Mutually Exclusive Interactions between Factors Binding to Adjacent Sp1 and AT-rich Elements Regulate Gastrin Gene Transcription in Insulinoma Cells

Abstract

The gastrin gene is transiently expressed in fetal pancreatic islets during islet neogenesis but then switched off after birth when islet cells become fully differentiated. Previous studies identified a cis-regulatory sequence between -109 and -75 in the human gastrin promoter which binds islet cell-specific activators and a nonspecific repressor and thus may act as a molecular switch. The present study identified another cis-regulatory sequence (-163ACACTAAATGAAAGGGCGGGGCAG-140) which bound two islet nuclear proteins in a mutually exclusive manner, as defined by gel shift competition, methylation interference, and DNase I foot-printing assays. The general transactivator Sp1 recognized the downstream GGGCGGGG sequence, but Sp1 binding was prevented when another islet factor bound to the adjacent AT-rich sequence (CTAAATGA). This gastrin AT-rich element is nearly identical to the binding site (ATAAATGA) for the islet-specific transcription factor beta TF-1. However, the gastrin AT-binding factor appeared to differ from beta TF-1 in its gel mobility shift pattern. Transfections of rat insulinoma cells revealed that mutations which blocked binding to the AT-rich element but allowed Sp1 binding up-regulated transcriptional activity. These results suggest that the gastrin AT-binding factor blocks transactivation by Sp1 and may have a role in the repression of gastrin transcription seen at the end of islet differentiation.

The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor– and MAP kinase–dependent interregulation

“…RasN17 suppressed both basal and ITF-induced hpS2-driven reporter activity. Suppression of Ras signaling by RasN17 was confirmed by the ability to block EGF-induced trans-activation of the human gastrin promoter (Figure 5), previously reported to be Ras dependent (30).Consistent with this data, cotransfection of wild-type c-Ha-Ras was able to strongly induce both basal and ITFinduced pS2 and SP reporter activity (data not shown).…”

“…The kinase-deficient EGF receptor (EGF-R) expression plasmid pRK5-HER653 (29) was a gift from M. Korc (University of California-Irvine, Irvine, California, USA). The human gastrin promoter fused to luciferase, containing Ras and EGF response elements (30), was a gift from D. Chung (Massachusetts General Hospital/ Harvard Medical School). The Renilla luciferase reporter vector pRL-TK was obtained from Promega Corp. Purified recombinant human SP and ITF, produced in yeast, have been described previously (9,31).…”

“…AGS cells were cotransfected with a human gastrin promoter-luciferase reporter plasmid (gas-luc) or the pS2 promoter luciferase construct with or without dominant-negative Ras (N17Ras). To confirm the effect of N17Ras, the effect of EGF (10 ng/mL) on Ras-dependent (30) activation was assessed. in this study but were present within the 820-bp and 1,100-bp SP and pS2 promoters, respectively.…”

Processing and proliferative effects of human progastrin in transgenic mice.

“…Studies based on transient transfections of human gastrin promoter constructs in cultured rat islet cell lines identified an islet-cell-specific enhancer (7)(8)(9). This enhancer sequence in the human gastrin promoter was located within 200 bp of the transcriptional initiation site.…”

Transcriptional Regulation of the Differentiation-linked Human K4 Promoter Is Dependent upon Esophageal-specific Nuclear Factors

“…Proteins-Additional EMSAs were performed utilizing EGTA to identify which factors may require Zn 2ϩ for DNA binding (31). These data revealed that complexes a and b, and to a lesser extent complex c, were attenuated (Fig.…”