A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoi ds in rat liver and kidney. Part of this regulation is mediated by an unusu al glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserve d half-site (half-sites 1 and 4 respectively) and a poorly conserved half-s ite (half-sites 2 and 3 respectively). The sequence binds cooperatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocor ticoid regulation of gene expression. Analysis of deletions of the cAspAT g ene promoter and subcloning of GRE A upstream of the thymidine kinase promo ter indicate that this sequence is responsive to glucocorticoids, but not t o androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nu cleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative bin ding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulati on of the thymidine kinase promoter. A single base modification in half-sit e 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respec tively, and restored transcriptional activation by androgens only in the la tter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevente d its binding to GRE A, indicating that the overlapping GRE A sequence of t he cAspAT gene promoter discriminates a glucocorticoid-mediated from an and rogen-mediated response.