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 experiments to characterize functionally that region and analyze the nuclear proteins interacting with it. Transient transfection assays were conducted in the fibroblast Rat2 cell line, in which PAP I is not expressed, and in the pancreatic cell line AR-42J, expressing PAP I, using the CAT gene as reporter. Experiments in Rat2 cells revealed that the sequence with silencer activity was located within the rep27 region (position -180/-153). Suppressor activity was observed when rep27 was inserted upstream from the core PAP I promoter, in both orientations. By contrast, inserting the rep27 region in front of the promoters of SV40 or thymidine kinase did not affect or weakly enhanced CAT activity. Suppressor activity is therefore position-independent and promoter-dependent. In pancreatic AR-42J cells, rep27 act as a positive element but 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 shifted complex migrated at the same position with both Rat2 and AR-42J nuclear extracts. Moreover, similar band shifts were obtained with rat nuclear extracts from healthy pancreas, pancreas with acute pancreatitis, liver, kidney, spleen, and small intestine. Results suggest that the rep27 cis-acting element contributes to the tissue specific expression of the PAP I gene. That activity could be mediated by the synergistic action of several transcription factors, one of which being present in all cells.
Ortiz
E M
EM
U.315 INSERM, Marseille, France.
Dusetti
N J
NJ
Dagorn
J C
JC
Iovanna
J L
JL
D000209:Acute-Phase Proteins / Q000235:genetics*; D000818:Animals; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D037181:Lectins, C-Type; D005810:Multigene Family; D009687:Nuclear Proteins / Q000378:metabolism; D010195:Pancreatitis / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism
D000209:Acute-Phase Proteins; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D037181:Lectins, C-Type; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; D014157:Transcription Factors