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

基本信息

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
项目类别：
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

项目摘要

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有力的区分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，自我与非自我的区分依赖于一系列积极的以及免疫受体（例如 T 细胞和 B 细胞受体）的阴性选择。在建立免疫学 T 细胞中对自身分子的耐受性、转录调节因子 Aire 发挥着核心作用。艾尔起来- 调节胸腺髓质上皮细胞中数千种外周组织抗原的表达，允许自身反应性 T 细胞进行克隆删除或发育成调节性 T 细胞。尽管其中心尽管 Aire 在 T 细胞发育中的作用，但其分子机制仍知之甚少。先前的研究表明 Aire的C端尾部（CTT）（AireCTT）在Aire靶基因的转录激活中起着关键作用。在我们努力剖析 AireCTT 如何作为转录激活域发挥作用时，我们最近发现 AireCTT 直接结合 Cyclin C (CycC)，它是 Mediator 的 CDK8 激酶模块的一个组成部分，而 CycC 是对艾尔的转录活性很重要。介体是一种大型多亚基转录共激活因子密集地装载超级增强子，可以调节广泛的基因。有趣的是，之前的研究表明 Aire 定位于超级增强子，改变整体染色质景观并调节目标基因激活。我们假设 Aire 利用 CTT 来结合介体并聚集在超级增强子上。此外，已知 Aire 在多个位点被磷酸化，其中一些位点符合共识 CDK8底物的序列。这也提出了 CycC 结合是否发挥额外作用的问题招募 CDK8 来磷酸化 Aire。为了测试这些假设并进一步定义 AireCTT:CycC 相互作用，我们在此提出两个具体的目标。首先，我们将确定 AireCTT 和 CycC 之间相互作用的结构基础，使用晶体学、生物化学和细胞检测的结合（目标 1）。其次，我们将定义 AireCTT:CycC 相互作用的功能结果，重点是测试其在超级中的潜在作用 Aire 的增强子定位和翻译后修饰（目标 2）。我们期望拟议的工作将为我们理解艾尔提供关键的缺失环节。此外，他们还将拥有广泛的对整个转录领域的影响。这是因为我们的工作准备确定以前 CDK8 激酶模块的功能和机制尚未被认识，这是 Mediator 中人们知之甚少的部分。