Supplemental Digital Content is available in the text. If unifying principles could be revealed for how the same genome encodes different eukaryotic cells and for how genetic variability and environmental input are integrated to impact cardiovascular health, grand challenges in basic cell biology and translational medicine may succumb to experimental dissection. A rich body of work in model systems has implicated chromatin-modifying enzymes, DNA methylation, noncoding RNAs, and other transcriptome-shaping factors in adult health and in the development, progression, and mitigation of cardiovascular disease. Meanwhile, deployment of epigenomic tools, powered by next-generation sequencing technologies in cardiovascular models and human populations, has enabled description of epigenomic landscapes underpinning cellular function in the cardiovascular system. This essay aims to unpack the conceptual framework in which epigenomes are studied and to stimulate discussion on how principles of chromatin function may inform investigations of cardiovascular disease and the development of new therapies. The tracks of the train point where it is going—they also reveal where it has been. The tracks of the leopard in the snow do only the latter. Which is the case for epigenetic processes in cardiovascular health and disease? Chromatin enables bespoke storage and retrieval of genetic information across the hundreds of cells in a multicellular organism. Chromatin is also perhaps the largest, in terms of physical size and list of component parts, and most functionally diverse molecular structure in existence, enabling the vast diversity of the multicellular world. The goals of this essay are (1) to review recent advancements in the concepts of epigenomics; (2) to summarize the evidence that epigenomic processes participate in normal and diseased cardiovascular physiology; and (3) to stimulate discussion about the prospects for novel basic science and translational investigations of epigenomes in the cardiovascular system.Online Data Supplement).In development, the setting in which epigenesis has been most extensively examined, the task of epigenetic processes is to guide the unidirectional differentiation of cells. In adulthood, epigenetic mechanisms provide stability, maintaining the blueprints laid down in development and resisting environmental stresses and stochastic intracellular changes. In cardiovascular disease, an area in which epigenetic processes are increasingly appreciated to operate and in which insights into human health are the most actionable, the task, teleologically, of chromatin is not all that clear. Are the epigenetic processes at work in the cell during disease always combating the insult, attempting to restore and preserve healthy adult phenotypes? Have epigenetic processes been hijacked by evolution to contribute to cell survival decisions? What are the operational principles of epigenetics and how are they distinguished from gene regulation? For this essay, we define an epigenome as a genome plus everything binding to and modifying it  genetic and pharmacological gain- and loss-of-function studies in the cardiovascular system targeting individual histone-modifying enzymes, chromatin remodelers, chromatin structural components, and regulatory RNAs can induce or prevent pathology; (2) descriptive epigenomic investigations from humans and animal models demonstrate that widespread reprogramming of histone modifications, DNA methylation, and protein binding occurs during the development of disease; (3) data have arisen that indicate epigenomic processes are interconnected with other cellular events  that are deranged in disease and that modulation of these epigenetic processes may hold potential for therapeutic intervention. Let us now examine recent advancements in our understanding of how chromatin controls gene expression and consider the implications for cardiovascular disease.1: bind histone H3 to histone H4 and combine 2 of these dimers to form a tetramer; then combine this tetramer with histone H2A and histone H2B, twice, and embrace this octamer in turn with ≈145 to 147 bp of DNA. Crystal structures2 of the nucleosome in 1997 revealed this octameric protein complex to be assembled through histone fold dimers of H2A with H2B and H3 with H4. The intervening years have seen the discovery of multiple histone isoform variants that can exert structural changes at the atomic level on the functional properties of the nucleosome: 1 example is histone H2A.z, an H2A variant associated with active transcription, which operates by altering the interfaces between H2A–H2B dimers, as well as among the entire tetramer, partially destabilizing the nucleosome core (thereby facilitating nucleosome eviction during transcription).3 Another example is centromeric histone H3, called CENP-A, which replaces H3 in centromeric nucleosomes, altering the interactions among histones but maintaining the octameric structure.4 Mammalian genomes encode multiple versions of core histones, with each family hosting multiple variants of unknown significance. These histone isoform variants have been shown to participate in development and disease through altered roles in DNA replication, chromosome packaging, and DNA accessibility.6 Proteomics experiments have demonstrated variable expression of these isoforms in different cell types, including in the heart,7 and the stoichiometry of histones changes with disease, such as in pressure overload hypertrophy.8 In general, however, the nucleosome is among the most conserved protein structures known.The epigenome has 2 fundamental components: the structural features of chromatin and the enzymes, RNAs, small molecules, and processes that modify these features. For example, a nucleosome is the chromatin structural unit, but an ATP-dependent chromatin remodeler is an enzyme complex that modifies this structural unit. Here is how you make a nucleosome9 An additional class