Hypertension. 2014 Feb;63(2):281-8.

Dynamic CCAAT/enhancer binding protein-associated changes of DNA methylation in the angiotensinogen gene.

Wang F, Demura M, Cheng Y, Zhu A, Karashima S, Yoneda T, Demura Y, Maeda Y, Namiki M, Ono K, Nakamura Y, Sasano H, Akagi T, Yamagishi M, Saijoh K, Takeda Y.

Department of Hygiene, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. m-demura@med.kanazawa-u.ac.jp.



DNA methylation patterns are maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not preserved. We demonstrate that stimulatory signals can change the DNA methylation status at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site and activate expression of the angiotensinogen gene (AGT). A CEBP binding site in the human AGT promoter was hypomethylated in tissues with high expression of AGT, but not in those with low expression. The transcriptional activity of AGT promoter sequences cloned into a reporter plasmid depended on DNA methylation. In cultured human cells, interleukin 6 stimulation caused DNA demethylation around a CEBP binding site and a transcription start site; demethylation was accompanied by increased CEBP-β recruitment and chromatin accessibility of the AGT promoter. DNA methylation activity decreased in the nucleus. Excess circulating aldosterone upregulated AGT expression and was accompanied by DNA hypomethylation around a CEBP binding site and a transcription start site in human visceral adipose tissue. High salt intake led to upregulation of Agt expression, DNA hypomethylation around 2 CEBP binding sites and a transcription start site, and decreased DNA methylation activity in rat visceral adipose tissue. Taken together, CEBP binding initiates chromatin relaxation and transcription, which are followed by DNA demethylation around a CEBP binding site and a transcription start site in the AGT promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of AGT expression from an inactive to an active state.

KEYWORDS: CCAAT-enhancer binding protein-β; CEBPB protein, human; DNA methylation; angiotensinogen; chromatin; transcription initiation site

PMID: 24191285



DNA methylation patterns are generally thought to stabilize after development and differentiation. However, recent progress in this field has revealed DNA methylation pattern dynamics in response to various environmental stimuli. Daily factors involved in the alteration of DNA methylation include chemicals, infection, smoking, exercise and learning. Although DNA demethylation almost always causes transcriptional activation, it is a consequence of transcriptional activation rather than a cause.

Angiotensinogen (AGT) is a precursor of angiotensin and a part of the renin-angiotensin-aldosterone system (RAAS) that regulates blood pressure, body fluids, and electrolyte homeostasis. DNA demethylation occurs upon stimulation, whereas remethylation occurs when the stimulus is removed in the AGT promoter (Figure 1). Dynamic changes in DNA methylation following stimulation occur in relaxed chromatin regions, both where transcription factors actively interact and where transcription is initiated (Figure 2).

Circulating AGT derived from adipose tissue is increased in obese patients with hypertension. High salt intake is associated with obese human subjects, suggesting a potential link between high salt intake and increased adipose AGT in humans. High salt intake increases Agt expression and demethylates the Agt promoter in visceral adipose tissue (VAT) from Wistar-Kyoto rats. DNA demethylation occurs around the TSS and these CEBP binding sites (Figure 3). Although a high salt intake suppresses the level of circulating RAAS, VAT Agt expression is paradoxically increased in rats fed a high-salt diet. Salt-dependent hypertension in humans may be partially dependent on increased VAT AGT expression.

Cortisol and aldosterone exert differential effects through the binding and activation of a subfamily of NR3C glucocorticoid receptors (GRs) (NR3C1) and mineralocorticoid receptors (MRs) (NR3C2), respectively. In our study that compared VAT surrounding cortisol-producing adenoma (Cushing’s syndrome, CS), aldosterone-producing adenoma (APA), and non-functioning adenoma (NFA), we have drawn a strong conclusion that both cortisol and aldosterone are able to stimulate VAT AGT transcription, with accompanying DNA demethylation of the AGT promoter in adult humans. However, the effect of cortisol on VAT AGT transcription was different from that of aldosterone in terms of agonist efficacy. Aldosterone, although ≈1000 times less abundant than cortisol, exerts a significant stimulatory effect on VAT AGT transcription, whereas cortisol does so to a much lesser extent.

In our study, however, VAT AGT expression in CS patients tended to be higher than that of NFA patients. As mentioned, DNA demethylation occurs in relaxed chromatin regions where transcription is initiated (Figure 2). Therefore, the low DNA methylation levels observed around a TSS clearly indicate that an excess of cortisol activates VAT AGT transcription in CS patients. Furthermore, low DNA methylation levels around the distal glucocorticoid response elements (GREs) strongly suggest that activated GRs are involved in transcriptional activation, through their interaction with the distal GREs (Figure 4). Taken together, these results suggest that cortisol has a weak but definite ability to stimulate VAT AGT transcription in adult humans.

