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Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1
H König1, H Ponta, H J Rahmsdorf
1Kernforschungszentrum Karlsruhe, Institut für Genetik und Toxikologie, Germany.
Abstract:
Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.
Insights
Glucocorticoid hormones do not affect AP-1 binding to DNA in living cells, despite their ability to inhibit gene expression. This suggests the hormone receptor acts indirectly, without direct DNA binding.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cell Signaling
Background:
- Phorbol esters and glucocorticoid hormones regulate gene expression through the transcription factor AP-1 (Fos/Jun).
- AP-1 controls promoters, including the collagenase gene, influencing cellular processes.
- Understanding the precise mechanisms of AP-1 regulation by these signaling molecules is crucial.
Purpose of the Study:
- To investigate the in vivo binding of AP-1 to the collagenase promoter upon phorbol ester treatment.
- To determine if glucocorticoid receptors interfere with AP-1 binding to its DNA site.
- To elucidate the mechanism by which glucocorticoid hormones down-regulate gene expression mediated by AP-1.
Main Methods:
- Genomic footprinting of the collagenase promoter in treated cells.
- In vitro DNA-protein binding assays using purified AP-1 and synthesized glucocorticoid receptor.
- Analysis of AP-1 binding site occupation in vivo under hormonal influence.
Main Results:
- Phorbol ester treatment induced in vivo binding of AP-1 to the collagenase promoter.
- In vitro, glucocorticoid receptor disturbed Jun homodimer binding but not Fos-Jun heterodimer binding.
- Glucocorticoid hormones at inhibitory doses did not alter AP-1 binding site occupation in vivo.
- The hormone receptor's inhibitory action occurred without direct DNA binding at the AP-1 site.
Conclusions:
- AP-1 (Fos/Jun) binds its cognate DNA site in vivo upon phorbol ester stimulation.
- Glucocorticoid hormones down-regulate AP-1-mediated transcription indirectly, without directly competing for the AP-1 DNA binding site.
- The glucocorticoid receptor influences AP-1 activity through a mechanism independent of direct DNA interaction at the AP-1 site.