CHARACTERIZATION OF A SILENCER REGULATORY ELEMENT IN THE RAT PAP-I GENE WHICH CONFERS TISSUE-SPECIFIC EXPRESSION AND IS PROMOTER-DEPENDENT

Previous analysis of the rat PAP I promoter indicated that the region between nt -180 and -81 possessed silencer activity in cells that did not express PAP I. Based on this finding, we performed a series of exp eriments to characterize functionally that region and analyze the nucl ear proteins interacting with it. Transient transfection assays were c onducted in the fibroblast Rata cell line, in which PAP I is not expre ssed, and in the pancreatic cell line AR-42J, ex pressing PAP I, using the CAT gene as reporter. Experiments in Rata cells revealed that the sequence with silencer activity was located within the rep27 region ( position -180/-153). Suppressor activity was observed when rep27 was i nserted upstream from the core PAP I promoter, in both orientations. B y contrast, inserting the rep27 region in front of the promoters of SV 40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-de pendent. In pancreatic AR-42J cells, rep27 act as a positive element b ut did not alter CAT expression when inserted in front of the core PAP I promoter or heterologous promoters. Electrophoretic mobility shift assays allowed identification of specific DNA-protein complexes. The s hifted complex migrated at the same position with both Rata and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with ra t nuclear extracts from healthy pancreas, pancreas with acute pancreat itis, liver, kidney, spleen, and small intestine. Results suggest that the rep27 cis-acting element contributes to the tissue specific expre ssion of the PAP I gene. That activity could be mediated by the synerg istic action of several transcription factors, one of which being pres ent in all cells. (C) 1997 Academic Press.