Differential regulation of the human insulin gene transcription by GG1 and GG2 elements with GG- and C1-binding factors.
Using a human growth hormone reporter system, the introduced mutations in GG1 alone or both GG elements of GG1 and GG2 in the human insulin promoter abolished 94 or 96% of the beta-cell-specific transcriptional activity in a pancreatic islet beta-cell line of MIN6, while the mutations in GG2 or its total deletion abolished 85 or 86% of the transcriptional activity. When linked to the thymidine kinase promoter, mutations in GG1 or both GG elements abolished 74% of the transcriptional activity in MIN6 cells, while the mutations in GG2 or its total deletion abolished 55 or 54%. In the electrophoretic mobility shift assay (EMSA), one nuclear factor was shown to interact with two GG elements, and another C1-binding factor with GG1 and C1. The differential effects of deletions or selective mutations in the GG2 or GG1 sequence in the oligonucleotide probes on the binding activity of GG- or C1-binding factors in EMSA proved the requirement of both GG1 and GG2 or both GG1 and C1, respectively, for the transaction of these two factors. The molecular size of the GG-binding factor was estimated about 30 kDa. Based on these, we conclude that two GG elements contribute, with GG1 more critically than GG2, to the beta-cell-specific transcription of the human insulin gene through transaction with the GG- and C1-binding factors.
Tomonari
A
A
Otsuka Department of Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Yoshimoto
K
K
Mizusawa
N
N
Iwahana
H
H
Itakura
M
M
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects*; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects
D004268:DNA-Binding Proteins; D007328:Insulin; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D019382:Human Growth Hormone; D013937:Thymidine Kinase