Epigenetic Mechanisms and Hypertension

Epigenetic mechanisms contribute to the regulation of physiology and disease by changing gene expression, and epigenetic changes occur as a result of the interplay between DNA and environmental factors (Figure 1). Unlike permanent changes in DNA sequences that change amino acid sequences or gene expression, epigenetic regulation can respond dynamically to environmental and lifestyle factors. On the other hand, this regulation may have more stable, longer-lasting effects than do transient changes in gene expression or cellular regulation independent of changes in gene expression (Figure 1).

Figure 1. Epigenetic mechanisms’ role in disease mechanisms.

A disease can arise from the expression of mutated proteins, stable changes in gene expression levels, or other abnormalities, including transient changes in gene expression, post-transcriptional or post-translational changes in proteins and changes in other chemical or physical factors. Stable changes in gene expression can occur because of variations in DNA regulatory sequences or effects of environment and lifestyle factors. Epigenetic mechanisms underlie stable changes in DNA function that variations in the sequence of the DNA do not determine directly. Such changes can respond to environment and lifestyle factors dynamically and can result from DNA-environment interactions.

The fundamental importance of epigenetic mechanisms in biology is illustrated best by striking epigenetic phenomena, such as the presence of roughly 200 cell types in the human body that are distinct molecularly and phenotypically, although nearly all of them share identical DNA sequences. Another striking illustration of epigenetic mechanisms’ biological importance is the inactivation of one of the X chromosomes in female cells.

Increasingly strong evidence supports epigenetic mechanisms’ significant role in the development of hypertension. Nevertheless, many fundamental questions remain to be addressed. In this review, I present a tiered approach to classifying evidence of epigenetic regulation and provide a brief overview of the biology of epigenetic mediators and the methods used to study them. I then summarize reports of changes in epigenetic mediators that occur in association with changes in blood pressure (BP) or hypertension. Next, I highlight emerging evidence of epigenetic mediators’ functional role in the development of hypertension. Finally, I outline key questions in the field and propose resources, technologies, and the concepts of a tree-like paradigm and systems molecular medicine that could help advance epigenetic research on hypertension.

I. A Tiered View of the Evidence for Epigenetic Regulation

Researchers in the epigenetics field still grapple with what qualifies as an epigenetic mechanism.^{1, 2, 3} Most investigators now agree to use the term epigenetics to refer to stable changes in DNA function that are not determined directly by the sequence of the DNA. However, what “stable” means is not always clear. Some would say it indicates that changes must be heritable across multiple generations of the organism, while many others would say the changes have to be heritable only through cell division. However, it can be challenging to test whether a change in DNA function is heritable, even through cell division alone, especially those changes that occur in mammalian species in vivo.

Rather than engaging in another debate about the definition of epigenetics, I propose a practical, tiered approach to examining evidence for epigenetic regulation in this article (Box). Tier 1 includes any evidence that suggests the involvement of any mediators that could underlie stable changes in DNA function, such as DNA methylation, histone modifications, non-coding RNA that regulates transcription, and chromatin conformation.

Box. A three-tier view of the evidence for epigenetic regulation.

Refer to the text for additional details.

Tier 1: Stable changes of genome function not determined directly by changes in DNA sequence

Regulatory mediators:

DNA methylation (mechanisms for reaching Tier 2 are known)

Histone modification

Regulatory RNA

Chromatin conformation

Tier 2: Tier 1 and heritable through mitosis

Examples:

Some long-term effects of early-life experiences

Some maternal effects

Tier 3: Tier 1 and heritable through germ cell reprogramming

Examples:

Transgenerational effects

Some paternal effects

Evidence would be considered as Tier 2 if heritability through cell division is proven. The mechanism underlying the inheritance of DNA methylation marks through cell division is known.⁴ During DNA replication, DNA methyltransferase 1 (DNMT1), together with other proteins, recognize methylated cytosines in the template strand of DNA and catalyze cytosine methylation in the DNA strand newly synthesized. Therefore, DNA methylation can be considered heritable through cell division unless proven otherwise. Mechanisms that mediate the inheritance of most histone marks, non-coding RNA-determined transcriptional activities, or chromatin conformation through cell division, have not been established.⁵

Physiological studies sometimes provide indirect or suggestive evidence for the involvement of epigenetic mechanisms across cell divisions. For example, the maternal environment and early-life experiences are known to influence physiology or diseases in adulthood (Box),⁶ and it has been shown that some of these effects are associated with changes in the epigenetic mediators listed under Tier 1^7,8. However, whether epigenetic mechanisms contribute to these effects functionally largely remains unknown.

