Identification of a novel Aire-regulating cis-regulatory element using H3K27ac ChIP-seq. Mus musculus

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. We used sequence conservation analysis and ChIP-seq against the enhancer-associated histone mark H3K27ac to identify a candidate Aire cis-regulatory element. There is enrichment of H3K27ac near this element, ACNS1, in mTECs and the element also has characteristics of being NF-κB-responsive. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance. Overall design: Two experimental groups (GFP neg mTECs and GFP pos mTECs), each with three samples, and one control sample (D10 Th2 cells).

胸腺中枢耐受（Thymic central tolerance）在抵御自身免疫发生的过程中发挥着不可或缺的作用。在髓质胸腺上皮细胞（medullary thymic epithelial cells, mTECs）内，自身免疫调节因子（Autoimmune regulator, Aire）通过驱动多种组织特异性抗原（tissue-specific antigens, TSAs）的表达，在该过程中扮演核心角色——这些抗原经呈递后，可介导自身反应性胸腺细胞的免疫耐受诱导。有趣的是，Aire的表达具有高度的组织限制性，在成年个体中，仅在mTECs与胸腺外外周表达Aire的细胞（peripheral extrathymic Aire-expressing cells, eTACs）中可检测到其可观表达水平。尽管Aire的组织特异性如此显著，但其表达的顺式调控元件至今仍未被阐明。本研究借助序列保守性分析与针对增强子相关组蛋白修饰H3K27ac的染色质免疫共沉淀测序（ChIP-seq），成功鉴定出一个候选的Aire顺式调控元件。该元件ACNS1在mTECs中的邻近区域存在H3K27ac富集，同时具备核因子κB（NF-κB）的应答特征。最终研究发现，该元件在体内对Aire的表达至关重要，且是预防自发性自身免疫反应的必要条件，这充分体现了该调控DNA元件在促进免疫耐受中的关键意义。整体实验设计：设置两组实验组（GFP阴性mTECs与GFP阳性mTECs），每组各含3份样本，外加1份对照样本（D10 Th2细胞）。