DCAF1(VprBP) regulates FoxO1 to promote Rag transcription

项目摘要 B和T淋巴细胞形成我们的适应性免疫系统的基础，这是基于特定的 通过结构多样的表面抗原受体识别外来分子。结构多样性在这些 受体起源于淋巴细胞增殖过程中抗原受体基因的特异性重排 发展这种重排过程称为V（D）J重组，当RAG 1/2蛋白 在抗原受体基因片段处引入DNA双链断裂（DSB），并且当 DSB通过非同源末端连接被感测和修复。这一过程受到许多层的影响， 调节，但限制V（D）J重组的基本手段是控制RAG的水平 蛋白质本身该实验室的工作表明RAG 1水平由RAG 1相互作用控制 我们鉴定的蛋白质称为Vpr结合蛋白（VprBP;也称为DCAF 1），它通过以下途径发挥其控制作用： 两种不同的机制：第一，通过促进RAG 1的及时蛋白酶体依赖性降解， VprBP与Cul 4-DDB 1 E3泛素连接酶复合物的结合;第二，通过调节Rag 在刺激V（D）J重组的条件下进行转录诱导。隐藏在 第二种机制尚不清楚。然而，最近的研究发现VprBP可能具有介导作用 与Rag表达所需的关键转录因子FoxO 1直接相互作用， 对FoxO 1乙酰化状态敏感。这些发现使我们假设VprBP（DCAF 1）介导 乙酰化依赖性关联与FoxO 1调节Rag转录诱导。为了验证这一 假设，我们将（i）确定VprBP是否介导与FoxO 1的乙酰化依赖性关联， 影响其定位;和（ii）测试FoxO 1乙酰化是否调节FoxO 1沉积到Rag基因座， Rag转录激活和V（D）J重组。建立 VprBP-FoxO 1相互作用的分子基础，它们对乙酰化的依赖，以及乙酰化对 Rag的表达将定义VprBP在V（D）J重组中的新的生理作用，并揭示了VprBP在V（D）J重组中的潜在作用。 Rag表达失调引起的免疫系统改变和基因组不稳定性的来源。这 这项工作也将为理解VprBP如何调节FoxO 1依赖的转录提供一个范例。 激活其靶基因，这将进一步扩大该项目的科学兴趣，并可能 揭示了治疗调节FoxO 1依赖性基因表达的新途径。

PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of the antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins introduce DNA double-strand breaks (DSBs) at antigen receptor gene segments, and is completed when the DSBs are sensed and repaired by non-homologous end-joining. This process is subjected to many layers of regulation, but an elementary means to constrain V(D)J recombination is to control the level of the RAG proteins themselves. Work in this laboratory suggests that RAG1 levels are controlled by a RAG1 interacting protein we identified called Vpr binding protein (VprBP; also called DCAF1), which exerts its control through two different mechanisms: first, by promoting timely proteasome-dependent degradation of RAG1 through VprBP’s association with a Cul4-DDB1 E3 ubiquitin ligase complex; and second, by regulating Rag transcriptional induction under conditions that stimulate V(D)J recombination. The details underlying the second mechanism remain unclear. However, recent studies have identified VprBP as potentially mediating direct interactions with a key transcription factor required for Rag expression, called FoxO1, in a manner that is sensitive to FoxO1 acetylation status. These findings lead us to hypothesize that VprBP(DCAF1) mediates acetylation-dependent association with FoxO1 to regulate Rag transcriptional induction. To test this hypothesis, we will (i) establish whether VprBP mediates acetylation-dependent association with FoxO1 and influences its localization; and (ii) test whether FoxO1 acetylation regulates FoxO1 deposition to the Rag locus, Rag transcriptional activation, and V(D)J recombination, in a VprBP-dependent manner. Establishing the molecular basis for VprBP-FoxO1 interactions, their reliance on acetylation, and the influence of acetylation on Rag expression would define a new physiological role for VprBP in V(D)J recombination, and exposes potential sources for altered immune repertoire and genomic instability caused by dysregulated Rag expression. This work would also provide a paradigm for understanding how VprBP regulates FoxO1-dependent transcriptional activation of its target genes, which would further broaden the scientific interest of the project and potentially reveal new avenues to therapeutically regulate FoxO1-dependent gene expression.