Tier 3 refers to epigenetic changes that are heritable through germ cell reprogramming (Box). Germ cells in species such as humans, mice, and rats, undergo two rounds of epigenetic reprogramming during early embryonic development: one after fertilization and the other during the formation of primordial germ cells.^{9, 10} This reprogramming, which was believed once to involve the complete erasure of epigenetic marks, could leave behind some marks.^11,12,13 The residual marks, perhaps working with trans regulatory factors, such as RNA,^14,15 could serve as the molecular basis of epigenetic memory following germ cell reprogramming.

A special type of epigenetic inheritance is transgenerational inheritance.⁹ An F0 generation’s environment during pregnancy can influence primordial germ cells in the embryo that become germ cells in F1 and individual organisms in F2. Therefore, typically, an epigenetic trait would have to be inherited from F0 to at least F3 to be considered inherited trans-generationally rather than a direct effect of the initial environmental exposure. However, fewer generations are required if the initial environmental exposure is limited to the paternal side or occurs before the formation of primordial germ cells on the maternal side.¹⁶

The Basic Biology of Epigenetic Mediators

II.1. DNA methylation

DNA methylation occurs primarily at cytosine in cytosine-guanine dinucleotides (CpG), and involves the addition of a methyl group to carbon number 5 in cytosine to form 5-methylcytosine (5mC).^{17, 18} Some parts of the genome are methylated heavily and remain in the transcriptionally silent state of heterochromatin generally. In other parts of the genome, the methylation levels can change dynamically in response to developmental or environmental cues. The level of methylation, particularly that of CpG islands (clusters of CpG sites) located in promoter regions, may be correlated inversely with the level of gene transcription activity. However, the relation between methylation and expression levels can be complex. ^19,20,21 In addition, DNA methylation also can occur at cytosine in CpH (H=A, C or T).²²

DNMT catalyzes the formation of 5mC.^{4, 23} As described above, DNMT1 is responsible primarily for maintaining DNA methylation patterns during DNA replication and cell division. DNMT3, which includes DNMT3A, 3B, and 3L, mediates de novo formation of 5mC. DNMT3A and 3B are catalytic and can methylate overlapping, but not identical, genomic loci. DNMT2 might be involved in tRNA methylation. Ten-eleven translocases (TET), which include TET1, 2, and 3, catalyze DNA de-methylation.²⁴ TETs convert 5mC to 5-hydroxymethylcytosine (5hmC), which can be converted further to 5-formylcytosine or 5-carboxylcytosine and replaced with unmodified C. These three TET isoforms act on overlapping genomic loci.

II.2. Histone modifications

DNA wraps around eight histone proteins, two each of H2A, H2B, H3, and H4, to form nucleosomes. The N-terminal tails of H3 and H4 are subject to post-translational modifications that influence chromatin structure and gene expression.^25,26 Common types of modification include acetylation at select lysine (K) residues, methylation at K or arginine residues, and phosphorylation at serine or threonine residues. Ubiquitylation and sumoylation also can occur.

Histone acetyltransferases (HATs) catalyze histone acetylation using acetyl CoA as the source of the acetyl group. Histone deacetylases (HDACs) catalyze the hydrolytic removal of acetyl groups, histone methyltransferases (HMTs) catalyze the transfer of one to three methyl groups from S-adenosyl-L-methionine to histones, and histone demethylases (HDMTs) remove methyl groups from histones. Each of these enzyme families includes multiple sub-families or members that target different amino acid residues or require different cofactors. Pharmacological inhibitors are available for several of these enzymes.^27,28

II.3. Non-coding RNA

The role of non-coding RNAs, such as microRNAs, in post-transcriptional regulation of mRNA stability and translation has been well-established. However, a molecule’s participation in posttranscriptional and translational regulation is not a sufficient basis for considering the molecule a bona fide epigenetic regulator. While several microRNAs have been reported to influence gene transcription activities and potentially could be involved in epigenetic regulation or inheritance, evidence that supports microRNAs’ contribution to stable changes in chromatin structure and genome function is limited in mammalian species. Long non-coding RNAs (lncRNAs) also participate in post-transcriptional regulation. However, some lncRNAs are well-established regulators of gene transcription activities, and part of this regulation might occur through epigenetic mechanisms.^{29, 30} For example, lncRNA Xist plays a key role in X chromosome inactivation.

Accordingly, this review will cover studies of lncRNAs in hypertension, but recognize that the mechanism underlying the physiological role of lncRNAs is not always clear and could involve both epigenetic and non-epigenetic actions. The reader is referred to other reviews for microRNAs’ role in hypertension^31,32.

