Abstract
Somatostatin is a peptide synthesized in the pancreatic islets, nervous system, gastrointestinal tract, and thyroid gland. Factors that control islet cell-specific expression of the somatostatin gene were analyzed by expression of fusion genes consisting of 5' rat somatostatin gene sequences linked to coding sequences of the receptor genes, bacterial chloramphenicol acetyltransferase, and human growth hormone. Fusion genes containing 900 and 250 base pairs (bp) of 5'-flanking DNA were preferentially expressed at 5-10-fold higher levels in somatostatin-producing islet cell lines, as compared with islet cell lines that produced insulin and glucagon, and in three non-islet cell lines. A deletional mutation consisting of only 65 bp of 5'-flanking sequence of the rat somatostatin gene expressed in all islet cell lines but not in non-islet lines, indicating the existence of a negative-acting islet cell-specific element located between nucleotides -250 and -65. The 65-bp sequence contains the octameric cAMP-responsive enhancer (CRE) TGACGTCA (nucleotides -48 to -41). Fine mapping of sequences responsible for islet-specific expression by substitution of synthetic oligonucleotide cassettes revealed full retention of expression by deletion to nucleotides -48 and complete loss of expression at nucleotides -42 of the CRE. Substitution of the 9 bp adjacent 3' to the CRE of the somatostatin gene (nucleotides -40 to -32) with the corresponding sequence located 3' to the CRE of the glucagon gene abolished expression. By gel mobility shift and DNaseI footprinting analyses, proteins in extracts of islet cells bound to the 24 bp including the CRE and downstream adjacent 9 bp (nucleotides -58 to -35). An additional upstream region of DNA was protected from DNase I digestion (nucleotides -110 to -80). Proteins from non-islet cells bound to the region from nucleotides -58 to -35, but patterns of DNase I protection differed from those using proteins from islet cells. These observations indicate that several DNA-binding proteins interact with cis-acting elements located between 35 and 58 bp upstream of the transcriptional start site of the rat somatostatin gene to determine islet cell-specific gene expression. CRE-binding protein(s) is ubiquitous among phenotypically different cells, and expression of the somatostatin gene in non-somatostatin-producing islet cells appears to be inhibited by a negative-acting element located upstream of the CRE.
Highlights
Somatostatin is apeptidesynthesizedin thepan- protein(s) is ubiquitous among phenotypicalldyifferent creatic islets, nervous system, gastrointestinal tract, cells, and expressionof the somatostatingene in nonand thyroid gland
Expression of a Somatostatin FusionGene Is Preferentially Restricted to Somatostatin-producing Islet Cell Lines-The somatostatin fragment from nucleotides -900 to +54 was fused to either thecoding sequence for the bacterial enzyme chloramphenicol acetyltransferase or for human growth hormone (Fig. lA).The somatostatinfusion gene was introduced in the rat islet cell lines RIN 1027-B2 and RIN 1056A and the threenon-islet cell lines BHK, JEG, andHeLa
The expression of the SMS-GH fusion gene was expressed as a ratio of thymidine kinase (TK)-GH fusion gene expression
Summary
Gel Mobility Shift Assay-Nuclear extracts from the RIN 1027-B2 cell line were incubated with somatostatin fragment -71 to +54 (labeled on the coding strand as described above) in the presence of 1 pgof poly(d1-dC) in 6.7 mM Tris, pH 8.0, 10 mM EDTA, 3 mM sodium acetate. DNase I Footprinting Analyses-The somatostatin fragment -250 to +54 was end-labeled as described above and incubated on ice with either bovine serum albumin or nuclear extracts from the RIN 1027B2, INRI-G9, JEG-3, HepG2, HeLa, and PC-12 cell lines (50 pgof total protein) in a totvalolume of 30 p1 with 1pg of poly(d1-dC)(final buffer concentration 20 mM Hepes, pH 7.9, 75 mM KCl, 5 mM MgC12, 1 mM EDTA, 10% (v/v) glycerol, and 2% polyvinyl alcohol). Nucleotide position and sequence were determined by parallel running of Maxam-Gilbert sequencing ladders (30)
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