of chromatin structural element includes non-nucleosome chromatin structural proteins, such as histone H1 family proteins, CTCF (CCCTC-binding factor), and HMG (high mobility group) proteins. Histone H1, the so-called linker histone , is not part of the nucleosome core particle but may participate in the formation of higher order chromatin fibers through interactions with nucleosome histones and DNA.10 Structures of nucleosomes with H1 (which has been termed the chromatosome11) show the latter nestled just outside of the core,13 making independent contacts with DNA, but whether linker histones are obligatory components of a structural feature in eukaryotic chromatin remains a matter of debate. Observations from a variety of eukaryotic systems implicate linker histone H1 in genomic functions, including DNA repair/stability, replication, and transcription.14 CTCF and HMG proteins each have likewise been attributed a wide range of possible functions, based mostly on gain-/loss-of-function studies in cell systems. As a general mechanism, CTCF binds DNA and facilitates chromatin looping 15; specificity may arise in different cell types to facilitate regulatory interactions. HMG proteins, of which there are many families, bind and stabilize distinct structural features in DNA, thereby contributing to high order chromatin anatomy and perhaps assemblies of nucleosomes. Interestingly, the HMGA family of these proteins has been implicated in cardiac disease, with both hetero- and homozygous knockout mice developing hypertrophy.16Where nucleosomes bind along the genome is influenced—certainly in reconstituted systems but likely also in vivo to some degree—by primary DNA sequence, with poly dA:dT–rich regions being comparably depleted of nucleosomes, due in part to the biophysical properties of these regions that resist bending necessary for nucleosome binding.How nucleosomes position along chromatin is largely specified, in multicellular eukaryotes, by ATP-dependent chromatin remodeling enzymes and the trio of protein classes responsible for the so-called the histone code, those being writers, erasers, and readers, that add, remove, or interpret histone modifications, respectively. Although all these processes exist in other organelles and compartments , the allegorical grammar terminology adopted for chromatin post-translational modification (PTMs) has been particularly helpful in conceptualizing experiments into how gene expression is regulated and particularly effective in biasing the interpretation of results.19 Histone post-translational modifications were originally described in the 1960s20 but only with the advent of chromatin immunoprecipitation and DNA sequencing  is highly dependent on the informatics tools used to map reads and subsequently call peaks23 . Thus, one does not know whether histone PTMs occupy the same nucleosome, which would be required for a histone PTM reader protein to distinguish a full, accurate code from a partial and inaccurate code .The histone code hypothesis (discussed in greater detail below), as originally articulated by Strahl and Allis,27 but has not been demonstrated to operate in mammals in a tissue-specific manner. Progress has recently been made in the area of heart failure, however, with the example of the BET  bromodomain protein BRD4 (bromodomain-containing protein 4), whose inhibition with the small molecule JQ1 prevents hypertrophy and associated transcriptional changes29 and whose pharmacological inhibition reverses some of the fibrotic and deleterious structural remodeling in the wake of infarction or pressure overload .30 These studies have raised the intriguing possibility that epigenetic readers may be novel targets of epigenetic therapy in cardiovascular disease31 given their cell type–specific expression patterns, ability to integrate the actions of multiple chromatin modifiers, and their role to program gene expression with high fidelity at individual loci.Histone writers and erasers are signal transduction processes that sometimes use histones as substrates. The concept of the reader, however, is an indispensable component of epigenetic language and one without obvious counterpart outside the context of the chromatin . To work as advertised—that is, to distinguish target region α from nontarget β, to bind α, and then to do something—readers must be able to simultaneously recognize and discriminate between histone PTMs, which has been shown in some model systems,32 When examining a new PTM, no good antibody, and no tractable cell system for transgenesis of a tagged surrogate, means no genome-wide address book, which means, for the present, the given modification is assumed to be noise. That histone modifications cannot be unequivocally examined by genetic approaches  is the reason why they are alternatively so frustrating and interesting.All 4 core histones are extensively post-translationally modified, with different histones (and different modifications) studied to varying extents and degrees of rigor and functional depth. The bane of accurate and reproducible proteomic mass spectrometry is that a truly unbiased experiment to detect PTMs will identify many, with no certainty about which ones are important versus otiose signaling noise. The most common answer to the question of which PTMs are important for chromatin regulation is some variation on “only the ones shown to be functionally involved in a phenotype.” Often the PTMs deemed most tantalizing are those previously published on in lower organisms and those for which commercially available antibodies exist. In the chromatin world, these PTMs and the associated antibodies are the ones getting the most game time in ChIP-seq experiments. A quantitative example: the silencing mark histone H3 lysine 27 trimethylation, which enriches around gene regulatory regions, is represented at the time of this writing by 403 ChIP-seq data sets in ENCODE (Encyclopedia of DNA Elements) and ≈2000 to 