II.4. Chromatin conformation

Chromatin conformation influences gene transcription by changing the spatial organization and accessibility of DNA regulatory elements.^{33, 34} DNA segments, such as enhancers and promoters, interact more frequently within than across topologically associating domains (TAD). A TAD may contain multiple DNA loops or sub-TADs ranging from thousands to millions of bp in size. CCCTC-binding factors (CTCF) that form homodimers and interact with other proteins, such as cohesin, maintain the DNA loops at the anchoring point. An important consequence of DNA looping is that enhancers or super-enhancers located thousands of bp from a promoter can be brought within its spatial proximity and regulate the transcriptional activity there. Further, some chromatin conformations bring DNA segments on different chromosomes together spatially.

Epigenetic regulatory mediators can influence each other. Changes in DNA methylation can induce changes in histone modifications, and vice versa. These changes, together with the assembly or disassembly of molecular complexes they induce, often change gene transcriptional activities by changing chromatin conformation and making regulatory DNA elements more or less accessible or active.

Methods Used to Study Epigenetics

Some of the major methods that can be used to detect and quantify epigenetic mediators and features, or study their functional roles are summarized in Table 1. For a more detailed description of several of these techniques, refer to reviews and methods published elsewhere.^35–50 The method chosen for a study depends on several factors, including whether the study’s focus is genome-wide or on specific loci and the availability of expertise and resources.

Table 1.

Major Methods Used to Study Epigenetic Regulation.

Goal of study	Epigenetic mark	Major Methods	Notes
Describing epigenetic mediators	DNA methylation	Whole genome bisulfite sequencing	Bisulfite treatment converts C, but not methylated C, to U. Single base resolution.
		RRBS and variants	Digestion-based enrichment, followed by bisulfite deep sequencing
		MeDIP and similar methods	Affinity enrichment, followed by deep sequencing or array-based analysis
		Cloning-based bisulfite sequencing	To analyze specific genomic loci
		Methylation-specific PCR	To analyze specific genomic loci
	Histone modification	ChIP-seq	Identifying DNA segments bound by a histone with specific modifications
	Regulatory RNA	RNA-seq or real-time PCR	Some lncRNAs do not have a poly(A) tail.
	Chromatin conformation	3C, 4C, Hi-C and variants	Mapping genomic proximity contacts globally or from a specific viewpoint
	Chromatin accessibility	DNase-seq, ATAC-seq	Identifying open chromatin
Testing functional role	DNA methylation	Pharmacological inhibitors	Inhibitors used commonly require DNA replication.
		Silencing or targeting DNMTs and TETs
		Targeted (de)methylation at specific genomic loci	Frequently adapting DNA recognition agents developed for genome editing
	Histone modification	Pharmacological inhibitors	Some histone modifying enzymes also can modify non-histone proteins.
		Silencing or targeting histone modifying enzymes
	Regulatory RNA	Modified anti-sense or genetic targeting	Some lncRNAs are located in the nucleus.
Examining transgenerational inheritance		Multi-generation breeding	The number of generations required could be reduced by using paternal or controlled maternal exposure.

RRBS, reduced representation bisulfite sequencing; MeDIP, methylated DNA immunoprecipitation; ChIP-seq, chromatin immunoprecipitation sequencing; 3C, chromatin conformation capture; ATAC, assay for transposase-accessible chromatin.

A key consideration in epigenetic analysis is cell type, because while some epigenetic features are shared across cell types, other features vary. In that respect, epigenetic analysis is similar to the analysis of gene expression and unlike genomic sequencing, although epigenetic analysis often involves DNA analysis. In some cases, epigenetic features detected in one cell or tissue type might serve as biomarkers or surrogates for epigenetic features of another, less accessible cell or tissue type. However, it is important to use caution in such extrapolations, especially in studies of a disease’s cellular mechanisms.

Epigenome-wide association studies (EWAS) can be performed to examine the association of epigenetic marks, especially DNA methylation, with a phenotype.⁵¹ The sample size EWAS requires depends on the degree of epigenetic variation and its effect size, but often, it is large and similar to genome-wide association studies (GWAS). In part because of the requirement for large sample sizes, EWAS have often used DNA from accessible peripheral blood. When interpreting results from such studies, it is important to distinguish biomarkers from mechanistic mediators and consider the tissue specificity of some epigenetic marks and whether blood cells are pathophysiologically relevant to the disease of interest.

