The molecular mechanism of Aire

AIRE通过控制胸腺髓质上皮细胞(MEC)的表达调节中枢T细胞耐受性 编码外周组织抗原的一组转录本。从很早的时候起，艾尔就被公认为 转录调节因子，但很快就发现它不像常规转录那样工作 因子，即与启动子结合并诱导转录启动。在过去一年内完成的研究 资金周期表明，Aire诱导的基因以染色体内或染色体间簇的形式出现在 单个细胞，Aire参与多个多蛋白复合体，并优先定位于 并激活所谓的“超级增强剂”。超级增强剂(也称为“拉伸”或“系列”增强剂)是 染色质的扩展区域超载了通用的和细胞类型特异的转录因子， 并被认为是协调和有效地将这些因素传递给目标推动者的仓库 通过染色质循环。此外，这里记录的初步数据表明，Aire Associates直接或 间接地，与粘附素，远程染色质相互作用的主要协调者之一(在合作中 与CTCF和/或调解人)。因此，这个拟议项目的总体目标是确定Aire如何整合 染色质的三维组织。这一目标将通过三个具体目标来实现： 要确定Aire是否与已知的协调3D染色质相互作用的蛋白质相关联： CTCF、粘附素、NIPBL和介体。这一目标下的实验将主要使用生物化学 确定Aire是否与三个主要的3D染色质组织元件(和 粘附素加载器，NIPBL)。 将Aire在MEC染色质上的分布与CTCF、粘附素和介体的分布进行整合； 确定AIRE如何影响CTCF/粘附素/介体的放置，反之亦然，如何粘合 影响艾尔的位置。这一系列实验将利用最新的芯片序列改进(和 其他全基因组)技术以绘制与Aire相关的三个染色质组织者的结合位点 结合和其他标志-在野生型、Aire基因敲除和可诱导的Smc1基因敲除小鼠中 明确Aire与染色质三维结构的关系。这套 实验将使用HIC技术来生成MEC染色质的全基因组相互作用图 Aire+/+和Aire-/-小鼠。 这些研究的成功完成将使我们对Aire控制T细胞耐受的了解 分子理解的新水平，并有可能解决与以下几个悬而未决的难题 AIRE函数。艾尔的故事继续激起人们的兴趣！

Aire regulates central T cell tolerance by controlling thymic medullary epithelial cell (MEC) expression of a battery of transcripts encoding peripheral-tissue antigens. From early on, Aire was recognized to be a transcriptional regulator, but it soon became apparent that it does not operate like a conventional transcription factor, i.e. binding to promoters and inducing initiation of transcription. Studies completed during the last funding-cycle demonstrated that Aire-induced genes occur as intra- or inter-chromosomal clusters within individual cells, that Aire participates in multiple multi-protein complexes, and that it preferentially localizes to and activates so-called “super-enhancers.” Super-enhancers (a.k.a “stretch” or “serial” enhancers) are extended regions of chromatin that are over-loaded with general and cell-type-specific transcription factors, and are thought to serve as depots for coordinate and efficient delivery of these factors to targeted promoters via chromatin looping. In addition, preliminary data documented herein indicate that Aire associates, directly or indirectly, with cohesin, one of the major orchestrators of long-range chromatin interactions (in collaboration with CTCF and/or mediator). Thus, the overall goal of this proposed project is to determine how Aire integrates into the three-dimensional organization of chromatin. This goal will be addressed via three Specific Aims: ● To determine whether Aire associates with proteins known to orchestrate 3D chromatin interactions: CTCF, cohesin, NIPBL and mediator. Experiments under this Aim will employ primarily biochemical approaches to determine whether Aire associates with the three major 3D-chromatin-organizing elements (and a cohesin-loader, NIPBL). ● To integrate Aire’s distribution along MEC chromatin with those of CTCF, cohesin and mediator; to determine how Aire impacts CTCF/cohesin/mediator placement and, vice versa, how cohesin influences Aire’s placement. This series of experiments will exploit recent improvements in ChIP-seq (and other whole-genome) technologies to map the binding sites of the three chromatin organizers in relation to Aire binding and other landmarks – in wild-type, Aire-knockout and inducible Smc1-knockdown mice ● To define the relationship between Aire and three-dimensional chromatin organization. This set of experiments will use HiC technology to generate whole-genome interaction maps for MEC chromatin from Aire+/+ and Aire-/- mice. Successful completion of these studies will bring our understanding of Aire control of T cell tolerance to a new level of molecular understanding, and is likely to resolve several of the outstanding conundrums related to Aire function. The Aire story continues to intrigue!