Both polymorphic variable number of tandem repeats and autoimmune regulator modulate differential expression of insulin in human thymic epithelial cells.
Both polymorphic variable number of tandem repeats and autoimmune regulator modulate differential expression of insulin in human thymic epithelial cells.
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作者：
Cai CQ;Zhang T;Breslin MB;Giraud M;Lan MS
多态性INS-VNTR在调节人胸腺中的胰岛素转录本表达中起重要作用，从而导致胰岛素自身免疫或耐受。INS-VNTR单倍型依赖性胰岛素表达的分子机制尚不清楚。在这项研究中，我们确定的机制组件的差异胰岛素基因表达在人胸腺上皮细胞，这应该有深远的影响胰岛素自身免疫耐受的诱导。一个被称为可变数目串联重复序列（VNTR）的重复DNA区域位于人胰岛素基因的上游，与1型糖尿病的发病率相关。我们产生了六个I类和两个III类VNTR结构连接到人胰岛素基础启动子或SV 40异源启动子/增强子，并证明了AIRE蛋白通过与VNTR区域结合来调节胰岛素启动子活性的差异。在这里，我们表明，在自身免疫调节（AIRE）的存在下，III类VNTR单倍型是负责平均三倍高的胰岛素表达比I类VNTR在胸腺上皮细胞。在蛋白质-DNA下拉实验中，AIRE蛋白能够与VNTR I类和III类探针结合。此外，AIRE对INS-VNTR的转录激活需要胰岛素基础启动子。当连接到异源启动子和/或增强子时，VNTR序列失去其激活活性。这些研究结果表明，1型糖尿病易感性编码的INS-VNTR基因座和AIRE发挥的关键功能，这构成了一个双重控制机制，调节胰岛素在人胸腺上皮细胞的定量表达。
Polymorphic INS-VNTR plays an important role in regulating insulin transcript expression in the human thymus that leads to either insulin autoimmunity or tolerance. The molecular mechanisms underlying the INS-VNTR haplotype-dependent insulin expression are still unclear. In this study, we determined the mechanistic components underlying the differential insulin gene expression in human thymic epithelial cells, which should have profound effects on the insulin autoimmune tolerance induction. A repetitive DNA region designated as a variable number of tandem repeats (VNTR) is located upstream of the human insulin gene and correlates with the incidence of type 1 diabetes. We generated six class I and two class III VNTR constructs linked to the human insulin basal promoter or SV40 heterologous promoter/enhancer and demonstrated that AIRE protein modulates the insulin promoter activities differentially through binding to the VNTR region. Here we show that in the presence of the autoimmune regulator (AIRE), the class III VNTR haplotype is responsible for an average of three-fold higher insulin expression than class I VNTR in thymic epithelial cells. In a protein-DNA pull-down experiment, AIRE protein is capable of binding to VNTR class I and III probes. Further, the transcriptional activation of the INS-VNTR by AIRE requires the insulin basal promoter. The VNTR sequence loses its activation activity when linked to a heterologous promoter and/or enhancer. These findings demonstrate a type 1 diabetes predisposition encoded by the INS-VNTR locus and a critical function played by AIRE, which constitute a dual control mechanisms regulating quantitative expression of insulin in human thymic epithelial cells.