It is important as well to consider the mechanisms of action and specificity when pharmacological inhibitors of enzymes that mediate changes of DNA methylation and histone modification are used (Table 1). DNA methylation inhibitors used commonly, such as 5-aza-2’-deoxycytidine (decitabine), are cytosine analogs that will prevent methylation after they are incorporated into DNA strands newly synthesized during DNA replication and cell division.⁵² Therefore, these inhibitors do not prevent de novo DNA methylation that occurs in non-dividing cells effectively. Most of the HDAC inhibitors used commonly inhibit multiple HDACs.²⁸ Moreover, the substrates of HDACs are not limited to histones, and inhibition of HDACs could alter the acetylation of non-histone proteins in addition to histones.

Association of Epigenetic Mediators with BP or Hypertension

Because of space limitations, this review focuses only on epigenetic mechanisms in the context of the pathogenesis of systemic hypertension, and does not address epigenetic mechanisms in pulmonary arterial hypertension or hypertensive end organ damage.

This section summarizes studies that have tested associations between changes in epigenetic mediators and BP or hypertension. Such evidence is available from both human and animal studies. Human studies can test the clinical relevance of epigenetic mediators directly, while animal studies may provide additional mechanistic insights by analyzing BP-relevant tissues that are less accessible in humans.

It is important to recognize that correlation does not prove causality. Studies that have demonstrated a functional role of epigenetic mediators in the regulation of BP or the development of hypertension will be reviewed in a later section in this article. Nonetheless, evidence of association is valuable, especially for epigenetic research of hypertension in its current stage of development, as it can provide biomarkers for diagnostic purposes and a basis for developing mechanistic studies.

IV.1. DNA methylation

IV.1a. Human studies

BP variations and the development of hypertension have been associated with changes in DNA methylation in several human studies. Richard et al., analyzed cross-sectional associations of systolic and diastolic BP with blood-derived genome-wide DNA methylation measured in 17,010 individuals of European, African American, and Hispanic ancestry,⁵³ and identified 13 replicated CpG sites that were associated significantly with BP. These replicated methylation sites explained 1.4% and 2.0% of the interindividual variations in systolic and diastolic BP, respectively, in addition to what known BP genetic variants can explain. In a GWAS of BP in up to 320,251 individuals of East Asian, European, and South Asian ancestry, the sentinel single nucleotide polymorphisms (SNPs) identified are enriched for association with DNA methylation at multiple CpG sites nearby.⁵⁴ A study of hypertensive and normotensive subjects reported possible differential methylation of some CpG sites in leukocytes.⁵⁵

Prenatal exposure to air pollutants was associated with lower or higher DNA methylation in long interspersed nuclear elements (LINE1) in newborn blood spots and higher systolic BP in 11-year-old children, depending on the type of pollutant and timing of the exposure.⁵⁶ The magnitude of associations varied according to genotype for 11 SNPs within DNMT1, DNMT3B, TET2, and thymine DNA glycosylase genes. Bellavia et al. exposed 15 healthy adult participants to concentrated ambient particles for 130 minutes,⁵⁷ and found that decreased Alu and TLR4 methylation in the blood was associated with higher diastolic BP postexposure and decreased TLR4 methylation was associated with higher systolic BP postexposure.

A study of 1,160 DNA trios from two established birth cohorts (the Cambridge Baby Growth and Wellbeing Studies) and 1367 Hyperglycemia and Adverse Pregnancy Outcome Study participants identified significant associations between polymorphic variants in several fetal imprinted genes and maternal mean arterial BP.⁵⁸

IV.1b. Animal model studies

Changes in DNA methylation have been associated with hypertension in several animal models used to study the effect of maternal diet on offspring health. Restricting the supply in the periconceptional diet of mature female sheep of methionine, vitamin B12, and folate, all of which are relevant to the methyl cycle metabolism, resulted in adult offspring with elevated BP, most obviously in males. The methylation status of 4% of 1,400 CpG islands examined in the fetal liver was altered, again, particularly in males.⁵⁹ In Sprague-Dawley rats, feeding the parents a vitamin-D depleted diet resulted in increased BP in the offspring, which was accompanied by hypermethylation of the promoter region of the Pannexin-1 (Panx1) gene in the kidney.⁶⁰

Embryo transfer experiments have shown that feeding Dahl salt-sensitive SS rats the grain-based 5L2F diet during gestation and lactation attenuated salt-induced hypertension in the offspring substantially compared to the casein-based AIN-76A diet. ⁶¹ Several hundred genomic loci exhibited differential methylation in the renal outer medulla of SS rats fed the two diets.⁶²

Liu et al. reported the first base resolution maps of 5mC and 5hmC in a model of hypertension.²⁰ They found that 11% and 1% of CpG sites in the renal outer medulla of SS rats that fell within CpG islands contained significant 5mC and 5hmC, respectively. Feeding SS rats and congenic, less salt-sensitive SS.13^BN26 rats a high-salt diet for just seven days changed the 5mC levels of several hundred CpG islands and the 5hmC levels of several dozen islands in a rat strain-specific manner. Many of the islands methylated differentially were intragenic. The study by Liu et al. also was one of the first to report 5hmC maps in any in vivo disease model.

