The chymotrypsin enhancer core: Specific factor binding and biological activity

Abstract

Abstract A 20-base pair (bp) conserved sequence present in the 5'-flanking regions of genes highly expressed in the exocrine pancreas forms part of the enhancers of the rat amylase 2A, chymotrypsin B, and elastase I genes. Factor(s) that interact with the conserved DNA sequence of the rat chymotrypsin B gene in vitro have been detected in extracts from acinar cells but not in three other cell lines tested. Transfection experiments suggest that the acinar cell factor(s) recognizing this enhancer core sequence are transcriptional activators. Multimers of a 28-bp sequence located by DNase I protection are capable of activating heterologous promoters in acinar cells. In vitro competition binding and methylation interference analyses indicate protein-DNA interactions at two distinct sites 10 base pairs apart on the DNA. This interaction is identical in extracts from cultured acinar cells as well as from whole pancreas tissue. The presence of two contiguous binding motifs on the same face of the DNA suggests that two (multiple) factors cooperate in the transcriptional regulation of this gene. We term these factors CACCTG pan-1 and TTTCCC pan-1. A factor with the binding specificity of the adenovirus major late transcription factor (MLTF) cross-reacts with the factor CACCTG pan-1 in vitro. However, the distribution of this factor in the various cells does not correlate with the activity of the enhancer core element in vivo. Further, conversion of the chymotrypsin sequence into a consensus MLTF site by three-point mutations abolished enhancer activity in acinar cells. Thus, the MLTF-like factor cannot substitute functionally for the factor CACCTG pan-1 and may act as an inhibitor of chymotrypsin enhancer function.