Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.
In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.
Knust
B
B
Institute of Genetics, University of Cologne, Federal Republic of Germany.
Brüggemann
U
U
Doerfler
W
W
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D015152:Blotting, Northern; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000235:genetics; D008745:Methylation; D009693:Nucleic Acid Hybridization; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection
D017456:Adenovirus Early Proteins; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D012367:RNA, Viral; D001374:Azacitidine