DNA methylation may regulate several genes relevant to BP regulation. For example, the expression pattern of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2), which is crucial to prevent abnormal stimulation of mineralocorticoid receptors by glucocorticoids in the distal nephron, is correlated inversely with the degree of methylation at its promoter region.⁶³ Other examples of BP-relevant genes that may be regulated by DNA methylation include angiotensinogen⁶⁴ and αENaC,⁶⁵ as well as glucocorticoid receptor⁶⁶ and angiotensin type 1 receptor^{67, 68} as reviewed previously.¹⁶

IV.1c. Studies related to preeclampsia

Preeclampsia is associated with changes in methylation in humans and animal models. A CpG island in the promoter region of serine protease inhibitor A3 is hypomethylated in placentas from patients with preeclampsia and intrauterine growth restriction, which is associated with differential expression of the gene.⁶⁹

DNA methylation of the promoter of the thromboxane synthase gene TBXAS1 was lower in omental arteries of preeclamptic women, which is associated with up-regulation of thromboxane synthase expression. Treatment with 5-aza-2’-deoxycytidine increased the expression of thromboxane synthase in a neutrophil-like cell line.⁷⁰

Methylation levels at IGF2 in umbilical cord blood from infants born to women with preeclampsia were significantly lower than those from a normal pregnancy.⁷¹ Preeclamptic placenta exhibited DNMT3A dysregulation and a low promoter methylation and high protein expression of insulin-like growth factor-binding protein 5 (IGFBP5). DNMT3A knockdown caused promoter hypomethylation and up-regulation of IGFBP5 and inhibited trophoblast cell migration and invasion, the latter of which IGFBP5 downregulation reversed.⁷²

Children of mothers with preeclampsia and those conceived by assisted reproductive technologies (ART) display vascular dysfunctions. ART mice and the progeny of male ART mice also exhibit vascular dysfunction and higher arterial BP. Methylation at the promoter of the eNOS gene in the aorta increased, and eNOS expression in the carotid artery and NO metabolites in the plasma decreased in ART mice. Butyrate, which is a deacetylase inhibitor, can reverse these changes, suggesting that a cross-talk between DNA methylation and protein acetylation is involved.⁷³

CpG methylation at some sites in the promoter region of estrogen receptor-α and large-conductance Ca²⁺-activated potassium channel β₁ subunit (BKβ1) in sheep uterine arteries was regulated differentially during pregnancy or chronic hypoxia during gestation,^{74, 75} and treatment with 5-aza-2’-deoxycytidine reversed the effects of hypoxia.⁷⁶ Pregnancy also increases TET1 expression in uterine arteries in sheep, and treating isolated uterine arteries with the TET inhibitor monomethyl fumarate reversed several effects of pregnancy.⁷⁷

IV.2. Histone modifications

Several treatments or genetic perturbations that change BP have been shown to change histone modifications. In mice, deletion of Af17, a putative transcription factor, leads to increased dimethylation of histone H3K79, reduced ENaC function, higher urine volume and sodium excretion, and lower BP.⁷⁸ Stimulation of β₂-adrenergic receptor in mice decreases the expression of WNK4, a regulator of sodium reabsorption, which contributes to the development of salt-sensitive hypertension in mice and rats.⁷⁹ Stimulation of β₂-adrenergic receptor in mouse cultured distal convoluted tubule cells results in cyclic AMP-dependent inhibition of histone deacetylase-8 (HDAC8) activity, increased histone acetylation, and the glucocorticoid receptor binding to a negative glucocorticoid-responsive element in the promoter region of WNK4.⁷⁹

Treating spontaneously hypertensive rats (SHR) with pentaerythritol tetranitrate during pregnancy and lactation reduced the female offspring’s BP, and several potentially anti-hypertensive genes are upregulated in the aorta of eight-month-old female offspring, which is associated with enhanced H3K27 acetylation and H3K4 trimethylation.⁸⁰ In mice, the expression of hGRK4γ(142V), a variant of G-protein-coupled receptor kinase type 4, results in hypertension. The hGRK4γ(142V) phosphorylates histone deacetylase type 1, promotes its nuclear export to the cytoplasm, and increases expression of angiotensin II type I receptor.⁸¹ db/db mice, which exhibit increased BP in addition to diabetes, express more RAGE and PAI-1 in glomeruli than do control db/+ mice. This is associated with increased RNA polymerase II recruitment and permissive histone marks and decreased repressive histone marks at these genes, as well as altered expression of relevant histone modification enzymes.⁸²

