Identification of a transcriptional regulatory region of the rat pancreatitis-associated protein I (PAP I) gene that confers tissue specificity.
We have previously characterized the rat pancreatitis-associated protein I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated with the DNA sequence comprising nt -1253 to + 10 of the gene. That sequence strongly promoted the transcription of the promotorless chloramphenicol acetyltranferase (CAT) gene in cells of pancreatic origin (AR-42J) but not in cells of non-pancreatic origin (Rat 2 and IEC 6). The influence on CAT expression of stepwise 5' deletions in the promoter sequence was monitored in the three cell lines. In pancreatic AR-42J cells, deletion down to position -926 did not affect significantly the expression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not have any additional effect, but a further deletion to nt -180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 resulted in a further reduction to about one-third of that. Finally, deletion down to nt -61 further reduced activity by a factor of 3, although it remained above background. These results suggest the presence of several positive cis-acting elements in the PAP I promoter. In non-pancreatic cells, CAT expression remained very low when the promoter was deleted down to nt -180. Yet, deletion from -180 to -118 significantly increased CAT expression, suggesting suppression of a negative cis-acting element. Further deletion down to nt -61 decreased CAT activity by a factor of 5. The region between nt -180 and -61 was subjected to footprint analysis. A similar pattern of DNase protection was obtained with AR-42J and Rat 2 nuclear extracts, the only protected region extending from nt -125 to -95. That region was further analysed by inserting the nt -180 to -81 fragment, in both orientations, upstream of thymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but reduced in Rat 2 cells. These results indicated the presence of cell-specific positive and negative elements within that region.
D000209:Acute-Phase Proteins / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D014408:Biomarkers, Tumor; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation; D005809:Genes, Regulator / Q000235:genetics*; D037102:Lectins / Q000235:genetics; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000235:genetics*
D000209:Acute-Phase Proteins; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D017931:DNA Primers; D037102:Lectins; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; C000612144:Reg3b protein, rat; D015500:Chloramphenicol O-Acetyltransferase