We have shown that transcriptional activation of the human urokinase gene via the PKC pathway in HepG2 cells leads to the formation of a nuclease hypersensitive site which identifies the phorbol ester-dependent enhancer at -2 kb from the transcription start site, the enhancer presents an octameric and an eptameric AP1 sites, which mediate PMA induction of the uPA gene. We show that the alteration in chromatin structure occurs very rapidly and is rapidly reversible after withdrawal of PMA. The enhancer region is occupied by nucleosomes before induction and after removal of PMA, but this pattern is perturbed upon addition of the phorbol ester. Restriction enzyme digestion of chromatin reveals that the region encompassing the enhancer becomes more accessible to restriction after 2/3 hours PMA treatment. The rapidity of chromatin changes implies that neither disruption nor reassembly of the nucleosomes is dependent on DMA replication. Analysis of the enhancer-promoter region of the gene by micrococcal nuclease reveals a discrete banding pattern consistent with precise nucleosome positioning. Two more nuclease hypersensitive sites corresponding to the silencer element and the start of transcription were identified, both remained unchanged after PMA stimulation, suggesting that the enhancer is the single most important site involved in the activation of uPA transcription through the PKC pathway. The loss of hypersensitivity on the enhancer resulting from PMA withdrawal suggests that maintenance of the HS requires the binding of AP1 to its cognate sequence. When deinduction takes place the enhancer reverts to a nucleosomal conformation. Our observations describe an inducible enhancer which is regulated by a dynamic balance between two types of protein-DNA complexes, one of which is nucleosomal.
|Number of pages||1|
|Issue number||SUPPL. 3|
|Publication status||Published - 1996|
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