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 p lasminogen activator (uPA) gene in HepG2 cells, in which gene expression ca n be induced by phorbol esters, The results reveal that, within the regulat ory region, the enhancer, the silencer and the minimal promoter become hype rsensitive 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 e lements of the enhancer, previously identified in vitro, are occupied in vi vo upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation of HepG2 ce lls, Micrococcal nuclease (MNase) cleavage of this region fails to reveal d iscrete 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 appr oximately 600 bp long region around the enhancer becomes more, but not full y, accessible to restriction endonucleases upon stimulation. A time-course experiment shows that this accessibility reaches a plateau after a Ih TPA t reatment suggesting the persistent presence of nucleosomes, These results i ndicate that TPA induces the binding of transcription factors to the uPA en hancer without chromatin remodelling of this region.