The –928 G/C and –362 G/C Single-Nucleotide Polymorphisms in the Promoter of MCP-1: Increased Transcriptional Activity and Novel Binding Sites

Abstract

Background: The –928 guanine (G)/cytosine (C) and –362 G/C single-nucleotide polymorphisms (SNPs) in the proximal promoter region of the monocyte chemoattractant protein 1 gene have been associated with an increased risk for intimal medial thickness and carotid atherosclerosis, respectively. We characterized the transcriptional activity of these two SNPs in vitro and identified transcription factors that bind to them. Methods: The proximal promoter region spanning bases –2746 to +440 was sequenced in subjects with carotid atherosclerosis. Two SNPs consisting of C-for-G substitution at bases –928 and –362 were characterized. Each observed haplotype was inserted into a luciferase reporter and transfected into mammalian cells. Results: Stimulation with 12-O-tetradecanoylphorbol 13-acetate increased transcriptional activity of the –928 C plasmid (p = 0.005). The basal transcription activities of the plasmids containing –928 C and –362 C were also increased (p < 0.001 and p < 0.0001, respectively). Electrophoretic mobility shift assay (EMSA) and DNA footprinting identified an Ah receptor nuclear translocator protein (ARNT) binding site at the –928 C SNP. EMSA data indicated signal transducers and activators of transcription (STAT) binding at the –362 G SNP. A nuclear binding protein, poly(ADP-ribose) polymerase (PARP) 1, was purified from the –928 C SNP site and identified by mass spectroscopy. Conclusion: The –928 C SNP and the –362 C SNP are associated with increased transcriptional activity in vitro, but the –362 G site is not. The –928 C SNP is associated with PARP-1 and ARNT binding, and the –362 G is associated with a STAT binding site.