Lovewell Thomas R J, McDonagh Andrew J, Messenger Andrew G, Azzouz Mimoun, Tazi-Ahnini Rachid
School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom.
Dermatology Department, Royal Hallamshire Hospital, Sheffield, United Kingdom.
PLoS One. 2015 May 15;10(5):e0127476. doi: 10.1371/journal.pone.0127476. eCollection 2015.
The autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus.
Therefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase.
AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions.
自身免疫调节因子（AIRE）在胸腺中表达，特别是在胸腺髓质上皮细胞（mTECs）中，mTECs异位表达多种外周组织抗原需要AIRE，这有助于其对自身反应性未成熟T细胞进行阴性选择。外周组织抗原的表达谱不仅受AIRE缺乏的影响，还受胸腺中AIRE活性变化的影响。
因此，我们筛选了AIRE转录起始位点上游591bp区域，包括AIRE最小启动子，以寻找单核苷酸多态性（SNP），并分别鉴定出两个SNP，即-655R（rs117557896）和-230Y（rs751032）。为了研究这些变异对AIRE启动子活性的影响，我们构建了一个Flp-In宿主细胞系，该细胞系用单拷贝报告载体进行稳定转染。通过比较不同报告基因AIRE启动子-报告基因构建体（包括AIRE-655G AIRE-230C、AIRE-655G AIRE-230T和AIRE-655A AIRE-230C）裂解物的荧光素酶比活性来估计相对启动子活性。分析表明，最常见的单倍型AIRE-655G AIRE-230C具有最高的荧光素酶比活性（p<0.001）。而AIRE-655G AIRE-230T的荧光素酶比活性值接近零。两个AIRE启动子多态性位点都有一个等位基因形成一个CpG甲基化位点，我们通过使用M.SssI CpG甲基转移酶的甲基化测定确定该位点可以被甲基化。
AIRE-230Y位于启动子的保守区域，与预测的WT1转录因子结合位点相邻，这表明AIRE-230Y通过影响生化因子与该区域的结合来影响AIRE表达。我们的研究结果表明，AIRE-655GAIRE-230T单倍型可显著改变AIRE转录，从而影响阴性选择过程，并影响自身免疫性疾病的易感性。
