Transgenic mice with -6A haplotype of the human angiotensinogen gene have increased blood pressure compared with -6G haplotype

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.     

Natural selection and population history in the human angiotensinogen gene (AGT): 736 complete AGT sequences in chromosomes from around the world

Several lines of evidence suggest that patterns of genetic variability in the human angiotensinogen gene (AGT) contribute to phenotypic variability in human hypertension. The A(-6) promoter variant of AGT is associated with higher plasma angiotensinogen levels and increased risk of essential hypertension. The geographic distribution of the A(-6) variant leads to the intriguing hypothesis that the G(-6) promoter variant has been selectively advantageous outside Africa. To test these hypotheses, we investigated the roles of population history and natural selection in shaping patterns of genetic diversity in AGT, by sequencing the entire AGT gene (14400 bp) in 736 chromosomes from Africa, Asia, and Europe. We found that the A(-6) variant is present at higher frequency in African populations than in non-African populations. Neutrality tests found no evidence of a departure from selective neutrality, when whole AGT sequences were compared. However, tests restricted to sites in the vicinity of the A(-6)G polymorphism found evidence of a selective sweep. Sliding-window analyses showed that evidence of the sweep is restricted to sites in tight linkage disequilibrium (LD) with the A(-6)G polymorphism. Further, haplotypes carrying the G(-6) variant showed elevated levels of LD, suggesting that they have risen recently to high frequency. Departures from neutral expectation in some but not all regions of AGT indicate that patterns of diversity in the gene cannot be accounted for solely by population history, which would affect all regions equally. Taken together, patterns of genetic diversity in AGT suggest that natural selection has generally favored the G(-6) variant over the A(-6) variant in non-African populations. However, important localized effects may also be present.     

Contribution of the hormone-response elements of the proximal ApoA-I promoter, ApoCIII enhancer, and C/EBP binding site of the proximal ApoA-I promoter to the hepatic and intestinal expression of the ApoA-I and ApoCIII genes in transgenic mice

We have generated and studied the pattern of expression of transgenic mouse lines carrying the human apoA-I and apoCIII gene cluster mutated at different sites. In two lines, we have either mutated the hormone-response element (HRE) of element G of the apoCIII enhancer or the C/EBP binding site of the proximal apoA-I promoter. In a third line, we have mutated the two HREs of the apoA-I promoter and the HRE of the apoCIII enhancer. Mutations in the HRE of element G reduced the hepatic and intestinal expressions of the reporter chloramphenicol acetyltransferase (CAT) gene (which substituted the apoCIII gene) to 4 and 13% of the wild-type (WT) control, whereas the hepatic and intestinal expressions of the apoA-I gene were reduced to 92 and 25% of the WT control, respectively. A mutation in the C/EBP site increased the hepatic and intestinal expressions of the apoA-I gene approximately 1.25- and 1.6-fold, respectively, and did not affect the expression of the CAT gene. The mutation in the three HNF-4 binding sites of the apoA-I promoter/apoCIII enhancer nearly abolished the expression of apoA-I and the reporter CAT gene in all tissues. These findings establish the importance of the HREs for the hepatic and intestinal expressions of the apoA-I and apoCIII genes and suggest that C/EBP does not play a central role in the expression of the apoA-I gene.     

Effect of the CCAAT/enhancer binding protein on expression of the gene for chicken malic enzyme

The gene for malic enzyme is expressed at a high level in chick embryo-hepatocytes (CEH) treated with triiodothyronine (T3) and at a low level in the absence of T3. In chick-embryo fibroblasts (CEF), expression of the malic enzyme gene is low and not regulated by T3. Specific nuclear proteins from both CEH and CEF bound to a consensus CCAAT/enhancer binding protein (C/EBP) site at -335 to -327 bp of the malic enzyme gene. The level of binding was much higher in extracts from CEH than in extracts of CEF, and the complexes formed had different mobilities. C/EBPalpha was present in the complex that bound to the C/EBP site in nuclear extracts from CEH but not in those from CEF. The C/EBP element was necessary and sufficient to bestow full T3 responsiveness to 5800 bp of 5'-flanking DNA of the malic enzyme gene in CEH. C/EBPalpha was not detectable in wild-type CEF, and deletion of the C/EBP binding site had no effect on expression of transgenes containing 5800 bp of 5'-flanking DNA of the malic enzyme gene. In CEF, overexpression of C/EBPalpha stimulated promoter activity of constructs that contained the C/EBP site linked to the malic enzyme promoter or a heterologous reporter. The results suggest that C/EBPalpha or a closely related isoform is involved in the tissue-specific expression of the malic enzyme gene.     

