The role of the proximal CTAAT-box of the rat glucokinase upstream promoter in transcriptional control in insulin-producing cells.
Sequence analysis of the 5'flanking region of the beta-cell specific transcription unit of the rat glucokinase gene (r beta GK) revealed the presence of sequence motifs very similar to the IEB-(Far)-box and a CT-motif which play a crucial role in transcriptional control of insulin genes. 5'deletional analysis of the r beta GK proximal promoter element (localized between nucleotides -278 and -49) as well as site directed mutagenesis showed that both motifs are mutationally sensitive and contribute to transcriptional control in HIT M2.2.2 cells. The combination of the IEB-(Far)-like motif with the CT-box was unable to form a "mini-enhancer" similar to the Far-FLAT-element of the rat insulin I gene promoter but rather functions as a beta-cell specific control element in r beta GK expression. Electrophoretic mobility shift assays (EMSAs) and competition studies using oligonucleotides containing CT-motifs of rat insulin genes promoters, human insulin gene promoter, and rat amylin gene promoter showed similar binding patterns with nuclear extracts isolated from insulin-producing cell lines. These studies indicate that CT-motifs of rat glucokinase, insulin, and amylin gene promoters may bind similar--probably identical--nuclear factor(s) and may play a central role in the coordinated expression of these genes in insulin-producing cells.
Leibiger
B
B
Institut für Biochemie, Klinikum, Ernst-Moritz-Arndt-Universität Greifswald, Germany.
Walther
R
R
Leibiger
I B
IB
D000682:Amyloid / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004742:Enhancer Elements, Genetic; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D058228:Islet Amyloid Polypeptide; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic
D000682:Amyloid; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D005941:Glucokinase