Sequences Downstream of the Glucocorticoid Regulatory Element Mediate Cycloheximide Inhibition of Steroid Induced Expression from the Rat α1-Acid Glycoprotein Promoter: Evidence for a Labile Transcription Factor

Abstract

The glucocorticoid induction of .alpha.1-acid glycoprotein (AGP) RNA in rat hepatoma cells is diminished by inhibiting protein synthesis. We now show that the AGP 5''-flanking region contains a DNA sequence (position-121 to -107), exhibiting a high degree of homology to the glucocorticoid regulatory element (GRE) consensus sequence ACAXXXTGTTCT, which serves to specifically bind purified rat glucocorticoid receptor in vitro. A 15 base pair oligonucleotide representing the AGP GRE confers glucocorticoid responsiveness on a heterologous promoter; such regulation is not diminished by concurrent inhibition of protein synthesis. However, inclusion of the AGP sequences immediately downstream of the AGP GRE (position -106 to -42) renders the hormonal induction sensitive to inhibition of protein synthesis. Furthermore, inclusion of these downstream sequences results in a more pronounced induction mediated by the AGP GRE. In vitro DNase-1 treatment using nuclear extracts prepared from HTC hepatoma cells generate footprints that indicate the presence of DNA-protein interactions spanning the region from -110 to -68 of the AGP gene. We propose that one or more labile factors acting within this domain, immediately downstream of the GRE, is required for efficient transcription at the AGP promoter.