title: Definition of the carbohydrate response element of the rat S14 gene. Evidence for a common factor required for carbohydrate regulation of hepatic genes.
abstract: The 5'-flanking region of the S14 gene from -4316 to +18 contains regulatory sequences responsible for activation of promoter activity in response to elevated carbohydrate metabolism in primary hepatocytes. To map these sequences, a series of constructs containing various internal deletions of the S14 5'-flanking sequence were assayed in primary hepatocytes. The region from -1601 to -1395 was found to be essential for this response. Comparison of the sequence of this S14 region to a region of the L-type pyruvate kinase gene that has been shown to mediate carbohydrate regulation (Thompson, K. S., and Towle, H. C. (1991) J. Biol. Chem. 266, 8679-8682) revealed a segment with 9 out of 10 identity. In both cases, this conserved sequence aligned with a DNase I footprint formed with hepatic nuclear extract. Oligonucleotides (approximately 30 base pairs) from either S14 or pyruvate kinase genes containing the conserved element bound to a hepatic nuclear factor(s) that gave identical complexes by mobility shift assay. Furthermore, these two oligonucleotides cross-competed for binding to the nuclear factor(s), suggesting that a common factor(s) binds to this conserved element. Reinsertion of the S14 oligonucleotide into an unresponsive S14 promoter construct restored the carbohydrate control. Moreover, this oligonucleotide could confer a glucose response when fused to a heterologous promoter. Thus, the S14 segment from -1457 to -1428 is a carbohydrate response element essential for the binding of nuclear factor(s) regulated by increased carbohydrate metabolism. This factor(s) may be common to the carbohydrate regulation of the S14 and pyruvate kinase genes.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis 55455.
authors.forename: H M
authors.initials: HM
authors.lastname: Shih
authors.forename: H C
authors.initials: HC
authors.lastname: Towle
mesh_terms: D000818:Animals; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D002855:Chromatography, Thin Layer; D016172:DNA Fingerprinting; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D009841:Oligonucleotides; D015500:Chloramphenicol O-Acetyltransferase; D011770:Pyruvate Kinase