Sirt1 is essential for Aire-mediated induction of central immunological tolerance

Autoimmune regulator (Aire) is a unique transcriptional regulator that induces promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for induction of immunological self-tolerance. Although several recent studies provided very important molecular insights into how Aire operates, a more comprehensive understanding of this process still remains elusive. Here we demonstrate that a lysine deacetylase Sirtuin-1 (Sirt1) is predominantly expressed in mature Aire+ mTECs, where it is required for expression of Aire-dependent TRA genes and a subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and uncovers a unique functional role for Sirt1 in preventing organ-specific autoimmunity. ~100ng of total RNA isolated by Trizol extraction from MHC-II low and high mTECs (pool of 3 mice) was used to generate poly-A-selected transcriptome libraries using the non-directionnal TruSeq V3 RNA Sample Prep Kit (without additional pre-amplification) following the manufacturer's protocols. Enrichment of DNA fragment with adapter molecules on both ends was done using 15 cycles of PCR amplification using the Illumina PCR mix and primer cocktail. Paired-end (2 x 100 bp) sequencing was performed using the Illumina HiSeq2000 machine.自身免疫调节因子（Autoimmune regulator, Aire）是一类独特的转录调控因子，可在髓质胸腺上皮细胞（medullary thymic epithelial cells, mTECs）中诱导数千种组织限制性抗原（tissue-restricted antigens, TRAs）的异位表达，该过程对免疫自耐受的诱导至关重要。尽管近期多项研究为Aire的作用机制提供了重要的分子层面见解，但学界对该过程的全面认知仍存在空白。本研究证实，赖氨酸去乙酰化酶Sirtuin-1（lysine deacetylase Sirtuin-1, Sirt1）主要在成熟Aire阳性mTECs中表达，且该蛋白对于Aire依赖型TRA基因的表达以及后续免疫自耐受的诱导不可或缺。本研究阐明了此前未知的Aire介导的转录调控分子机制，并揭示了Sirt1在预防器官特异性自身免疫病中的独特功能。本研究通过Trizol提取法，从3只小鼠的MHC-II低表达与高表达mTECs混合样本中分离得到总RNA，每份总量约100 ng；随后按照制造商说明书，采用非定向TruSeq V3 RNA样本制备试剂盒（未进行额外预扩增）构建polyA富集转录组文库。通过Illumina PCR预混液与引物混合物，经15轮PCR扩增完成两端带有接头分子的DNA片段富集。最终使用Illumina HiSeq2000测序仪完成双端（2×100 bp）测序。