A Functional Glucocorticoid-Responsive Unit Composed of Two Overlapping Inactive Receptor-Binding Sites: Evidence for Formation of a Receptor Tetramer

• Published in: Molecular and cellular biology Vol. 14; no. 12; pp. 8007 - 8017
• Main Authors: Garlatti, Michèle, Daheshia, Massoud, Slater, Emily, Bouguet, Jacqueline, Hanoune, Jacques, Beato, Miguel, Barouki, Robert
• Format: Journal Article
• Language: English
• Published: Taylor & Francis 01.12.1994
• ISSN: 1098-5549; 1098-5549
• DOI: 10.1128/mcb.14.12.8007-8017.1994

An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (ΔMTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.