Histone acetylation and acetyltransferases have been reported to be important in determining renin cell identity.^{83, 84} The histone acetyltransferase activity of p300 contributes to its effect of increasing the promoter activity of guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1).⁸⁵ All-trans retinoic acid increases H3 and H4 acetylation, enhances their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter, and increases promoter activity.⁸⁶ Transcriptional factor Yin Yang 1 associates with histone deacetylase 2 and increases the promoter activity of human B-type natriuretic peptide gene, while treatment with the histone deacetylase inhibitor trichostatin A decreases hBNP reporter activity.⁸⁷ Other examples of BP-relevant genes that histone modifications may regulate include ENaC^{88, 89} and Angiotensin converting enzyme 1,⁹⁰ as reviewed previously.¹⁶

IV.3. lncRNA

Several dozen lncRNAs are expressed differentially in the renal outer medulla in SS and SS.13^BN26 rats fed a high-salt diet.⁹¹ Two of these are located within the congenic region of SS.13^BN26. Several hundred lncRNAs are expressed differentially in the kidney between SS and Dahl salt-resistant rats or SHR⁹² or in the aorta between SHR and Wistar-Kyoto rats.⁹³

Several lncRNAs participate in the regulation of processes relevant to BP regulation.⁹⁴ For example, treating rats’ vascular smooth muscle cells with angiotensin II results in the differential expression of several lncRNAs.⁹⁵ LncRNA hexokinase 2 pseudogene 1 (HK2P1) is dysregulated in the decidua of severe preeclampsia patients, and HK2P1 promotes human endometrial stromal cells’ proliferation and differentiation and appears to regulate hexokinase 2 expression via competition for the shared miR-6887-3p.⁹⁶

IV.4. Chromatin conformation

Stodola et al. used a 4C-seq approach to map the chromatin conformation in the physical proximity of the proximal promoter of the renin gene Ren in rat cardiac endothelial cells.⁹⁷ Many of the contact regions are located on rat chromosome 13, which harbors Ren, including a Ren enhancer 6 kbp from the proximal promoter identified previously (Figure 2). Other contact regions are distributed across all chromosomes except chromosome X. These contact regions are enriched significantly for genes of which the expression is correlated with Ren and for quantitative trait loci associated with BP, cardiovascular, and renal phenotypes.

Figure 2. Several DNA regions spanning chromosome 13 are in physical proximity of the renin proximal promoter.

The contact map shown derives from a 4C-seq analysis of rat cardiac microvascular endothelial cells. Top: Blue lines indicate contacts between Ren proximal promoter and distant interacting regions on chromosome 13. Bottom: Contacts with the Ren proximal promoter within 1 Mb of the viewpoint. Location of the Ren proximal promoter viewpoint is indicated with a dashed red line. Red fragments labeled “Exact Interaction” have significant contact with the Ren proximal promoter in multiple samples analyzed. Reproduced from Stodola et al.⁹⁷

A genetic fine mapping analysis prioritizes rs9349379, a common and intronic SNP, as the putative causal variant in a locus that has been associated with hypertension and four other vascular diseases.⁹⁸ Genome editing in cultured cells demonstrates that rs9349379 regulates the expression of the endothelin 1 (EDN1) gene located 600 kb upstream of the genetic locus. Chromatin conformation mapping using 4C-seq did not identify substantial contacts between rs9349379 and EDN1 promoter. However, the EDN1 promoter contacts multiple sites extending up to 500 kb in both the 3’ and 5’ directions, which overlap with rs9349379’s contact region. The overlap region appears to contain a super-enhancer.⁹⁸

Functional Role of Epigenetic Mechanisms in Hypertension

Compared to the numerous reports correlating changes in epigenetic mediators with BP or hypertension, clear evidence of epigenetic changes’ functional role in the regulation of BP or the development of hypertension remains scarce. Some of this evidence is summarized in Table 2 and discussed below.

Table 2.

Evidence of Epigenetic Mediators’ Functional Role in Hypertension.

