Abstract 162: Increased Allele-Specific Expression of Angiotensinogen Derived From the -20C vs. -20A In Subcutaneous Adipose Tissue From Anonymous Human Donors
The mechanism by which single nucleotide polymorphisms (SNP) regulate angiotensinogen (AGT) expression in humans has been extensively studied but remains unclear. The E box (CACGTG) at position -20 in the human AGT promoter binds USF1 and USF2 in vitro. Recently, we published that USF1 and USF2 are both essential for human AGT expression in double transgenic mice carrying a -20C SNP in human AGT and a gene trap USF1 knockdown. Therefore, we hypothesize that selective binding of USF1/2 to -20C versus -20A (the rs5050 SNP) in the promoter region underlies the enhanced expression of AGT in humans containing the rs5050 SNP. We genotyped subcutaneous adipose tissue from 89 anonymous human donors for both the A-20C SNP and a coding region SNP known to be in linkage disequilibrium with the A-20C (C620T; rs4762). Based on sequencing and TOPO TA cloning, 22% of the samples were heterozygous for A-20C and linkage disequilibrium between -20A with 620C and -20C with 620T was confirmed in 80% of the samples. AGT and USF2 mRNA was elevated significantly (2.0±0.3 and 2.2±0.4 fold, n=8, respectively) in the male -20AC compared to the -20AA group, whereas expression of fatty acid synthase, a known USF target gene, was unchanged. These changes in expression were not observed in females, suggesting sexual dimorphism in the regulation of both USF1/2 and AGT. We developed and validated a quantitative PCR assay using Taqman probes selective for the mRNA separately corresponding to 620T and 620C. We used this assay on RNA derived from 16 heterozygous (-20AC and 620CT) subcutaneous adipose samples. Expression derived from the -20C promoter (620T mRNA) was 4.1±0.6 fold higher compared to the -20A promoter (620C mRNA) (n=16). We have also developed a quantitative allele-specific assay to measure USF1/2 binding to -20AC for use in ChIP. We conclude that USF1/2 and the -20C SNP play physiological roles in AGT regulation in human adipose tissue which we hypothesize is due to increased USF recruitment at -20C.
- © 2012 by American Heart Association, Inc.