Angiotensinogen Gene Polymorphism at -217 Affects Basal Promoter Activity and is Associated with Hypertension in African-Americans

-6A Our laboratory is interested in understanding the role of single nucleotide polymorphisms (SNPs) in the AGT gene on human hypertension. The nucleotide sequence of the human AGT gene promoter contains an A/G polymorphic site at -217. In this paper we show that the -217A allele of the AGT gene is associated with hypertension in African-American population (p=.0017) and not in Caucasian population (p=.12). The nucleotide sequence of the human AGT gene containing the -217 A/G polymorphic site has partial homology with a consensus C/EBP binding site. We show that an oligonucleotide containing human AGT gene promoter with nucleoside A at -217 binds more strongly to recombinant C/EBP α , C/EBP β and DBP. In addition, we show that reporter constructs containing human AGT gene promoter with nucleoside A at -217 have increased basal promoter activity on transient transfection in HepG2 cells as compared to reporter constructs containing nucleoside G at -217. Furthermore, we show that IL-6 treatment in the presence or absence of over-expressed C/EBP β increases the promoter activity of reporter constructs containing nucleoside A at -217 as compared to reporter constructs containing nucleoside G at -217. homology with the binding site of C/EBP family of transcription Our gel shift assays have shown that recombinant C/EBP α , C/EBP β , and DBP bind more strongly to an oligonucleotide containing human AGT gene promoter with nucleoside A at -217 compared to the same oligonucleotide containing nucleoside G at -217. Since C/EBP family of transcription factors play an important role in IL-6 induced expression of a number of genes, we studied the effect of IL-6 and C/EBP β on the promoter activity of reporter constructs containing either -217A or -217G by transient transfection in HepG2 cells. Results of our experiments have shown that over-expression of C/EBP β in the presence or absence of IL-6, or IL-6 treatment alone increases the overall promoter activity of reporter constructs containing the -217A variant as compared to the -217G variant. In addition, our data show that treatment of cells with IL-6 enhances the promoter activity of the -217A variant as compared to the -217 variant, particularly in the case of over-expressed C/EBP β . Since IL-6 and C/EBP β enhance the expression of human AGT gene together, our data suggest that modification of C/EBP β or another interacting factor by IL-6 is involved in selective up-regulation of the -217A variant of this gene.


Summary
Hypertension is a serious health problem in Western society, in particular for the African-American population. Although previous studies have suggested that the angiotensinogen (AGT) gene locus is involved in human essential hypertension, the molecular mechanisms involved in hypertension in African-American population remain unknown.
We show that an A/G polymorphism at -217 in the promoter of the AGT gene plays a significant role in hypertension in African-American population. The frequency of -217A allele is increased significantly in African-American hypertensive subjects as compared to normotensive controls. We also show that the nucleotide sequence of this region of AGT gene promoter binds strongly to the C/EBP family of transcription factors when nucleoside A is present at -217. In addition, we show that reporter constructs containing human AGT gene promoter with nucleoside A at -217 have increased basal transcriptional activity on transient transfection in HepG2 cells as compared to reporter constructs with nucleoside G at -217. Finally, we show that IL-6 treatment in the presence or absence of over-expressed C/EBPβ increases the promoter activity of reporter constructs containing nucleoside A at -217 as compared to reporter constructs containing nucleoside G at -217. Since, the AGT gene is expressed primarily in liver and adipose tissue and C/EBP family of transcription factors plays an important role in gene expression in these tissues, we propose that increased transcriptional activity of the -217A allele of the human AGT gene is associated with hypertension in African-Americans.
However, since amino acid 235 is located far away from the renin cleavage site, this polymorphism does not explain the mechanism involved in increased plasma AGT level.
The human AGT gene also has an A/G polymorphism at -6. It has been shown recently that : (a) molecular variants 235T and -6A are in complete linkage disequilibirium and (b) reporter constructs containing human AGT gene promoter with nucleoside A at -6 have increased promoter activity on transient transfection in human liver derived HepG2 cells compared to reporter constructs containing nucleoside G at -6 (14). Results of these experiments suggest that increased plasma AGT level by allele 235T may be due to increased transcriptional activity of the human AGT gene by nucleoside A at -6.
Although hypertension is more prevalent in the black population and complication rates, particularly for renal failure, are many times higher in blacks than whites, relatively little work has been done to understand the molecular mechanism involved in hypertension in this population. Plasma AGT level is generally higher in black population (15). It has been shown that: (a) plasma AGT level is about 19% higher in black children as compared to white children, (b) blood pressure is normally higher and increases faster over time in black children as compared to white children, and (c) plasma AGT level is associated with AGT gene in black children (16)(17)(18). Caulfield et al., have found an association between the AGT gene locus and high blood pressure in 63 affected sibling pairs of African-Carribbean origin using CA dinucleotide marker (19). However, these by guest on March 24, 2020 http://www.jbc.org/ Downloaded from workers could not find an association between variants M235T or A/G at -6 and hypertension in African-American population. Other studies have also suggested that although the frequency of -6A allele is increased in African-American population, there is no association between -6A allele and hypertension in this population (20).
Our laboratory is interested in understanding the role of single nucleotide polymorphisms (SNPs) in the AGT gene on human hypertension. The nucleotide sequence of the human AGT gene promoter contains an A/G polymorphic site at -217. In this paper we show that the -217A allele of the AGT gene is associated with hypertension in African-American population (p=.0017) and not in Caucasian population (p=.12). The nucleotide sequence of the human AGT gene containing the -217 A/G polymorphic site has partial homology with a consensus C/EBP binding site. We show that an oligonucleotide containing human AGT gene promoter with nucleoside A at -217 binds more strongly to recombinant C/EBPα, C/EBPβ and DBP. In addition, we show that reporter constructs containing human AGT gene promoter with nucleoside A at -217 have increased basal promoter activity on transient transfection in HepG2 cells as compared to reporter constructs containing nucleoside G at -217. Furthermore, we show that IL-6 treatment in the presence or absence of over-expressed C/EBPβ increases the promoter activity of reporter constructs containing nucleoside A at -217 as compared to reporter constructs containing nucleoside G at -217.

