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 prote in I (PAP I) gene by nucleotide sequencing. We describe in this paper its promoter region by analysing the regulatory functions associated w ith the DNA sequence comprising nt -1253 to +10 of the gene. That sequ ence strongly promoted the transcription of the promotorless chloramph enicol acetyltransferase (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 s equence was monitored in the three cell lines. In pancreatic AR-42J ce lls, deletion down to position -926 did not affect significantly the e xpression of the reporter gene. Deletion to nt -685 caused about a 30% decrease in expression. Extending the deletion to nt -444 did not hav e any additional effect, but a further deletion to nt - 180, resulted in a reduction to about 25%. Moreover, deletion from nt -180 to -118 r esulted in a further reduction to about one-third of that. Finally, de letion down to nt -61 further reduced activity by a factor of 3, altho ugh it remained above background. These results suggest the presence o f several positive cis-acting elements in the PAP I promoter. In non-p ancreatic cells, CAT expression remained very low when the promoter wa s deleted down to nt -180. Yet, deletion from -180 to -118 significant ly 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 obtaine d with AR-42J and Rat 2 nuclear extracts, the only protected region ex tending from nt -125 to -95. That region was further analysed by inser ting the nt -180 to -81 fragment, in both orientations, upstream of th ymidine kinase (TK) or simian virus 40 (SV40) promoter-CAT constructs. In all cases CAT expression was increased in pancreatic cells but red uced in Rat 2 cells. These results indicated the presence of cell-spec ific positive and negative elements within that region.