Stimulation of transcription in vitro from a liver-specific promoter by human glucocorticoid receptor (hGRalpha).
The rat tyrosine aminotransferase (TAT) gene is a liver-specific and glucocorticoid-inducible gene. Previous studies have shown that the TAT promoter (TAT0.35; nt -350 to +1) is able to sustain liver-specific gene expression both in transient transfection and in a transcription assay in vitro [Schweizer-Groyer, Groyer, Cadepond, Grange, Baulieu and Pictet (1994) Nucleic Acids Res. 22, 1583-1592]. Here we report that the basal transcriptional activity generated from TAT0.35 in the presence of crude liver nuclear extracts is enhanced by added human glucocorticoid receptor (hGRalpha), provided that TAT0.35 sequences were flanked (5') with a glucocorticoid responsive unit (GREII of the TAT gene, including its 5'-CCAAT flanking sequence). Two sources of hGRalpha were used: nuclear extracts prepared from Sf9 insect (Sf9-NEs) cells over-expressing hGRalpha, and hGRalpha from pRShGRalpha-transfected COS-7 cells, enriched by high-performance ion-exchange chromatography. The enhancement of transcription in vitro (1.5-4.5-fold) was dependent on the amount of added hGRalpha and independent of the nature (agonist or antagonist) of the ligand. Moreover, the hGRalpha-mediated stimulation of transcription was (i) dependent on GRE/progesterone response element (PRE) (it was inhibited by a 25-fold excess of GRE/PRE but not by a 100-fold excess of oestrogen response element) and (ii) receptor-dependent (Sf9-NEs prepared from uninfected Sf9 cells or from Sf9 cells infected with wild-type baculoviral DNA did not enhance transcription). Taken together, these experiments support the conclusions that in vitro the glucocorticoid receptor is able to enhance transcription from genomic, liver-specific, promoter sequences (those of the TAT gene), and that this enhancement of transcription from the liver-specific TAT0.35 promoter is dependent both on the glucocorticoid receptor and on the latter's interaction with its cognate response elements.
Schweizer-Groyer
G
G
Inserm U.33, Laboratory of Hormones, 80, rue du Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France.
Cadepond
F
F
Jibard
N
N
Neau
E
E
Segard-Maurel
I
I
Baulieu
E E
EE
Groyer
A
A
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006801:Humans; D008099:Liver / Q000378:metabolism*; D015735:Mifepristone / Q000378:metabolism / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014158:Transcription, Genetic
D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D015735:Mifepristone