Target	Species/model	Approach	Effect on BP	Reference
Cg08035323 methylation site	Humans	Bidirectional Mendelian randomization	Influencing BP	53
DNA de novo (de)methylation in the kidney	SS Rats	Intra-renal injection of GapmeRs targeting Dnmt3a and Tet3	Attenuating salt-induced hypertension	62
DNA methylation	CBS+/− mice	5-aza-2'-deoxycytidine treatment	Attenuating hypertension	99
DNA methylation	Rats, long-term intermittent hypoxia	Decitabine treatment	Attenuating hypertension	100
Histone acetylation*	SHR	Valproic acid treatment	Attenuating hypertension	102
Histone acetylation*	DJ-1/park7−/− mice	Valproic acid treatment	Attenuating increase in systolic BP	107
Histone methylation	Mice	LSD1 heterozygous knockout	Increasing BP	108
lncRNA Rffl-lnc1*	Rats	Gene targeting and knockin	A 19bp indel increases BP	110
lncRNA AK098656*	Rats	Transgenic overexpression	Resulting in hypertension	111

^*Whether epigenetic changes are involved remains to be tested.

BP, blood pressure; SS, Dahl salt-sensitive rats; Dnmt, DNA methyltransferase; Tet, ten-eleven translocase; CBS, Cystathionine-β-synthase; SHR, spontaneously hypertensive rats; LSD1, lysine-specific demethylase-1.

V.1. DNA methylation

In Richard et al.’s study, Mendelian randomization analysis suggested the presence of causal regulatory relations among select methylation sites, BP, and gene expression.⁵³ Methylation at cg08035323 (TAF1B-YWHAQ) appeared to influence BP, while BP influenced methylation at cg00533891 (ZMIZ1), cg00574958 (CPT1A), and cg02711608 (SLC1A5). In addition, the expression of TSPAN2 was a putative mediator of the association between methylation at cg23999170 and BP.

The renal outer medulla consists primarily of non-dividing cells that have differentiated terminally. Changes in DNA methylation in the renal outer medulla of rat models occurred after only a few days on a high-salt diet^{20, 62} and likely were the consequence of de novo DNA methylation or de-methylation. Liu et al. targeted Dnmt3a and Tet3 by administering anti-Dnmt3a and anti-Tet3 GapmeR’s directly into the renal interstitium in SS rats. The treatment attenuated salt-induced hypertension and prevented the significant differential expression of 76% of the genes that would respond to the high-salt diet otherwise. The study was one of the first to demonstrate a functional role of de novo DNA methylation or de-methylation in the development of diseases primarily involving non-dividing cells.⁶²

Cystathionine-β-synthase heterozygous knockout mice exhibit hyperhomocysteinemia, high BP, global hypermethylation, and up-regulation of Dnmt1 and Dnmt3a, and treatment with 5-aza-2’-deoxycytidine normalizes BP in these mice.⁹⁹ Sprague-Dawley rats exposed to long-term (30-day) intermittent hypoxia develop hypertension, higher DNA methylation of genes encoding anti-oxidant enzymes, and other abnormalities. These abnormalities do not normalize after recovery, but they can be prevented or normalized by treatment with decitabine either during intermittent hypoxia or the recovery period.¹⁰⁰

Treating mice with a compound that attenuates aging-related increases in BP enhances DNA demethylase activity, decreases methylation of Klotho gene in the kidneys, and abolishes aging-associated downregulation of secreted Klotho levels in both kidneys and serum.¹⁰¹

V.2. Histone modification

Treatment with HDAC inhibitor valproic acid attenuates hypertension in SHR¹⁰² and a rat model of DOCA-salt hypertension.¹⁰³ HDAC inhibitors trichostatin A, butyric acid, and MS-275 decrease eNOS protein levels in endothelial cells.¹⁰⁴ However, it is unclear whether changes in histone acetylation mediate these effects. HDAC can influence eNOS activity and mineralocorticoid receptors by changing the acetylation of eNOS and mineralocorticoid receptor proteins.^103,105 However, in several non-endothelial cell types, treatment with trichostatin A and sodium butyrate induces eNOS expression, which is accompanied by increased acetylation of histone H3 associated with the eNOS 5’-flanking region. Combined treatment with trichostatin A and 5-aza-2’-deoxycytidine synergistically induces eNOS expression in non-endothelial cells.¹⁰⁶

DJ-1/park7(−/−) mice exhibit lower eNOS expression, which is associated with increased HDAC1 recruitment and decreased H3 acetylation at the eNOS promoter, as well as higher systolic BP, and treatment with HDAC inhibitor valproic acid attenuated the increase of systolic BP in these mice.¹⁰⁷ Mice with heterozygous knockout of lysine-specific demethylase-1 (LSD1, Kdm1a), which induces demethylation of H3K4 or H3K9, have higher BP than do wild-type mice when they are fed a 4% NaCl diet, but not when fed a 0.08% NaCl diet.¹⁰⁸ African-Americans and Hispanics, but not Caucasians, who carry the minor allele of one or two LSD1 SNPs, display greater changes in systolic BP in response to changes from a low to liberal salt diet.¹⁰⁹

V.3. lncRNA

Several lncRNAs have been reported to influence BP. Targeted disruption of the rat Rffl-lnc1 locus, which is homologous to a human QT-interval locus, causes aberrant, short QT-intervals and elevated BP, while a CRISPR/Cas9 based targeted knock-in rescues the aberrant QT-interval and BP phenotypes in the strain that has a deletion polymorphism.¹¹⁰ Transgenic overexpression of lncRNA AK098656 in rats results in spontaneous development of hypertension.¹¹¹ However, it is unclear whether these lncRNAs influence BP by regulating stable changes in DNA function.

