cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element.

The cis-acting elements that are functionally important for the basal, the growth hormone (GH), and the glucocorticoid hormone (GC) regulation of expression of the rat serine protease inhibitor 2.1 gene (spi 2.1) were mapped. Normal rat hepatocytes were transiently transfected with constructs harboring deleted or mutated versions of the spi 2.1 proximal promoter region fused to the chloramphenicol acetyltransferase gene. A purine-rich sequence (GAGA box, nucleotides -57 to -45), whose mutation or deletion almost completely knocks out both basal and hormone-stimulated promoter activities, plays the role of a key control element. A positive GC response element, spanning nucleotides -88 to -74, confers GC responsiveness to a heterologous promoter. Two structurally unrelated GH-response elements (GHRE) were identified. GHRE-II (nucleotides -136 to -104) contains a CCAAT enhancer binding protein binding site whose mutation completely abolishes its GH-dependent enhancer function. GHRE-I, which spans nucleotides -61 to +8, is not an enhancer element. Its GH-dependent activity depends on the preservation of the distance separating the GAGA box and elements of the basic transcriptional machinery. Taken together, these results have revealed the existence of an apparently new type of promoter functioning that strictly depends on the integrity of a key regulatory (G + A) motif.