EXPERIMENTAL PROCEDURES
Plasmid construction-Reporter construct pHAGT1.3luc was constructed by PCR amplification of human AGT gene (21,22) using TATGCTAGTCGAGTGAGTCCCTA TCTATAGTGAACA as the forward primer and CAAGTACCAGTAAGTGAGTCTGA GTGGGGCCCCGCTTA as the reverse primer. The amplified fragment contained the nucleotide sequence -1206 to +70 and was subcloned in the pGL3 basic vector that lacks eukaryotic promoter and enhancer sequences (Promega, Madison, WI). Reporter construct pHAGT303luc was constructed by PCR amplification of human AGT gene (22) using ACACACCTAGGGAGATGCTCCCGTTTCTGG as the forward primer and CAAGTACCAGTAAGTGAGTCTGAGTGGGGCCCCGCTTA as the reverse primer.
The amplified fragment contained the nucleotide sequence -303 to +70 and was subcloned in the pGL3 basic vector. These reporter constructs had nucleoside A at -6 and -217. Nucleoside A at -217 in these reporter constructs was mutated to G by site specific mutagenesis using CCTGCACCAGTCTCACTCTGTTCAGTCAGTG and its complementary oligonucleotide by Stratagene kit (Stratagene, La Jolla, CA). Nucleotide sequences of mutated reporter constructs were confirmed by sequence analysis. Reporter constructs (223A) 2  with recombinant human IL-6 (10 ng/ml of the media). Cells were harvested 48 h posttransfection and whole cell extracts were prepared by resuspension in 100 µl of lysis buffer (Promega). An aliquot of the cell extract was used to measure luciferase activity by Turners Design Luminometer TD 20/20 using a luciferase assay system (Promega) as described by the manufacturer. Luciferase activity was normalized with the β-gal activity.
Gel Mobility Shift Assay -The probes for electrophoretic mobility shift assay (EMSA) were chemically synthesized, annealed and radiolabeled at the 5'-ends by polynucleotide polyacrylamide gel in a cold room. After 2-3 h, the gel was dried under vacuum and protein-nucleic acid complexes were identified by autoradiography. For supershift assay, 1 µl of antibody was added to the reaction mixture that was incubated for 30 min and analyzed by EMSA. Radioactive oligonucleotides were purified by polyacrylamide gel electrophoresis followed by electroelution for quantitative gel shift assay. Nuclear extracts for gel mobility shift assays were prepared by modification of a previously described method (24). Recombinant C/EBPα and C/EBPβ were obtained through bacterial expression of histidine-tagged proteins as described previously (25).
Recombinant DBP was obtained using an in vitro coupled transcription-translation system obtained from rabbit reticulocytes as described previously (23). Antibodies against C/EBPα and C/EBPβ were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).   test for contingency table where appropriate. Genetic data were analyzed for allele frequency by gene-counting method.

Oligonucleotides-
Hardy-Weinberg equilibrium was tested by using the computer program GDA

