A Polymorphism in Intron I of the Human Angiotensinogen Gene (hAGT) Affects Binding by HNF3 and hAGT Expression and Increases Blood Pressure in Mice

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin-II. Genome-wide association studies (GWAS) have shown that two A/G polymorphisms (rs2493134 and rs2004776) located at +507 and +1164 in intron I of human AGT (hAGT) gene are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with hepatocyte nuclear factor 3 (HNF3)-binding site than does +1164G. Here, we found that (a) an oligonucleotide containing +1164A binds HNF3beta more strongly than does +1164G, and (b) Hap I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the HPRT locus, we generated transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor (GR), CCAAT enhancer-binding protein beta (C/EBPbeta), and HNF3beta bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure

Age Related Expression of Human AT1R Variants and Associated Renal Dysfunction in Transgenic Mice

Background: The contribution of single nucleotide polymorphisms in transcriptional regulation of the human angiotensin receptor type I (hAT1R) gene in age related chronic pathologies such as hypertension and associated renal disorders is not well known. The hAT1R gene has SNPs in its promoter that forms two haplotypes (Hap), Hap-I and Hap-II. Hap-I of AT1R gene is associated with hypertension in Caucasians. We have hypothesized here that age will alter the transcriptional environment of the cell and will regulate the expression of hAT1R gene in a haplotype-dependent manner. This could likely make subjects with Hap-I increasingly susceptible to age associated, AT1R mediated complications. Method: We generated transgenic (TG) mice with Hap-I and Hap-II. Adults (10-12 weeks) and aged (20-24 months) TG male mice containing either Hap-I or Hap-II were divided into four groups to study (a) the age associated and haplotype specific transcriptional regulation of hAT1R gene, and (b) their physiological relevance. Results: In aged animals, TG mice with Hap-I show increased expression of hAT1R and higher blood pressure; suppression of antioxidant defenses (HO1, SOD1) and antiaging molecules (ATRAP, Klotho, Sirt3); increased expression of pro-inflammatory markers (IL-6, TNF, CRP, NOX1); and increased insulin resistance. In vivo ChIP assay shows stronger binding of transcription factor USF2 to the chromatin of Hap-I mice. Conclusion: Our results suggest, that in aged animals, as compared to Hap-II, the TG mice with Hap-I over-express hAT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their blood pressure and associated renal disorders