GGAAAT motifs play a major role in transcriptional activity of the human insulin gene in a pancreatic islet beta-cell line MIN6.
The insulin gene is specifically expressed in pancreatic islet beta cells. Various cis-acting DNA elements in the 5'-flanking region of the human insulin gene were examined for their contribution to the transcriptional activity using sensitive human growth hormone (hGH) reporter plasmids. The hGH constructs, having successively deleted human insulin promoter sequences, were transfected to a pancreatic islet beta-cell line MIN6. The deletion of two GGAAAT (GG) motifs, GG2 at -145 to -140 bp and GG1 at -134 to -129 bp, decreased the transcriptional activity to 6.5% of that of the promoter sequence from -156 to +1 bp. The selective mutations in both GG motifs also decreased the transcriptional activity to 5.5%. One-base mutations of GG2 and GG1 decreased the transcriptional activity to 82 and 11%, respectively. The two-base mutations between GG2 and GG1 affected the transcriptional activity more strongly than those just outside the GG motifs. A single set of GG motifs in the upstream of thymidine kinase promoter increased the transcriptional activity to 216% compared to that of thymidine kinase promoter alone in MIN6 cells. With an electrophoretic mobility shift assay (EMSA), a nuclear factor in MIN6 cells was shown to bind the DNA fragments containing two GG motifs. This factor did not bind to another GGAAAT-like sequence at -313 to -305 bp in the human insulin gene. These results suggested that the GG motifs contributed to the cell-specific transcription of the human insulin gene in association with the binding of the sequence-specific nuclear factor.
Tomonari
A
A
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Yoshimoto
K
K
Tanaka
M
M
Iwahana
H
H
Miyazaki
J
J
Itakura
M
M
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics*; D007515:Islets of Langerhans / Q000166:cytology / Q000502:physiology*; D051379:Mice; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection
D015342:DNA Probes; D007328:Insulin; D019382:Human Growth Hormone