Goblet-cell-specific transcription of mouse intestinal trefoil factor generesults from collaboration of complex series of positive and negative regulatory elements

Intestinal trefoil factor (ITF) is expressed selectively in intestinal gobl et cells. Previous studies of the rat ITF gene identified one cis-regulator y element, designated the goblet-cell-response element (GCRE), present in t he proximal region of the promoter. To identify additional cis-regulatory e lements responsible for goblet-cell-specific expression, a DNA fragment con taining 6353 bp of the 5'-flanking region of the mouse ITF gene was cloned and its promoter activity was examined extensively. In human and murine int estinal-derived cell lines (LS174T and CMT-93), the luciferase activities o f a 6.3-kb construct were 5- and 2-fold greater than the smaller 1.8-kb con struct, respectively. In contrast, the activity in non-intestinal cell line s (HepG2 and HeLa) was 2-4-fold lower than the smaller construct. In the re gion downstream from the 1.8-kb position, strong luciferase activities in L S174T and HepG2 cells were observed using a 201-bp construct. Interestingly , increased activity was almost completely suppressed in cells transfected with a 391-bp construct. Detailed analyses of this region revealed the exis tence of a 11-bp positive regulatory element(-181 to -170; ACCTCTTCCTG) and a 9-bp negative regulatory element (-208 to -200; ATTGACAGA) in addition t o the GCRE. All three elements were well conserved among human, rat and mou se ITF gene promoters. In addition, a mutant 1.8-kb construct in which the negative regulatory region was deleted yielded the same approximate lucifer ase activity as a 6.3-kb construct, suggesting binding of a goblet-cell-spe cific silencer inhibitor (SI) between -6.3 and -1.8 kb. The SI present in g oblet cells may block the silencers' binding to the pre-initiation complex and allow increased transcriptional activity driven by specific and non-spe cific enhancers. High-level expression of the mouse ITF gene specifically i n intestinal goblet cells may be achieved through the combined effects of t hese regulatory elements.