Laboratory of Endocrine Genetics, Department of Pediatrics, The McGill University Health Center, 4060 Ste-Catherine West, Montreal, Quebec, Canada H3Z 2Z3.
Mol Immunol. 2013 Dec;56(4):637-42. doi: 10.1016/j.molimm.2013.05.238. Epub 2013 Aug 1.
Insulin self-tolerance is, to a large extent, assured by the expression of small quantities of insulin by medullary thymic epithelial cells (mTECs). Regulation of thymic insulin expression differs from that in pancreas and its therapeutic manipulation could play an important role in the prevention of type 1 diabetes (T1D). Knowledge of the transcriptional regulators involved in the mTEC nuclear environment is essential for the development of such therapeutics. The yeast one-hybrid (Y1H) approach was used in order to identify such mTEC-specific nuclear proteins. We used a target composed of the human insulin gene promoter joined to the upstream class III VNTR allele, which is associated with both protection from T1D and higher thymic insulin expression, and a cDNA library from our insulin-producing mouse mTEC line. The Y1H screening allowed the identification of eleven proteins. An in vitro assay was used to confirm and quantify protein-DNA binding to the human insulin gene promoter alone or joined to a class I or class III VNTR allele, and identified the transcription factors ZBTB7A, JUN and EWSR1 as strong interacting partners. All three proteins could induce insulin expression in transfected HEK-293 cells, but ZBTB7A provided the most robust results especially in the presence of AIRE, with an additional 11-fold increase of the insulin mRNA levels from a co-transfected reporter driven by the class III VNTR allele. Thus, ZBTB7A is identified as a strong candidate for regulation of thymic insulin expression.
胰岛素自身耐受性在很大程度上是由髓质胸腺上皮细胞（mTECs）表达少量胰岛素来保证的。胸腺胰岛素的表达调控与胰腺不同，其治疗性操作可能在 1 型糖尿病（T1D）的预防中发挥重要作用。了解参与 mTEC 核环境的转录调节因子对于此类治疗方法的开发至关重要。我们使用酵母单杂交（Y1H）方法来鉴定这种 mTEC 特异性核蛋白。我们使用了一个由人胰岛素基因启动子与上游 III 类 VNTR 等位基因连接而成的靶标，该等位基因与 T1D 的保护和更高的胸腺胰岛素表达有关，以及来自我们的胰岛素产生小鼠 mTEC 系的 cDNA 文库。Y1H 筛选鉴定出了十一种蛋白。体外测定用于确认和定量蛋白与单独的人胰岛素基因启动子或与 I 类或 III 类 VNTR 等位基因连接的 DNA 结合，鉴定出转录因子 ZBTB7A、JUN 和 EWSR1 是强相互作用的伙伴。这三种蛋白都可以在转染的 HEK-293 细胞中诱导胰岛素表达，但 ZBTB7A 提供了最稳健的结果，特别是在 AIRE 存在的情况下，来自共转染报告基因的胰岛素 mRNA 水平增加了 11 倍，该报告基因由 III 类 VNTR 等位基因驱动。因此，ZBTB7A 被确定为调节胸腺胰岛素表达的强有力候选者。
