A common polymorphism in the 5' region of the human protein c gene binds USF1.

TitleA common polymorphism in the 5' region of the human protein c gene binds USF1.
Publication TypeJournal Article
Year of Publication2012
AuthorsThain, KR, Nakada, T-aki, Boyd, JH, Russell, JA, Walley, KR
JournalThromb Res
Volume130
Issue3
Pagination451-7
Date Published2012 Sep
ISSN1879-2472
Keywords5' Flanking Region, Genetic Variation, Hep G2 Cells, Humans, Polymorphism, Single Nucleotide, Protein Binding, Protein C, Upstream Stimulatory Factors
Abstract

INTRODUCTION: Genetic variation in the Protein C gene (PROC) is associated with altered risk of adverse outcome for a number of diseases. Common single nucleotide polymorphisms (SNPs) in the promoter region and the adjacent 5' region of PROC are associated with Protein C expression. We tested the hypothesis that common SNPs (minor allele frequency >10%) between the frequently studied promoter SNPs -1654 (rs1799808) and -1641 (rs1799809), and the end of PROC intron 2 alter nuclear transcription factor binding.MATERIALS AND METHODS: We used electrophoretic mobility shift assays with 25-mer oligonucleotides centered on each of the 10 SNPs assessed in this potential regulatory region of the Protein C gene to test for differential binding to nuclear factors isolated from Hep-G2 cells.RESULTS: We found that the G-allele oligo of the intron 2 SNP rs2069915[G/A] bound nuclear factors more avidly than the A-allele (p=1.9 × 10(-9), n=24). Similarly, we found that the C-allele oligo of the intron 2 SNP rs2069916[C/T] bound nuclear factors more avidly than the T-allele, (p=3.7 × 10(-6), n=19). Cold competition and supershift assays suggested that the protein differentially binding to the C-allele of rs2069916 was USF1. Notably, we observed minimal nuclear factor binding to oligos containing haplotypes of the previously reported -1654 and -1641 SNPs. Luciferase reporter assays that showed the A-T haplotype of rs2069915 and rs2069916 drives transcription significantly more than the C-G haplotype (t-test, P=0.015, n=12).CONCLUSION: Differential transcription factor binding occurs for common SNPs in the 5' intronic regions of PROC which may contribute to PROC regulation and reported PROC SNP - phenotype associations.

DOI10.1016/j.thromres.2012.02.045
Alternate JournalThromb. Res.
PubMed ID22425321
Grant List / / Canadian Institutes of Health Research / Canada