1. Identification of upstream stimulatory factor binding sites in the human IGFBP3 promoter and potential implication of adjacent single-nucleotide polymorphisms and responsiveness to insulin.
- Author
-
Paquette J, Bessette B, Ledru E, and Deal C
- Subjects
- Base Sequence, Binding Sites genetics, Cell Line, Tumor, Chromatin Immunoprecipitation, DNA Methylation, Electrophoretic Mobility Shift Assay, Haplotypes, Humans, Insulin-Like Growth Factor Binding Protein 3, Luciferases genetics, Luciferases metabolism, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transcription, Genetic drug effects, Insulin pharmacology, Insulin-Like Growth Factor Binding Proteins genetics, Polymorphism, Single Nucleotide, Promoter Regions, Genetic genetics, Upstream Stimulatory Factors metabolism
- Abstract
The actions of IGFs are regulated at various levels. One mechanism involves binding to IGF-binding protein-3 (IGFBP-3) for transport, thus governing bioavailability. IGFBP3 transcription is modulated by many hormones and agents that stimulate or inhibit growth. We have previously shown in pediatric and adult cohorts a correlation between IGFBP-3 serum levels and two single-nucleotide polymorphisms (SNPs) located within the minimal promoter (-202 A/C and -185 C/T). Functionality of these SNPs was further explored in hepatic adenocarcinoma-derived SK-HEP-1 cells using transient transfections of luciferase constructs driven by different haplotypes of the IGFBP3 promoter. Basal luciferase activity revealed a significant haplotype-dependent transcriptional activity (at nucleotides -202 and -185, AC > CC, P < 0.001; AC > CT, P < 0.001; AC > AT, P < 0.001). Insulin treatment produced a similar haplotype dependence of luciferase activity (AC > CC, P = 0.002; AC > CT, P < 0.001; AC > AT, P = 0.011). However, induction ratios (insulin/control) for CC and AT were significantly higher compared with AC and CT (CC > AC, P = 0.03; CC > CT, P = 0.03; AT > AC, P = 0.03; AT > CT, P = 0.04). Gel retardation assays were used to identify upstream stimulatory factor (USF-1 and USF-2) methylation-dependent binding to E-box motifs located between the SNPs. Mutation of the USF binding site resulted in a significant loss of insulin stimulation of luciferase activity in the transfection assay. Chromatin immunoprecipitation with anti-USF-1/-2 showed an enrichment of IGFBP3 promoter in insulin-treated cells compared with unstimulated cells. Bisulfite sequencing of genomic DNA revealed that CpG methylation in the region of USF binding was haplotype dependent. In summary, we report a methylation-dependent USF binding site influencing the basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter.
- Published
- 2007
- Full Text
- View/download PDF