Yeast one-hybrid screen of a thymus epithelial library identifies ZBTB7A as a regulator of thymic insulin expression

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.