locus and a proto-oncogene, underscoring the vital importance of tightly regulating V(D)J recombination. My thesis lab discovered that RAG or genotoxic DSBs rapidly repress transcription of the Rag1/2 locus by signaling via the ATM kinase, a key regulator of the cellular DSB response. Atm-/- mice have higher frequencies of developing B cells with RAG DSBs at both Ig alleles and of mature B lymphocytes with Ig translocations. Moreover, B cell-specific Atm deletion in mice increases the incidence of B lineage lymphomas with Ig translocations. These data are consistent with DSB-induced repression of Rag1/2 being critical to protect from Ig translocations; yet, given the multifunctional roles of ATM in the DSB response, these phenotypes cannot be directly attributed to DSB-induced repression of Rag1/2 expression. Genotoxic DSBs induce ATM-dependent phosphorylation of the NFκB essential modulator (Nemo) protein to activate NFκB transcription factors. I show that Nemo-/- developing B cells have impaired repression of Rag1/2 in response to DSBs and display increased RAG DSBs at Ig loci. My data are consistent with prior Atm-/- studies, but unlike Atm-/- cells, Nemo-/- cells retain normal DSB repair and checkpoint/apoptosis activation. Thus, the Nemo-/- model provides me an opportunity to more directly test my central hypothesis that DSBs induce Nemo/NFκB-mediated transcriptional repression of Rag1/2 to suppress Ig translocations and resultant lymphoid cancers. I propose to elucidate the mechanism of Nemo-dependent Rag1/2 repression. Aim 1 of this proposal will test the hypothesis that NFκB factors directly bind the Rag1/2 Erag enhancer to mediate DSB-induced repression of Rag1/2 by inhibiting transcriptional elongation. This may reveal novel mechanisms of NFκB- mediated transcriptional repression, which are largely undefined. I will determine DSB-induced NFκB binding sites and their functional role in Rag1/2 repression. Furthermore, I will define the transcriptional state of Rag1/2 in the absence or presence of DSBs to determine the mechanistic levels at which DSBs repress Rag1/2. Aim 2 will test the hypothesis that DSB-induced repression of Rag1/2 limits RAG DSBs and thereby suppresses oncogenic Ig translocations. Mice with B cell specific deletion of Nemo will be used to i) quantify RAG DSBs at Ig alleles in pro-B and pre-B cells, ii) quantify Ig translocations in non-malignant B lineage cells, and iii) evaluate predisposition to B lineage cancers with oncogenic Ig translocations. The successful completion of these studies should elucidate DSB-induced mechanisms of RAG repression, provide new insights into NFκB-mediated transcriptional repression, and may uncover pathological mechanisms underpinning B lineage cancers.