Sequences downstream of the glucocorticoid regulatory element mediate cycloheximide inhibition of steroid induced expression from the rat alpha 1-acid glycoprotein promoter: evidence for a labile transcription factor.
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.
Klein
E S
ES
Department of Pharmacology, Stanford University School of Medicine, California 94305.
DiLorenzo
D
D
Posseckert
G
G
Beato
M
M
Ringold
G M
GM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D003513:Cycloheximide / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005938:Glucocorticoids / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured / Q000378:metabolism
D005938:Glucocorticoids; D009961:Orosomucoid; D003513:Cycloheximide