Frequency of -217A allele of AGT gene is increased in African-American hypertensive patients
To understand the role of A/G polymorphism at -217 in the promoter of AGT gene in hypertension, we have analyzed genomic DNA from 186 hypertensive and 156 normotensive African-American subjects. All patients and control subjects were in Hardy-Weinberg equilibirium. The genomic DNA was amplified by PCR and the product was analyzed for the A/G polymorphic site at -217 by restriction analysis (Fig. 1). The frequency of the -217A allele in hypertensive patients was 0.29 as compared to 0.19 in normotensive population which is highly significant (p= 0.0017 and OR =1.792) ( Table   I) is not significant (p= .12) (Table I). Statistical analysis based on -217 A/G genotype (using A allele as a dominant model) also suggested a significant role of the -217A allele in hypertension in African-Americans (p= 0.0021 and OR = 2.015) and not in Caucasians (Table II). Since an A/G polymorphism at -6 has been previously associated with hypertension, we also analyzed genomic DNA from the African-American and Caucasian populations for this polymorphism. The frequency of -6A allele was 0.87 in African-American hypertensive subjects and 0.85 in normotensive controls which was not significant (p=0.58) (Table III). However, the frequency of -6A allele was marginally significant in Caucasian subjects (p=0.06). These experiments suggested that -217A allele of the human AGT gene plays a significant role in essential hypertension in African-Americans and not in Caucasians. Results of this experiment confirmed our previous observation that oligonucleotide 223A

Reporter constructs containing human AGT gene promoter with nucleoside
forms a stronger complex with r-C/EBPα as compared to oligonucleotide 223G.
Since DBP also plays an important role in transcriptional regulation of liver specific genes especially during circadian rhythm and binds to C/EBP binding sites (27), it was of interest to determine whether DBP also binds to this region of the human AGT gene

Discussion
To date, the AGT gene locus is the only locus that has been associated with human essential hypertension. We have presented evidence that an A/G polymorphism at -217 may be involved in hypertension in the African-American population. The frequency of the -217A allele was significantly increased in African-American hypertensive subjects as compared to normotensive controls. On the other hand, the frequency of -217A allele was not significantly different in Caucasian hypertensive and normotensive subjects. We have also found that 65% of African-American normotensive controls were GG homozygotes whereas 48% of African-American hypertensive subjects were GG homozygotes (p = .0021). This observation suggests that -217G allele may be partially responsible for protection of African-American subjects from hypertension. On the other hand 80% of normotensive Caucasian controls were GG homozygotes and 72% of Caucasian hypertensive subjects were GG homozygotes, which is not significantly different (p = .14). In accordance with previous studies, we also found that although the frequency of -6A allele is increased in African-American subjects, this difference is not significant between hypertensive and normotensive subjects.
In order to understand the biological significance of this polymorphic site, we constructed three types of reporter constructs containing either nucleoside A or G at -217 and used these reporter constructs in transient transfection assays in human liver derived HepG2 cells. Transient transfection of these reporter constructs indicated statistically significant increased basal promoter activity of reporter constructs containing nucleoside A at -217 as compared to reporter constructs containing nucleoside G at -217.  (30)(31)(32). The leucine zipper is a heptad of leucine repeats that intercalate with repeats of the dimerization partner forming a coil of α-helices in parallel orientation (33,34). This dimerization is essential for binding of the C/EBP family of transcription factors to cis-acting DNA elements.
AGT is an acute phase protein and its expression is increased by LPS, IL-6, and glucocorticoid treatment (35)(36)(37)(38). An acute phase response unit (APRU) located between -470 and -554 has been identified in the rat AGT gene (39). This region of the promoter contains a composite NF-κB and C/EBP binding site located between -531 and -557, one full GRE located between -570 and -584 and a half GRE located between -470 and -477.
All of these sites are required for maximum acute phase response of this gene. Although, expression of both rat and human AGT genes is increased in response to acute phase reaction, the APRU observed in the rat gene promoter is absent in the human gene promoter. Similarly, nucleotide sequence around the A/G polymorphic site at -217 of the human AGT gene is not conserved in the rat gene. We have previously shown that nucleotide sequence located between -99 and -91of the human AGT gene binds to the C/EBP family of transcription factors and this region of the promoter plays an important role in DBP and C/EBPβ induced expression of this gene (23). We have also shown that CREB binds to the nucleotide sequence located between -840 and -830 of the human AGT gene and this sequence is involved in cAMP induced expression of the human AGT gene (40). It has been shown previously that human AGT gene has a C/A polymorphic site at -20 (located between TATA box and transcriptional initiation site shown that USF binds to this sequence when nucleoside C is present at -20 and ER binds to this sequence when nucleoside A is present at -20 (41). Orphan receptor Arp-1 also binds to this sequence and reduces ER induced promoter activity (42). Yanai et al., have shown that the nucleotide sequence located between the TATA box and transcriptional initiation site of the human AGT gene binds to USF and plays a critical role in its expression (43). They have also shown that the liver enriched transcription factor HNF4 binds to the human AGT gene promoter and regulates expression of this gene in hepatocytes (44). In addition, we have shown that the liver enriched transcription factor HNF-3 binds to the nucleotide sequence located between +10 and +20 of the human AGT gene promoter (45). All of these transcription factors including C/EBP (that differentially binds to A/G polymorphic site at -217) may interact with transcriptional co-activator CBP and co-ordinately regulate the expression of this gene.
In conclusion, our data suggest that an A/G polymorphism at -217 of the human AGT