title: A common polymorphism in the 5' region of the human protein c gene binds USF1.
abstract: BACKGROUND
  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.
  
  
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Critical Care Research Laboratories, Institute for Heart + Lung Health at St. Paul's Hospital, University of British Columbia, Vancouver, Canada.
authors.forename: Katherine R
authors.initials: KR
authors.lastname: Thain
authors.forename: Taka-Aki
authors.initials: TA
authors.lastname: Nakada
authors.forename: John H
authors.initials: JH
authors.lastname: Boyd
authors.forename: James A
authors.initials: JA
authors.lastname: Russell
authors.forename: Keith R
authors.initials: KR
authors.lastname: Walley
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011485:Protein Binding; D011486:Protein C / Q000235:genetics* / Q000378:metabolism; D051720:Upstream Stimulatory Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D011486:Protein C; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors