Modulation of glucocorticoid induction of stably transfected tyrosine aminotransferase gene constructs involves elements up-stream of the glucocorticoid-responsive element.
Previous studies have documented that the amount of agonist activity expressed by the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) for tyrosine aminotransferase (TAT) induction in two rat hepatoma cell lines (Fu5-5 and HTC) is greater in Fu5-5 cells and could be varied in each cell line with changes in cell density. We have proposed that both phenomena are mediated by the binding of a trans-acting factor, the concentration or activity of which is lower in HTC cells. We have now used DNase-I hypersensitivity studies to identify a possible binding site for this factor at around -3.6 kilobases (kb) of the TAT gene. Fu5-5 and HTC cells were then stably transfected with hybrid constructs either with (3.9TATCAT) or without (2.9TATCAT) this region of the TAT gene fused up-stream of a chloramphenicol acetyltransferase (CAT) reporter gene. High levels of Dex-Mes agonist activity for the induction of CAT activity in Fu5-5 cells were seen only with the 3.9TATCAT construct, indicating that the 0.97-kb region unique to this construct controlled the high levels of Dex-Mes agonist activity. Furthermore, variations in Fu5-5 cell density caused major quantitative changes in the amount of Dex-Mes agonist activity only in cells containing the 3.9TATCAT construct, consistent with the same 0.97-kb sequences also controlling the variations in Dex-Mes agonist activity. Additional studies at high and low cell densities revealed that the modulation of Dex-Mes agonist activity for both the endogenous TAT gene and the transfected TAT/CAT gene was not due to changes in the start site of gene transcription. These studies both support our previous hypothesis that modulation of Dex-Mes agonist activity results from changes in a trans-acting factor and localize a necessary cis-acting element to sequences between -3.9 and -2.9 kb of the TAT gene. These studies, thus, define a potentially new element for glucocorticoid regulation of TAT gene transcription.
Szapary
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D
Steroid Hormones Section, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Oshima
H
H
Simons
S S
SS
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D003907:Dexamethasone / Q000031:analogs & derivatives* / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005809:Genes, Regulator; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014444:Tyrosine Transaminase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase; D014444:Tyrosine Transaminase; D003850:Deoxyribonuclease I; C031710:dexamethasone 21-methanesulfonate