Defining the molecular mechanism of Aire in T-cell tolerance

定义 Aire 在 T 细胞耐受中的分子机制

• 批准号: 9814890
• 负责人: Sun Hur
• 金额: $ 26.55万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-10 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9814890
• 关键词: Adaptive Immune System; Address; Antigens; Architecture; Area; Autoimmune Diseases; Autoimmune Process; Binding; Biochemistry; Biological Assay; C-terminal; Cells; ChIP-seq; Chromatin; Clonal Deletion; Collaborations; Complex; Consensus Sequence; Crystallography; DNA Polymerase II; DNA Sequence; DNA-Directed RNA Polymerase; Data; Discrimination; Enhancers; Gene Activation; Genes; Genetic; Genetic Transcription; Goals; Immune Tolerance; Immune system; Immunologic Receptors; Impairment; In Vitro; Individual; Left; Link; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Molecular; Mutation; Organism; Peripheral; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Polyglandular Autoimmune Syndrome Type I; Post-Translational Protein Processing; Public Health; Receptors, Antigen, B-Cell; Regulatory T-Lymphocyte; Research; Role; Series; Signal Transduction; Site; Specificity; Structure; Syndrome; T-Cell Development; T-Lymphocyte; Tail; Testing; Therapeutic; Thymic epithelial cell; Tissues; Transcription Coactivator; Transcription Elongation; Transcriptional Activation; Transcriptional Activation Domain; Work; X-Ray Crystallography; base; cyclin C; falls; global health; insight; molecular assembly/self assembly; promoter; recruit; transcription factor

Robust discrimination between self and non-self molecules is central to the immune system of all organisms. In vertebrate adaptive immune system, self vs. non-self discrimination relies on a series of positive and negative selections of immune receptors (e.g. T cell and B cell receptors). In establishing immunological tolerance against self molecules in T cells, a transcriptional regulator, Aire, plays a central role. Aire up- regulates the expression of thousands of peripheral tissue antigens in medullary thymic epithelial cells, allowing self-reactive T cells to undergo clonal deletion or to develop into regulatory T cells. Despite its central role in T cell development, molecular mechanism of Aire remains poorly understood. Previous studies showed that C-terminal tail (CTT) of Aire (AireCTT) plays a key role in the transcriptional activation of Aire target genes. In our effort to dissect how AireCTT functions as a transcriptional activation domain, we recently found that the AireCTT directly binds Cyclin C (CycC), a component of the CDK8 kinase module of Mediator, and that CycC is important for Aire's transcriptional activity. Mediator is a large, multi-subunit transcriptional co-activator that is densely loaded at super-enhancers and can regulate a broad spectrum of genes. Intriguingly, previous studies suggest that Aire is localized at super-enhancers, altering global chromatin landscape and mediating the target gene activation. We hypothesize that Aire utilizes CTT to bind Mediator and cluster at super-enhancers. Furthermore, Aire is known to be phosphorylated at multiple sites, some of which match the consensus sequence for CDK8 substrates. This also raises the question whether CycC binding plays an additional role of recruiting CDK8 for phosphorylation of Aire. To test these hypotheses and to further define the AireCTT:CycC interaction, we here propose two specific aims. First, we will determine the structural basis for the interaction between AireCTT and CycC, using a combination of crystallography, biochemistry and cell-based assays (Aim 1). Second, we will define the functional consequence of the AireCTT:CycC interaction, with a focus on testing its potential role in super- enhancer localization and post-translational modifications of Aire (Aim 2). We expect that the proposed work would provide key missing links in our understanding of Aire. Furthermore, they would also have a broad impact on the field of transcription in general. This is because our work is poised to identify previously unrecognized functions and mechanisms of the CDK8 kinase module, the poorly understood part of Mediator.

自我和非自我分子之间的强大歧视是所有免疫系统的核心 有机体。在脊椎动物适应性免疫系统中，自我与非自我歧视依赖于一系列积极 以及免疫受体（例如T细胞和B细胞受体）的阴性选择。在建立免疫学方面 对T细胞中自我分子的耐受性，转录调节剂AIRE起着核心作用。 aire up- 调节髓质胸部上皮细胞中数千种外周组织抗原的表达， 允许自反应性T细胞经历克隆缺失或发展为调节性T细胞。尽管它的中心 在T细胞发育中的作用，AIR的分子机制仍然很少理解。先前的研究表明 AIRE（AIRECTT）的C端尾（CTT）在AIR靶基因的转录激活中起关键作用。 为了剖析AIRECTT如何作为转录激活域的作用，我们最近发现 AIRECTT直接结合Cyclin c（Cycc），Cyclin c（Cycc）是介体CDK8激酶模块的一个成分，该Cycc为 对于Aire的转录活动很重要。介体是一个大型的，多支出的转录共激活器，是 密集地在超级增强剂上加载，可以调节一系列基因。有趣的是，先前的研究 表明AIRE位于超级增强剂，改变全球染色质景观并介导目标 基因激活。我们假设AIRE利用CTT在超级增强剂上结合介体和聚类。 此外，已知AIRE在多个位点被磷酸化，其中一些与共识匹配 CDK8底物的序列。这也提出了一个问题CYCC结合是否扮演的额外角色 募集CDK8用于AIRE的磷酸化。 为了检验这些假设并进一步定义了AIRECTT：CYCC相互作用，我们在这里提出了两个特定的特定 目标。首先，我们将使用A AIRECTT和CYCC之间相互作用的结构基础 晶体学，生物化学和基于细胞的测定法的组合（AIM 1）。第二，我们将定义 AIRECTT：CYCC相互作用的功能后果，重点是测试其在超级中的潜在作用 AIRE的增强剂定位和翻译后修饰（AIM 2）。我们期望拟议的工作 将在我们对AIR的理解中提供关键的缺失链接。此外，他们还将有一个广泛的 总体上对转录领域的影响。这是因为我们的工作准备以前识别 CDK8激酶模块的未识别功能和机制，这是介体的一部分知识。