CIS-ACTING ELEMENTS CONTROLLING TRANSCRIPTION FROM RAT SERINE-PROTEASE INHIBITOR 2.1 GENE PROMOTER - CHARACTERIZATION OF 2 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) regulati on of expression of the rat serine protease inhibitor 2.1 gene (spi 2. 1) were mapped. Normal rat hepatocytes were transiently transfected wi th constructs harboring deleted or mutated versions of the spi 2.1 pro ximal promoter region fused to the chloramphenicol acetyltransferase g ene. A purine-rich sequence (GAGA box, nucleotides -57 to -45), whose mutation or deletion almost completely knocks out both basal and hormo ne-stimulated promoter activities, plays the role of a key control ele ment. A positive GC response element, spanning nucleotides -88 to -74, confers GC responsiveness to a heterologous promoter. Two structurall y unrelated GH-response elements (GHRE) were identified. GHRE-II (nucl eotides -138 to -104) contains a CCAAT enhancer binding protein bindin g site whose mutation completely abolishes its GB-dependent enhancer f unction. 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 transcript ional machinery. Taken together these results have revealed the existe nce of an apparently new type of promoter functioning that strictly de pends on the integrity of a key regulatory (G + A) motif.