Key result
A methylation-dependent USF binding site influences basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter, with significant haplotype-dependent differences (e.g., AC > CC, P<0.001).
p-value: p=<0.001
The study identifies a methylation-dependent USF binding site that influences the basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter in a haplotype-dependent manner.
Should not alter clinical care; hypothesis-generating for haplotype-specific IGFBP3 promoter regulation in metabolic contexts.
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.
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Paquette et al. (2007) studied this question. Insulin treatment and IGFBP3 promoter haplotypes vs. Unstimulated cells and alternative haplotypes was evaluated on Transcriptional activity (luciferase activity) (p=<0.001). A methylation-dependent USF binding site influences basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter, with significant haplotype-dependent differences (e.g., AC > CC, P<0.001).
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