An excess of aldosterone in APA elicits low DNA methylation levels at all CpG dinucleotides in the AGT promoter, with an accompanying upregulation of VAT AGT expression (Figure 4). A CpG dinucleotide exists in a DNA sequence that functions as both a GRE and a CEBP binding site, implying that the activation of MRs (NR3C2), but not that of GRs (NR3C1), initiates chromatin remodeling along with CEBP (Figure 4). Members of the NR3C subfamily, and many other members of the nuclear receptor superfamily, act cooperatively with other DNA-binding transcription factors. The interaction of their target DNA sequences can integrate the hormonal response to other regulatory pathways. Collectively, these results suggest that activated MRs bind the DNA sequence serving as both a GRE and a CEBP binding site, and play a leading role in the transcriptional activation of AGT, thereby recruiting other transcription factors (Figure 4).

RAAS is a system of major importance in the control of fluid and electrolyte homeostasis. This positive feedback effect of aldosterone allows the efficient retention of fluids, especially effective in the context of dehydration in humans. In addition, this effect may contribute to the development of hypertension in cases of obesity, as well as in cases of CS and APA, despite decreased circulating renin levels in these pathologies.

High DNA methylation around a TSS is associated with the absence of RNA polymerase II. Low DNA methylation around a TSS is associated with the presence of RNA polymerase II, either active (high transcription levels) or stalled (low transcription levels). DNA methylation patterns of the VAT AGT promoter in NFA are high, whereas those around the TSS in NFA are not (Figure 4). The combination of high DNA methylation in the promoter region and intermediate DNA methylation around the TSS suggest that RNA polymerase II is stalled in the VAT AGT gene in NFA. As excessive salt in contemporary diets reduces the level of circulating RAAS, the DNA methylation patterns of VAT AGT in NFA reflects the reduced levels of circulating RAAS in contemporary humans, and the readiness to produce AGT. Collectively, these results suggest that VAT is one of the major sites of AGT production in humans. Aldosterone efficiently stimulates VAT AGT production in humans.

Using the AGT gene as a typical example, we show in vitro and vivo dynamic changes in DNA methylation in association with stimulatory signals. Stimulatory signals switch the gene expression phenotype within a tissue, from an inactive to an active state. (Figure 5). DNA demethylation is observed within dozens of minutes to days of transcriptional activation and reaches a maximum level in hours to years. Thus, sustained stimulation keeps DNA methylation patterns hypomethylated over days or years. By contrast, DNA remethylation gradually increases within the promoter and around the TSS, following the removal of a stimulus (Figure 6).

DNA methylation patterns act as memory to maintain the responsiveness of gene expression to additional signals. DNA methylation patterns have a profound effect on gene transcription, and thus affect constitution in a tremendous way. A wide variety of stimuli in daily life continues to slowly and dynamically change DNA methylation patterns throughout life.

Figure 1Figure 1 Dynamics of DNA demethylation and remethylation of the AGT promoter following interleukin 6 stimulation. TSS, transcription start site; CEBP, CCAAT/enhancer binding protein; RNA pol II, RNA polymerese II; TF, transcription factor.

Figure 2Figure 2 Chromatin regions where DNA demethylation takes place. RNA pol II, RNA polymerese II; TF, transcription factor; TFBS, transcription factor binding site.

Figure 3Figure 3 Salt-induced Agt transcription in the rat visceral adipose tissue. TSS, transcription start site; CEBP, CCAAT/enhancer binding protein.

Figure 4Figure 4 Differential effects of excess cortisol and aldosterone on the activation of AGT transcription in visceral adipose tissue. GR, glucocorticoid receptor; MR, mineralocorticoid receptor; CEBP, CCAAT/enhancer binding protein; CS, Cushing syndrome; APA, aldosterone-producing adenoma, NFA, non-functioning adenoma; TSS, transcription start site; RNA pol II, RNA polymerese II; Ang, angiotesin.

Figure 5Figure 5 Conversion of the gene expression phenotype. Stimulatory signals switch the gene expression phenotype from an inactive to an active state within a tissue. Following stimulation, the gene expression phenotype is gradually restored to its former state. TF, transcription factor; TFBS, transcription factor binding site.



Figure 6Figure 6 Schematic representation of dynamic changes in DNA methylation patterns. Stimulation can induce DNA demethylation. DNA remethylation takes place following the removal of the stimulus. The degree of changes in DNA methylation depends on the local chromatin structure. RNA pol II, RNA polymerese II; TF, transcription factor; TFBS, transcription factor binding site.

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