Key Questions, Concepts, and Technologies to Advance the Field

While increasing evidence supports epigenetic mediators’ role in hypertension, how great the role is remains to be assessed and likely will vary between patients and models. Transgenerational epigenetic inheritance and its role in hypertension remain fascinating, but largely untested, possibilities. In conditions in which fetal programming and early life experiences affect adult hypertension, the specific epigenetic mechanisms involved and the way they contribute to hypertension need to be elucidated further. A large majority of the BP-associated SNPs identified and replicated by GWAS are non-coding SNPs, nearly all of which overlap with potential enhancers, or are in linkage disequilibrium with SNPs that overlap them.¹¹² It is important to understand whether and the way in which epigenetic mechanisms might mediate these SNPs’ effect on gene expression and BP.

Changing the activity of one DNMT or TET or the level of just one histone modification potentially can change many genes’ expression. However, if a patient’s development of hypertension involves a large number of molecular changes, it will be necessary to identify and understand all of those molecular changes to understand truly the way hypertension develops in that patient and the best way to treat it. The tree-like paradigm depicted in Figure 3 can be used as a framework to understand the way numerous molecular changes converge to form regulatory networks that underlie a complex trait or disease.^{16, 113, 114} An increasing number of investigators are working in the emerging discipline of systems molecular medicine (Figure 4) with the goal to construct the tree of regulatory mechanisms underlying complex physiological traits and diseases.^{115, 116}

Figure 3. A tree-like paradigm for understanding the mechanisms underlying complex traits and diseases.

The large number of molecular changes, including epigenetic changes, involved in a complex trait, such as BP, are like leaves on a tree that will merge gradually into small and then large branches. Cellular signaling and metabolic pathways are the small branches, and physiological pathways are the large branches. In the case of BP, the pathways ultimately will influence cardiac output or peripheral vascular resistance, the final determinants of BP. The arrows in the graph illustrate the concept of convergence that is fundamental to this tree-like paradigm. Divergence, cross-talks and feedback loops are not depicted, but could contribute importantly to the regulatory network.

Figure 4. The emerging discipline of systems molecular medicine.

To construct the regulatory network underlying a complex physiological trait or disease, comprehensive molecular profiles, including epigenetic profiles, with sufficient spatial and temporal resolution need to be obtained from patients and clinically relevant models. The molecular compendia will have to be analyzed with a systems view, with respect to both the relations among molecules and in the context of organ systems function. Informative perturbations will need to be applied, followed by reiterations of molecular and systems analyses, to reveal and validate the underlying regulatory network. The discipline of systems molecular medicine is emerging from the intersection of several concepts and disciplines including systems biology, physiology, genomics, translational research, and precision medicine.

In applying the concept of systems molecular medicine to epigenetic research on hypertension, it would be highly valuable to obtain epigenetic profiles from human tissues that are physiologically important in BP regulation and hypertension.^{117, 118, 119} The epigenetic and expression profiles available from studies such as the Encyclopedia of DNA Elements (ENCODE) Project and the Genotype-Tissue Expression (GTEx) Project are helpful, but the anatomical resolution of the tissues used and their relevance to BP regulation are limited. Data on genome-wide epigenetic profiles in tissues, such as resistance arterioles and nephron segments, from healthy humans and patients in various stages of hypertension development would be a powerful resource to advance epigenetic research on hypertension.^{112, 120, 121}

Using oligonucleotides or conditional knockout to target specific DNA or histone modifying enzymes in a specific tissue or at a specific stage of disease development, coupled with genome-wide analysis of epigenetic mediators, will continue to be a powerful approach to study epigenetic mediators’ functional role in vivo.⁶² Development or further improvement of experimental techniques to induce methylation and de-methylation at specific genomic loci, alter specific histone modifications, and form or break specific chromatin loops, would be of great value in the study of specific epigenetic features’ functional role. Further, these techniques would have to be adapted for use in whole organisms to study traits such as BP.^{122, 123}