Abstract
The human pancreatic secretory trypsin inhibitor (PSTI) genes introduced into mice are specifically expressed in pancreas. The 1.0 kilobase pairs of PSTI 5'-flanking sequence directed preferential expression of a linked reporter chloramphenicol acetyltransferase, which was active in a PSTI-expressing pancreatic cell line (AR42j) but not in a PSTI-nonexpressing fibroblast cell line (XC). Two positively acting elements were found, Region I (-161/-116) and Region II (-103/-74), as defined by transfection and binding assays with AR42j cells. Region II is sufficient for the pancreas-specific expression, but the presence of both Regions I and II is needed for the maximum activity. Sequence studies also revealed that these two elements differ from the previously identified recognition sequence for pancreas transcription factor 1 (PTF1). When the same set of experiments was done with XC cells, one negatively acting element was identified, Region IV (-154/-137). Interestingly, Regions I and IV share a core sequence (-149/-139), CAATCAATAAC. These results suggest that this novel element regulates the human PSTI gene expression positively in pancreatic cells but negatively in nonpancreatic cells.
Highlights
The differentiation and maintenance of phenotype in eukaryotic cells depends on the regulated expression of selected genes, most of which relies on interaction of specific regulatory protein(s) and its cognate cis-acting DNA elements including a promoter and enhancer(s)
1 The abbreviations used are: bp, base pair(s); PTF1, pancreas-specific transcription factor 1; Pancreatic secretory trypsin inhibitor (PSTI), pancreas secretory trypsin inhibitor; kbp, kilobase pair(s); CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. This sequence interacts with a pancreas-specific transcription factor 1 (PTF1) [5], which consists of a 64-kDa protein subunit that binds to Box A and a 48-kDa protein subunit that binds to Box B [6]
The Human PSTI Gene Is Expressed in the Pancreas of Transgenic Mice—Transgenic mice were prepared by introducing a human PSTI gene containing the 8.3 kbp of the 5Ј-flanking sequence (Fig. 1A)
Summary
Identification of the Integrated PSTI Gene—A 27-kbp human PSTI genomic DNA that contains 8.3 kbp of the 5Ј-flanking sequence has been cloned into pWE16 by Horii et al [14]. PSTI(AccI) CAT [22], which carries a fragment containing from about Ϫ4.8 kbp to Ϫ1 bp of the 5Ј-flanking region of the human PSTI gene, served as the parental plasmid for the following clones. DNase I Footprinting Assay—A probe containing the nucleotides Ϫ179 to Ϫ1 of the human PSTI gene was excised from PSTI[179]CAT by digestion with HindIII (for a coding strand) or BssHII (a noncoding strand), end-labeled with [␣32P]dCTP by the Klenow fragment, digested again with BssHII or HindIII. The end-labeled fragment (3 fmol; specific activity, 3 ϫ 105 cpm) was incubated with 20 ng of nuclear protein for 30 min at room temperature in a volume of 50 l in the presence of 12 mM HEPES-KOH, pH 7.9, 60 mM KCl, 0.12 mM EDTA, 1 mM MgCl2, 12% glycerol, 1 mM DTT, and 1 mM PMSF with 1 or 2 g of poly(dI-dC). Cell-type specificity represents the CAT activity obtained in AR42j relative to that in XC
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