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)-resp onse element were found to regulate activity of the rat serine proteas e inhibitor 2.1 gene (spi 2.1) promoter in vitro. To assess the physio logical relevance of these observations, we have investigated the rela tionship existing between the level of spi 2.1 gene transcription, str uctural modifications of the chromatin, and in vivo nuclear protein-pr omoter interactions monitored by genomic footprinting, in control, hyp ophysectomized, and inflamed rats. We also addressed the mechanism of inflammation-mediated gene downregulation. 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 i n GR action due to its absence (i.e. hypophysectomized animals) or to the appearance of a cytokine-mediated GH-resistant state (i.e. inflame d 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 trans cription. Alterations in DNase I reactivity of the OC-response element region suggest that GC receptor-OC complexes may associate, in a tran sient manner, with the promoter in the actively transcribing control s tate. Taken together, our studies suggest a mechanism of spi 2.1 gene activation in vivo whereby the OR-dependent chromatin remodeling cause d by or concomitant to the recruitment of GAGA-box binding proteins is the first compulsory and presumably predominant step.