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

项目概述：B细胞介导的适应性免疫依赖于DNA双链的程序化诱导 断裂（DSB）以产生不同的免疫球蛋白（IG）基因库。RAG 1/RAG 2（RAG）内切核酸酶 通过重组IG基因座的可变（V）、多样性（D）和连接（J）基因片段组装IG基因。 这一过程对适应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谱系 癌症包含涉及IG基因座和原癌基因的克隆易位，强调了重要的 严格调控V（D）J重组的重要性。我的论文实验室发现RAG或基因毒性DSB 通过ATM激酶的信号传导快速抑制Rag 1/2位点的转录，ATM激酶是细胞凋亡的关键调节因子， DSB响应。Atm-/-小鼠在两个IG等位基因上都具有RAG DSB的发育B细胞的频率较高， IG易位的成熟B淋巴细胞。此外，小鼠中B细胞特异性Atm缺失增加了 B系淋巴瘤伴IG易位的发生率。这些数据与DSB诱导的抑制一致 Rag 1/2对于防止IG易位至关重要;然而，考虑到ATM在DSB中的多功能作用， 反应，这些表型不能直接归因于DSB诱导的Rag 1/2表达的抑制。 遗传毒性DSB诱导NFκB必需调节因子（Nemo）蛋白的ATM依赖性磷酸化， 激活NFκB转录因子。我发现Nemo-/-发育的B细胞对Rag 1/2的抑制受损 并在IG基因座上显示增加的RAG DSB。我的数据与之前的Atm-/-研究一致， 但与Atm-/-细胞不同，Nemo-/-细胞保留正常的DSB修复和检查点/凋亡激活。因此 Nemo-/-模型为我提供了一个机会来更直接地测试我的核心假设，即DSB会诱导 Nemo/NFκ B介导的Rag 1/2转录抑制，以抑制IG易位和产生的淋巴细胞 癌的我建议阐明Nemo依赖的Rag 1/2抑制的机制。本提案的目标1 将检验NFκB因子直接结合Rag 1/2 Erag增强子介导DSB诱导的 通过抑制转录延伸抑制Rag 1/2。这可能揭示了NFκB-κ B的新机制。 介导的转录抑制，这在很大程度上是不确定的。我将测定DSB诱导的NFκB结合 位点及其在Rag 1/2抑制中的功能作用。此外，我将定义Rag 1/2的转录状态 在不存在或存在DSB的情况下，以确定DSB抑制Rag 1/2的机制水平。目的2 将检验DSB诱导的Rag 1/2抑制限制RAG DSB从而抑制RAG DSB的假设。 致癌性IG易位。Nemo的B细胞特异性缺失的小鼠将用于i）定量RAG DSB， 前B细胞和前B细胞中的IG等位基因，ii）定量非恶性B谱系细胞中的IG易位，和iii）评估 具有致癌IG易位的B谱系癌症的易感性。这些研究的成功完成 阐明DSB诱导的RAG抑制机制，为NFκ B介导的 转录抑制，并可能揭示支持B谱系癌症的病理机制。

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.