Human angiotensinogen +11525 C/A polymorphism modulates its gene expression through microRNA binding

Hypertension is a serious risk factor for cardiovascular disease. Like other complex disease, hypertension is caused by a combination of genetic and environmental factors. The renin-angiotensin system plays an important role in the regulation of blood pressure. Angiotensinogen (AGT) gene is associated with essential hypertension in Caucasians, Japanese, and Asian-Indian subjects. AGT gene may also be associated with cardiac hypertrophy, coronary atherosclerosis, and microangiopathy related cerebral damage. Human AGT gene has a C/A polymorphism at nucleoside 11525 (rs7079) that is located in the 3′-untranslated region (3′-UTR) and is modestly associated with increased blood pressure. We show here that miR-31 and miR-584 bind strongly to the hAGT 3′-UTR containing 11525C allele compared with 11525A allele. We also show that transfection of miR-31 and miR-584 downregulates the hAGT mRNA and protein levels in human liver cells. These studies may provide new therapeutic approach to reduce hypertension. </jats:p

Abstract 129: The Role of Intron-2 Conversion Polymorphism in the Regulation of Human Aldosterone Synthase Gene Expression

The hCYP11B2 gene encodes aldosterone synthase, the rate-limiting enzyme in the biosynthesis of aldosterone. Inappropriate excess levels of aldosterone induces positive sodium balance and predisposes to hypertension and other cardiovascular problems. hCYP11B2 has a T/C polymorphism located at -344 in its promoter region. This variant is in close linkage disequilibrium with a complex hCYP11B2 Intron 2 conversion polymorphism (IC), which replaces a segment of DNA within intron 2 with the corresponding region of hCYP11B1. Epidemiological studies have suggested that -344 T/C and the IC polymorphism associating with hypertension. The IC polymorphism always occur when the promoter contains -344T variant. In the present study we have for the first time sequenced the full length hCYP11B2 intron 2 from randomly selected hypertensive samples from the Caucasian cohort and identified the segment of hCYP11B2 replaced by that of hCYP11B1 Intron2. It is interesting to note that in these genomic DNA samples there is no change in the hCYP11B1 intron2 sequence. In silico transcription factor binding site predictions indicate that there are several transcription factors which can bind more strongly to the hCYP11B2 IC region as compared to wild type intron2 (wt). We have cloned the IC polymorphism and the wt intron nucleotide sequences in front of the 1kb hCYP11B2 promoter containing -344T. These reporter constructs showed differential transcriptional activation. Basal promoter activity of IC intron was increased by 1.94 fold (p&lt;0.05) compared to that of wt intron2. Further co-transfection studies of these reporter constructs along with transcription factors HNF3B and NFkB showed significantly increased relative luciferase activity of 2.9 and 3.26 fold (p&lt;0.05) in IC construct as compared to wt intron respectively. These results strongly suggest that IC in hCYP11B2 has strong regulatory elements as compared to the wt intron leading to enhanced transcriptional activity and thus increased hCYP11B2 gene expression. This would increase tissue or plasma aldosterone levels in human subjects containing IC polymorphism. The long term consequence of altered regulation of aldosterone production can lead to an increase in blood pressure and cardiovascular complications. </jats:p

Abstract 048: Epigenetic Regulation of the Human Angiotensinogen Gene: Role of Individual SNPs and Implication for Hypertension

Hypertension is a complex disease caused by a combination of genetic and environmental factors. Renin angiotensin system (RAS) dysfunction is a frequent accompaniment of essential hypertension. In this regard, polymorphisms in the angiotensinogen (AGT) gene increase RAS activity and cause blood pressure elevation. We have identified two distinct haplotypes of the human AGT gene constituted by a group of four SNPs in linkage disequilibrium. Variants -1670A, -1562C, and -1561T always occur with -6A and form the haplotype-I (Hap-I), while variants -1670G, -1562G, and -1561G always occur with -6G and constitute haplotype-II (Hap-II). We hypothesized that these SNPs, when present together as Hap-I or Hap-II in transgenic mice (TG) may lead to haplotype-dependent DNA methylation of CpG sites in the promoter of the hAGT gene. Methylation patterns alter gene transcription at an epigenetic level and this, in turn, could be dependent on individual polymorphisms. hAGT promoter CpG sites in the kidney are more methylated as compared to liver and fat. In the Kidney, the hAGT promoter CpG sites are methylated at -460, -434, -386, -346, -282, -261, -245, -229, -218, -185, -144, -18, -11, -7, +10, +42, +65 in Hap-II whereas, in Hap-I the promoter DNA methylation is observed at -460, -434, -401, -386 positions. Further, in both liver and fat the CpG sites were methylated at -11, +42, +65 positions in Hap-II TG mice, whereas in the Hap-I there were no methylation sites detected. The hAGT gene mRNA levels from these tissues were quantitated by qPCR and were 2.1 (kidney), 2.5 (liver) and 4.28 (fat) fold higher in haplotype-I vs. haplotype-II (p&lt;0.05). Our results indicate that, in Hap-II TG mice liver, kidney and fat tissues having more CpG sites methylated in the promoter of the hAGT, and thus having less gene expression. On the other hand, Hap-I has either less or no methylation of CpG sites in the hAGT promoter and hence high gene expression. Thus, we show here for the first time that SNP blocks in the hAGT gene alter its methylation pattern in a tissue-dependent manner. This shifts the paradigm in favor of an interdependent system where epigenetic regulation is reliant on the gene haplotypes; together, these regulate protein expression and associated physiological outcomes.</jats:p

