Effects of Intron Conversion in the Human CYP11B2 Gene on its Transcription and Blood Pressure Regulation in Transgenic Mice

The human cytochrome P450 family 11 subfamily B member 2 (hCYP11B2) gene encodes aldosterone synthase, the rate-limiting enzyme in the biosynthesis of aldosterone. In some humans, hCYP11B2 undergoes a unique intron conversion whose function is largely unclear. The intron conversion is formed by a replacement of the segment of DNA within intron 2 of hCYP11B2 with the corresponding region of the hCYP11B1 gene. We show here that the intron conversion is located in an open chromatin form and binds more strongly to the transcriptional regulators histone acetyltransferase P300 (p300), NFκB, and CCAAT enhancer-binding protein α (CEBPα). Reporter constructs containing the intron conversion had increased promoter activity on transient transfection in H295R cells compared with WT intron 2. We generated humanized transgenic (TG) mice containing all the introns, exons, and 5\u27- and 3\u27-flanking regions of the hCYP11B2 gene containing either the intron conversion or WT intron 2. We found that TG mice containing the intron conversion have (a) increased plasma aldosterone levels, (b) increased hCYP11B2 mRNA and protein levels, and (c) increased blood pressure compared with TG mice containing WT intron 2. Results of a ChIP assay showed that chromatin obtained from the adrenals of TG mice containing the intron conversion binds more strongly to p300, NFκB, and CEBPα than to WT intron 2. These results uncover a functional role of intron conversion in hCYP11B2 and suggest a new paradigm in blood pressure regulation

Metabolic Syndrome Induces Over Expression of the Human AT1R: A Haplotype-Dependent Effect With Implications on Cardio-Renal Function

BACKGROUND: The transcriptional regulation of the human angiotensin receptor subtype 1 (AT1R) gene in pathophysiologies, like the metabolic syndrome, is poorly understood. The human AT1R gene has polymorphisms in its promoter that can be arranged in 2 haplotypes. Variants -810T, -713T, -214A, and -153A always occur together (Hap-I) and variants -810A, -713G, -214C, and -153G form Hap-II. We have hypothesized that high fat diet will alter cellular transcriptional milieu and increase hAT1R gene expression in a haplotype-dependent manner. This will set up an AT1R-mediated feed-forward loop promoting inflammation, oxidative stress, and hypertension in Hap-I mice. METHOD: Since Hap-I of the human AT1R gene is associated with hypertension in Caucasians, we generated transgenic (TG) mice with Hap-I and Hap-II and studied the physiological significance of high fat diet (HFD) on haplotype specific gene expression. Animals were fed with HFD for 20 weeks followed by blood pressure (BP) analysis and collection of their tissues for molecular and biochemical studies. RESULTS: After HFD treatment, as compared to Hap-II, TG mice with Hap-I show increased expression of hAT1R gene and higher BP; suppression of antioxidant defenses (HO1, SOD1) and increased expression of IL-6, TNFalpha, IL-1beta, NOX1. In vivo ChIP assay has shown that transcription factors CEBPbeta, STAT3, and USF bind more strongly to the chromatin obtained from Hap-I TG mice. CONCLUSIONS: Taken together, our results suggest, that after HFD treatment, as compared to Hap-II, the TG mice with Hap-I overexpress the AT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their BP

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