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.
Highlights
From the Gastrointestinal Unit and Department ofMedicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Gastrin Gene Regulation digested with Bglll (New England Biolabs) and transformed into the mismatch repair deficient muts- Escherichia coli strain [16], miniprep DNA was prepared via a modified alkaline lysis procedure (Qiagen), and mutants were selected by digestion again with Bglii and re-transformation into E. coli DH5-a cells
Prior deletional analysis demonstrated that 200 bp of 5'-flanking DNA of the human gastrin gene contained the regulatory elements that conferred most of the transcriptional activity in gastrinexpressing RIN 38A insulinoma cells [11, 12]
Summary
The general transactivator Sp1 recognized the downstream GGGCGGGG sequence, but Spl 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 /3TF-l. Pancreatic islet endocrine cells share common evolutionary and developmental origins with gut endocrine cells [1] This relationship is emphasized by the transient expression of hormones such as gastrin, peptide-YY, and secretin in fetal islets even though these peptides are expressed largely in the luminal gastrointestinal tract in the adult [2,3,4]. Nisms controlling gastrin gene transcription in islet cells represent an interesting example of developmental control of gene expression
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