STIMULATION OF TRANSCRIPTION IN-VITRO FROM A LIVER-SPECIFIC PROMOTER BY HUMAN GLUCOCORTICOID RECEPTOR (HGR-ALPHA)

The rat tyrosine aminotransferase (TAT) gene is a liver-specific and g lucocorticoid-inducible gene. Previous studies have shown that the TAT promoter (TAT(0.35); 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 a nd Pictet (1994) Nucleic Acids Res. 22, 1583-1592]. Here we report tha t the basal transcriptional activity generated from TAT(0.35) in the p resence of crude liver nuclear extracts is enhanced by added human glu cocorticoid receptor (hGR alpha), provided that TAT(0.35) sequences we re flanked (5') with a glucocorticoid responsive unit (GREII of the TA T gene, including its 5'-CCAAT flanking sequence). Two sources of hGR alpha were used: nuclear extracts prepared from Sf9 insect (Sf9-NEs) c ells over-expressing hGR alpha, and hGR alpha from pRShGR alpha-transf ected COS-7 cells, enriched by high-performance ion-exchange chromatog aphy. The enhancement of transcription in vitro (1.5-4.5-fold) was dep endent on the amount of added hGR alpha and independent of the nature (agonist or antagonist) of the ligand. Moreover, the hGR alpha-mediate d stimulation of transcription was (i) dependent on GRE/progesterone r esponse 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 tra nscription). Taken together, these experiments support the conclusions that in vitro the glucocorticoid receptor is able to enhance transcri ption from genomic, liver-specific, promoter sequences (those of the T AT gene), and that this enhancement of transcription from the liver-sp ecific TAT(0.35) promoter is dependent both on the glucocorticoid rece ptor and on the latter's interaction with its cognate response element s.