The -928 G/C and -362 G/C single-nucleotide polymorphisms in the promoter of MCP-1: Increased transcriptional activity and novel binding sites.
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.


CONCLUSIONS
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.
Nyquist
Paul
P
Division of Neurosciences Critical Care and Departments of Neurology, Anesthesiology-Critical Care Medicine and Neurosurgery, Johns Hopkins University School of Medicine, Bethesda, MD, USA. pnyquis1@jhmi.edu
Zhang
Jianhua
J
De Graba
Thomas J
TJ
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism; D001665:Binding Sites; D016893:Carotid Stenosis / Q000235:genetics* / Q000378:metabolism; D018932:Chemokine CCL2 / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D017930:Genes, Reporter; D020022:Genetic Predisposition to Disease; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012307:Risk Factors; D050791:STAT Transcription Factors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
C497396:ARNT protein, human; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D050791:STAT Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D013755:Tetradecanoylphorbol Acetate