AGT基因单倍型与原发性高血压

选取血管紧张素原（angiotensinogen, AGT）基因启动子区 -217, -152, -20, -6, 内含子1 的+31, 第二外显子T174M（3889）和 T235M（4072）共7个位点,对497例的样本（高血压患者298例,血压正常对照199例） 用PCR-RFLP、和最大期望值（expectation maximization,EM）算法为基础的最大似然法(maximum likelihood estimate,MLE)检测和估算,本群体AGT基因A-6G,C+31T,T235M三位点两两存在完全连锁不平衡(D^,=1);G-217A和G-152A位点,G-152A和3889T位点平衡传递。存在7种单倍型,单倍型H2(-217: A, -152: G, -20: A, -6: G, +31: T, 174: T, 235: M) 在正常血压个体中的频率高于高血压组。研究结果提示AGT基因中H2单倍型可能与控制血压的保护性因素连锁不平衡。此外,本研究结果支持基因剂量效应可能存在于单倍型中,而不与单个位点直接关联。     

Identification of a novel variant in the phosphoenolpyruvate carboxykinase gene promoter in Japanese patients with type 2 diabetes.

Phospho enolpyruvate carboxykinase (PEPCK) plays an important role in gluconeogenesis and hepatic glucose production. To test the hypothesis that mutations of the PEPCK gene promoter contribute to the increased hepatic glucose production that leads to diabetes, we screened for polymorphisms of the PEPCK promoter region in 252 Japanese type 2 diabetic patients and 188 non-diabetic control subjects. A novel variant at position - 232 (C to G) was found at a similar frequency in type 2 diabetes patients (32 %) and control subjects (35 %) (p = 0.26). However, patients with the - 232 G/G genotype had an earlier age of onset than those with the - 232 C/C or - 232 C/G genotypes (p = 0.028). As the variant might well otherwise influence hormonal action, we transfected PEPCK-luciferase fusion gene constructs with the variant into human hepatoma cells and examined the response to dexamethasone, insulin, and cAMP. The reporter assay showed no significant difference in hormonal responses with the fusion gene containing the variant. Accordingly, the single-base variant at position - 232 of the PEPCK gene promoter is most probably not a major contributor to the pathogenesis of type 2 diabetes. However, this variation may be useful as a genetic marker for other metabolic disorders, especially in Japanese.     

Upregulation of human angiotensinogen (AGT) gene transcription by interferon-gamma: involvement of the STAT1-binding motif in the AGT promoter

Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5' side suggested the major IFN gamma responsive DNA element to be between -303 and -103. This region contained a candidate STAT1-binding site between -271 and -279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection.     

A nucleoprotein complex containing CCAAT/enhancer-binding protein beta interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 gene and contributes to insulin-regulated gene expression

Highly related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK). Gel shift studies reveal that oligonucleotide probes containing an IRS from the IGFBP-1 or PEPCK gene form a similar complex with hepatic nuclear proteins. Unlabeled competitors containing the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit the formation of this complex. Antibody against C/EBPbeta (but not other C/EBP proteins) supershifts this complex, and Western blotting of affinity purified proteins confirms that C/EBPbeta is present in this complex. Studies with affinity purified and recombinant protein indicate that C/EBPbeta does not interact directly with the IRS, but that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS reveal that the ability to interact with factors required for the formation of this complex correlates well with the ability of insulin to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene constructs with a C/EBP-binding site (but not an HNF-3/forkhead site or cAMP response element) maintains the effect of insulin on promoter activity. Together, these findings indicate that a nucleoprotein complex containing C/EBPbeta interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression.     

A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression

We have identified previously a novel complex mutant allele in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient affected with cystic fibrosis (CF). This allele contained a mutation in CFTR exon 11 known to cause CF (S549R(T>G)), associated with the first alteration described so far in the minimal CFTR promoter region (-102T>A). Studies on genotype-phenotype correlations revealed striking differences between patients carrying mutation (S549R(T>G)) alone, who had a severe disease, and patients carrying the complex allele (-102(T>A)+S549R(T>G)), who exhibited milder forms of CF. We thus postulated that the sequence change (-102T>A) may attenuate the effects of the severe (S549R(T>G)) mutation through regulation of CFTR expression. Analysis of transiently transfected cell lines with wild-type and -102A variant human CFTR-directed luciferase reporter genes demonstrates that constructs containing the -102A variant (which creates a Yin Yang 1 (YY1) core element) increases CFTR expression significantly. Electrophoretic mobility shift assays indicate that the -102 site is located in a region of multiple DNA-protein interactions and that the -102A allele recruits specifically an additional nuclear protein related to YY1. The finding that the YY1-binding allele causes a significant increase in CFTR expression in vitro may allow a better understanding of the milder phenotype observed in patients who carry a severe CF mutation within the same gene.