Effect of Dietary Salt Excess on DNA Methylation and Transcriptional Regulation of Human Angiotensinogen Gene Expression

BACKGROUND: Hypertension is caused by a combination of genetic and environmental factors. Angiotensinogen (AGT) is a component of renin-angiotensin-aldosterone system, which regulates blood pressure. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are linked to hypertension. AGT polymorphisms result in two haplotypes, Hap-I a pro-hypertensive, whereas Hap-II is normotensive. Previous studies support the role of epigenetics in blood pressure regulation. In this study, we generated transgenic mice (TG) with hAGT containing Hap-I and Hap-II variants to investigate the effect of high salt diet (HSD) on epigenetics and transcriptional regulation. METHODS: We treated Hap-I and Hap-II TG mice with 4% HSD and identified DNA methylation patterns. We measured hAGT mRNA and protein by qPCR and immunoblot, respectively. Chromatin immunoprecipitation assay and RNA sequencing were performed. RESULTS: hAGT gene expression is increased by HSD in both Hap-I and Hap-II TG mice. In the liver and kidney, we observed significantly higher DNA demethylation (less CpG\u27s) and stronger binding of transcription factors in the promoter of Hap-I TG mice as compared to Hap-II post HSD. RNA-Seq identified differentially expressed genes, novel target genes, canonical pathways, and upstream regulators associated with hypertension. CONCLUSIONS: Our findings identified a novel high salt-sensitive risk haplotype, novel CpG sites and DNA methylation patterns, potential gene targets, and pathways implicated in hypertension. Combining epigenetic and transcriptional analysis allows for a more holistic understanding of the regulatory mechanisms that govern the hAGT gene

Abstract 239: MicroRNAs hsa-miR-584 and hsa-miR-31 Regulate Expression of Human Angiotensnogen Gene

Background: Hypertension is a risk factor for stroke, myocardial infarction, and congestive heart failure. A single nucleotide polymorphism (SNP) (rs7079) in 3’UTR of human angiotensinogen (hAGT) gene is associated with elevated blood pressure. We have used TargetScan V6.0, miRanda miRNA prediction algorithms and found that two microRNAs hsa-miR-584 and hsa-miR-31 may bind differentially to rs7079 and alter the expression of hAGT gene. METHODS: The effect of hsa-miR-584 and hsa-miR-31 miRNAs on endogenous AGT expression levels in Hep3B cells were studied by transfection of individual miRNA mimics followed by quantitative real time PCR (Q-RT-PCR) of the hAGT gene. In addition, the 600 bp 3’UTR of hAGT gene containing either rs7079C or rs7079A was PCR amplified and cloned in to the multiple cloning site of pMIR-REPORT™ miRNA Expression Vector containing luciferase gene. These reporter constructs were then co-transfected with either miRNA mimics or inhibitors to study the effect of miRNAs on luciferase activity in Hep3B cells since these cells express hAGT gene. These experiments were also performed in HEK293 cells which do not express the hAGT gene. Results: Q-RT-PCR showed that hsa-miR-584 and hsa-miR-31 mimics at 50nM concentration reduced endogenous hAGT mRNA levels by 38% and 30% respectively compared to the mock (without any microRNA) or negative control (which does not have any binding site for eukaryotic 3’UTRs) in Hep3B cells. Furthermore hsa-miR-584 and hsa-miR-31 showed 40% and 25% reduced luciferase activity of the construct containing rs7079 C allele. On the other hand these miRNAs did not affect the luciferase activity in the presence of rs7079 A. When dose dependent anti miRNA inhibitors were transfected along with miRNA mimics, these mimics abolished the microRNA induced down-regulation of luciferase activity. Conclusion: hsa-miR-584 and hsa-miR-31 miRNAs bind strongly to the hAGT 3’UTR containing rs7079 C allele as compared to rs7079 A allele and may down regulate the expression of AGT gene containing rs7079 C allele. This may be one of the possible mechanism involved in association of rs7079 A allele with human hypertension. </jats:p

Abstract 101: The Role of MicroRNAs in the Regulation of Human Aldosterone Synthase Gene Expression

The human aldosterone synthase ( hCYP11B2 ) gene encodes a key enzyme for the biosynthesis of aldosterone which is part of the renin-angiotensin-aldosterone system. Molecular variants in this gene have been associated with essential hypertension. MicroRNAs (miRNAs) are a family of small, noncoding RNAs that bind to the 3’-untranslated region (3’UTR) of the gene and regulate its expression. Our main objective in this study was to investigate whether miRNAs bind at the polymorphic sites present in the 3’UTR of hCYP11B2 gene and modulate its expression. Previous studies have suggested that -344T/C (rs1799998) polymorphism in the hCYP11B2 promoter is associated with hypertension. Here, we compared nucleotide sequences of the promoter and 3’UTR of hCYP11B2 gene from hypertensive and normotensive subjects and found that rs1799998 is in linkage disequilibrium with 735G/A (rs28491316). Thus, variant -344T almost always occurs with variant 735G; whereas variant -344C almost always occurs with variant 735A. Bioinformatic algorithms of the full-length 3’UTR for miRNAs revealed that hsa-miR-766 was possibly binding at the 735G allele. To examine if this miRNA regulates hCYP11B2 gene expression, reporter constructs were made where luciferase gene was attached to 1.4 kb of hCYP11B2 3’UTR containing either 735G, or 735A and co-transfected into HEK293 cells with either pre-miR-766 or anti-pre-miR-766. Our results showed that pre-miR-766 reduced luciferase activity in a dose dependent manner in the presence of 735G allele. There was a significant decrease (65+2%) in the luciferase activity with pre-miR-766 (50 nM) in the presence of 735G allele, whereas it was insignificant (4+5%) in the presence of 735A allele. In H295R cells that express hCYP11B2 gene, pre-miR-766 (50nM) reduced luciferase activity by 81+1% in the presence of 735G allele. We also showed that transfection of pre-mir-766 reduced hCYP11B2 mRNA level by about 23.6% in H295R cells. In corroboration with these results, anti-pre-miR-766 did not decrease luciferase activity in presence of either allele in both cell cultures. Our results suggest that hCYP11B2 gene expression may be regulated by differential binding of miR-766 at 735G/A polymorphism present in its 3’UTR. </jats:p

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

Abstract 021: Haplotype-Dependent Differential Regulation Of The Human AT1R Gene Is Exacerbated By Age: Effects On Tissue Inflammatory And Redox Milieu

Age-associated inflammation and redox imbalance underlie etiopathogenesis of cardiovascular-renal diseases, including hypertension and end organ damage. Angiotensin II (Ang II), via activation of the AT1R, contributes to the development and progression of these pathophysiologies. We have identified two haplotype blocks of single nucleotide polymorphisms (SNPs) in the hAT 1 R gene: haplotype II (Hap II: -810A, -713G, -214C, -153G) and I (Hap I: -810T, -713T, -214A, -153A). In clinical studies, Hap I is linked to human hypertension. This study examines haplotype-dependent and age-associated transcriptional regulation of the hAT1R gene. In this regard, we have engineered transgenic (TG) mice with either haplotype of the hAT1R gene using a 166-kb bacterial artificial chromosome. ChIP assay shows increased RNA-Pol II binding (~1.6 fold higher) to the chromatin extracts from renal tissues of adult (4-6 months) male Hap I-TG mice with increased hAT1R expression (~6 fold higher). This was accompanied by higher baseline blood pressure in Hap I-TG mice (Hap I- 129±3 vs. Hap II- 116±4, p&lt;0.05). Next, we examined the effects of age on this haplotype-dependent regulation of the hAT1R. Aged (&gt;18 months), male mice were used for this part of the study. hAT1R expression increases with age in both haplotypes; however, this increase is significantly higher in Hap I-TG mice (3.17±0.5 to 5.5±0.75 fold) as opposed to mice with Hap II (1.1±0.2 to 1.8±0.1 fold). Age-associated change (Δ) in inflammatory and redox markers was significantly (p&lt;0.05) greater in TG mice with Hap I including, IL1 (4.6±0.8 vs. 2.1±0.49 fold), IL6 (4.0±0.69 vs. 2.1±0.2 fold) and NOX1 (8.3±0.4 vs. 2.5±0.6 fold). This is accompanied by age-associated reduction in levels of antioxidant defenses (SOD1: 0.97±0.0 vs. 1.4±0.1 fold; HO1: 0.77±0.1 vs. 1.3±0.2 fold) and pro-survival genes including, NAMPTS (2.1 folds lower in Hap I vs. Hap II) and SIRT1 (1.8 fold lower in Hap I vs. Hap II). Thus, haplotype-dependent transcriptional regulation of the hAT 1 R gene causes increased hAT1R expression and blood pressure, in Hap I TG mice. Importantly, aging exacerbates this differential gene-expression regulation, further increasing hAT1R and promoting a prooxidant/inflammatory milieu in mice with Hap I. </jats:p