In vivo analysis of the state of the human uPA enhancer following stimulation by TPA.
We have analysed in vivo the -2.0 kb enhancer of the human urokinase-type plasminogen activator (uPA) gene in HepG2 cells, in which gene expression can be induced by phorbol esters. The results reveal that, within the regulatory region, the enhancer, the silencer and the minimal promoter become hypersensitive to deoxyribonuclease I (DNase I) upon induction of transcription. The hypersensitivity of the enhancer can be reversed after removal of the inducer. In vivo footprinting analysis indicates that all the cis-acting elements of the enhancer, previously identified in vitro, are occupied in vivo upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation of HepG2 cells. Micrococcal nuclease (MNase) cleavage of this region fails to reveal discrete nucleosomal boundaries in vivo in close proximity of the enhancer, either before or after stimulation. Furthermore, this region does not lose its nucleosomal configuration after TPA induction of transcription. An approximately 600 bp long region around the enhancer becomes more, but not fully, accessible to restriction endonucleases upon stimulation. A time-course experiment shows that this accessibility reaches a plateau after a 1 h TPA treatment suggesting the persistent presence of nucleosomes. These results indicate that TPA induces the binding of transcription factors to the uPA enhancer without chromatin remodelling of this region.
Ibañez-Tallon
I
I
Laboratory of Molecular Genetics, DIBIT- Ospedale. S. Raffaele, Milano, Italy.
Caretti
G
G
Blasi
F
F
Crippa
M P
MP
D001665:Binding Sites; D002273:Carcinogens / Q000494:pharmacology*; D002843:Chromatin / Q000235:genetics / Q000648:ultrastructure; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*
D002273:Carcinogens; D002843:Chromatin; D016328:NF-kappa B; D009707:Nucleosomes; D012333:RNA, Messenger; D014157:Transcription Factors; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate