Somatostatin gene expression in pancreatic islet cells is directed by cell-specific DNA control elements and DNA-binding proteins.
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.
Powers
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AC
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston.
Tedeschi
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F
Wright
K E
KE
Chan
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JS
Habener
J F
JF
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D000242:Cyclic AMP / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D005796:Genes; D005809:Genes, Regulator; D007515:Islets of Langerhans / Q000378:metabolism*; D051381:Rats; D013004:Somatostatin / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013004:Somatostatin; D000242:Cyclic AMP