Defining the molecular mechanism of Aire in T-cell tolerance

自我和非自我分子之间的强大区别是所有人免疫系统的核心 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 免疫受体(如T细胞和B细胞受体)的阴性选择。在建立免疫学方面 T细胞对自身分子的耐受性，转录调节因子Aire起着核心作用。准备好了- 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为调节性T细胞。尽管它的中心 Aire在T细胞发育中的作用，其分子机制尚不清楚。先前的研究表明 Aire的C末端尾部(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如何作为转录激活域发挥作用的过程中，我们最近发现 AireCTT直接结合细胞周期蛋白C(Cyclin C，CycC)，这是Mediator的CDK8激酶模块的一个组成部分，并且CycC是 对Aire的转录活动很重要。介体是一种大的多亚基转录共激活因子，它是 密集地装载在超级增强子上，可以调节广泛的基因。耐人寻味的是，之前的研究 提示Aire是本地化的超级增强子，改变了全球染色质格局，并调节了靶点 基因激活。我们假设AIRE利用CTT在超级增强子上绑定介体和集群。 此外，已知Aire在多个位点被磷酸化，其中一些与共识相符 CDK8底物的序列。这也提出了一个问题，CycC绑定是否在 招募CDK8用于Aire的磷酸化。 为了检验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 目标。首先，我们将使用AireCTT和CycC之间的 结晶学、生物化学和基于细胞的分析相结合(目标1)。其次，我们将定义 AireCTT的功能结果：CycC相互作用，重点是测试其在超级 Aire的增强子定位和翻译后修饰(Aim 2)。我们期望拟议的工作 将为我们对Aire的理解提供关键缺失的环节。此外，他们还会有一个广泛的 对整个转录领域的影响。这是因为我们的工作已经准备好识别之前的 CDK8激酶模块的功能和机制尚不为人所知，它是Mediator中知之甚少的部分。

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.