Defining the molecular mechanism of Aire in T-cell tolerance

自我和非自我分子之间的强大区分是所有免疫系统的核心 有机体在脊椎动物的适应性免疫系统中，自我与非自我的区分依赖于一系列积极的免疫反应。 和免疫受体（例如T细胞和B细胞受体）的阴性选择。在建立免疫学 在T细胞对自身分子的耐受中，转录调节因子Aire起着核心作用。准备好- 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细胞经历克隆缺失或发育成调节性T细胞。尽管它的中心 在T细胞发育中的作用，Aire的分子机制仍然知之甚少。以前的研究表明 Aire的C-末端尾（C-terminal tail，CTT）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作为转录激活结构域的功能时，我们最近发现， AireCTT直接结合细胞周期蛋白C（CycC），其是Mediator的CDK 8激酶模块的组分，并且CycC是 对艾尔的转录活性很重要介体是一种大的多亚基转录共激活因子， 密集地装载在超级增强子上，并且可以调节广泛的基因。有趣的是，以前的研究 这表明Aire定位于超级增强子，改变全局染色质景观并介导靶向 基因激活我们假设Aire利用CTT结合Mediator并聚集在超级增强子上。 此外，已知Aire在多个位点被磷酸化，其中一些位点与共有序列相匹配。 CDK 8底物的序列。这也提出了一个问题，即CycC结合是否发挥了额外的作用， 募集CDK 8用于Aire的磷酸化。 为了验证这些假设并进一步定义AireCTT：CycC相互作用，我们在这里提出了两个具体的方法。 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 结晶学、生物化学和基于细胞的测定的组合（目标1）。其次，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细胞中的潜在作用， Aire的增强子定位和翻译后修饰（Aim 2）。我们希望拟议的工作 将为我们理解Aire提供关键的缺失环节。此外，他们还将有一个广泛的 对整个转录领域的影响。这是因为我们的工作是准备确定以前 CDK 8激酶模块的未被认识的功能和机制，介体的知之甚少的部分。

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.