Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.
We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.
Cao
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SX
Laboratory of Chemical Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, Bethesda, MD 20892.
Mishoe
H
H
Elion
J
J
Berg
P E
PE
Schechter
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AN
D017456:Adenovirus Early Proteins; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005914:Globins / Q000235:genetics*; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*
D017456:Adenovirus Early Proteins; D000952:Antigens, Polyomavirus Transforming; D009856:Oncogene Proteins, Viral; D005914:Globins; D015500:Chloramphenicol O-Acetyltransferase