A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.
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 (delta MTV-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.
U-99 INSERM, Hôpital Henri Mondor, Créteil, France.
M
M
Garlatti
M
M
Daheshia
E
E
Slater
J
J
Bouguet
J
J
Hanoune
M
M
Beato
R
R
Barouki
D001219:Aspartate Aminotransferases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D001219:Aspartate Aminotransferases