Transcription of the rat serine protease inhibitor 2.1 gene in vivo: correlation with GAGA box promoter occupancy and mechanism of cytokine-mediated down-regulation.

Two GH-response elements (GHREs) and a single glucocorticoid (GC)-response element were found to regulate activity of the rat serine protease inhibitor 2.1 gene (spi 2.1) promoter in vitro. To assess the physiological relevance of these observations, we have investigated the relationship existing between the level of spi 2.1 gene transcription, structural modifications of the chromatin, and in vivo nuclear protein-promoter interactions monitored by genomic footprinting, in control, hypophysectomized, and inflamed rats. We also addressed the mechanism of inflammation-mediated gene down-regulation. We found that a high level of spi 2.1 gene transcription correlates with hypersensitivity of the promoter to deoxyribonuclease I (DNase I) and maximal occupancy of the GAGA box (GHRE-I). The failure of GAGA-box binding proteins (GAGA-BPs) to interact with the GAGA box appears to result from an impairment in GH action due to its absence (i.e. hypophysectomized animals) or to the appearance of a cytokine-mediated GH-resistant state (i.e. inflamed rats) in liver. Unlike the GAGA box, signal transducer and activator of transcription (STAT) factor-binding sites included in the GHRE-II were never found to be protected against DNase I attack but displayed a differential DNase I reactivity depending on the level of gene transcription. Alterations in DNase I reactivity of the GC-response element region suggest that GC receptor-GC complexes may associate, in a transient manner, with the promoter in the actively transcribing control state. Taken together, our studies suggest a mechanism of spi 2.1 gene activation in vivo whereby the GH-dependent chromatin remodeling caused by or concomitant to the recruitment of GAGA-box binding proteins is the first compulsory and presumably predominant step.