Identification of upstream stimulatory factor binding sites in the human IGFBP3 promoter and potential implication of adjacent single-nucleotide polymorphisms and responsiveness to insulin.
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.
Paquette
Jean
J
Endocrine Service, Department of Pediatrics, Ste-Justine Hospital, 3175 Côte Ste-Catherine, Montreal, Quebec, Canada.
Bessette
Benoit
B
Ledru
Elisabeth
E
Deal
Cheri
C
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D006239:Haplotypes; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D051720:Upstream Stimulatory Factors / Q000378:metabolism*