3000 publications in PubMed ; those numbers for H3 K27 acetylation, a euchromatic mark (note: on the same residue of the same protein), were 384 ChIP-seq datasets and ≈500 publications. In contrast, a recently identified succinylation on human histone H3 K79, also found to enrich in the transcription start sites of active genes, has 1 publication and 1 ChIP-seq data set.33 These early studies did not directly measure histone PTMs—although they (the PTMs) likely were reflected in the electrophoretic patterns reported in these investigations—because of the absence of accurate and quantitative mass spectrometry of proteins and PTMs. More recently, quantitative analysis of nuclear proteins34 or chromatin-associated proteins in the heart revealed the proteins regulating cardiac epigenomes7 and described changes in histone protein isoform stoichiometry8 in the setting of pressure overload hypertrophy, yet these studies did not characterize histone PTMs. Renal nuclear proteomes have also been explored,36 with physiological implications for cardiovascular disease. Across eukaryotes, ≈500 post-translational individual modifications have been identified just on the core nucleosome particle.22 Few of these have been identified, much less quantified and proficiently interrogated, in the cardiovascular system . Recent studies have used large libraries of histone modifications, in some cases representing >100 different nucleosome combinations , to investigate the ability of particular histone PTM combinations to influence the activity of human ATP-dependent chromatin remodelers.38 These libraries39 allow for the effects of individual recombinant nucleosomes with designer histone modifications (and combinations of modifications) to be tested in vitro for their ability to influence a host of chromatin properties, such as the binding preferences for transcription factors or chromatin-modifying enzymes.What about other changes in protein levels in the nucleus? The first attempts to understand proteomic remodeling of the cardiomyopathic nucleus were conducted in hamsters.41 (with implications for atherosclerosis42), to attenuate hypertension,43 to ameliorate ischemic/reperfusion injury and postischemic remodeling,47 and to block cardiac hypertrophy in the setting of heart failure.50 Molecular dissection of these phenomena, particularly in the setting of cardiac growth, have revealed HDACs to be powerful hypertrophic modulators: loss of HDAC9 leads to prodigious cardiac growth,51 HDAC4 and 5 regulate CamKII -dependent gene regulation,53 HDAC2 regulates GSK3β (glycogen synthase kinase 3 beta)-Akt–dependent fetal gene activation in hypertrophy,54 to name just a few examples. Indeed, the literature on the role of HDAC targeting by drugs or genetic manipulation in the cardiovascular system is sufficiently vast to devour entire, authoritative reviews articles.56Investigations into the enzymes responsible for depositing and removing histone modifications have demonstrated striking phenotypes in a range of cardiovascular syndromes. Histone deacetylases (HDACs) are one of the most extensively studied families of histone-modifying enzymes in the cardiovascular system. HDACs consist of 4 families, each with distinct isoforms which in turn have distinct histone and—in some cases—nonhistone targets, distinct cellular locations and distinct biological functions. Inhibition of HDACs has been shown pharmacologically  to prevent proliferation of vascular smooth muscle cells57 Multiple sirtuin isoforms have been shown to facilitate DNA repair through homologous recombination. Unlike other HDACs, sirtuins require nicotinamide adenine dinucleotide as a cofactor for activity. Intriguingly, these proteins have been shown to participate in an isoform-specific manner in myocardial ischemia, oxidative stress, apoptotic cell death, and cardiac hypertrophy.57 Sirtuin isoforms have also been shown to exert salubrious effects on fatty acid, amino acid, and glucose metabolism in mouse studies, which have led to clinical trials.58 A search at clinicaltrials.gov with the keywords cardiovascular disease and epigenetics revealed 29 studies in the active or recently completed phase. These studies range in their employment of epigenetic measurements or interventions. For example, some describe measuring epigenetic end points in response to treatments targeting known cardiovascular risk factors in common cardiovascular disease . Another example is a trial targeting rare genetic diseases with epigenetic manifestations in the cardiovascular system , which measured DNA methylation, histone PTMs, and coding/noncoding RNAs expression in blood, white cells, and other tissues. Other examples investigate use of hypomethylating agents, including 5′azacytidine and 5-aza-2′-deoxycytidine, in adult patients with acute myeloid leukemia and atrial fibrillation  or study DNA methylation as a marker for high blood pressure in the setting of pregnancy . The Human Epigenetic Drug Database (hedds.org) is a useful, searchable resource for information on epigenetic drugs, their targets, and associated data sets including, when relevant, links to clinical trials. Several epigenetic drugs are Food and Drug Administration approved for clinical use, including HDAC inhibitors and DNA methyltransferase (DNMT) inhibitors,59 and several of these have been explored in cardiovascular disease .Class III HDACs are also known as sirtuins and have been shown to exert powerful effects in the cardiovascular system after their initial identification and association with longevity in yeast. There are 7 sirtuin isoforms, each with varied subcellular localization and some of which, such as sirtuins 1, 2, 6, and 7, localize to nucleus and thus are poised to use DNA-bound histones as substrates, including in the heart.62 One of the best characterized histone acetyltransferases is p300 which, in addition to its common residence at enhancer elements, has been shown to regulate genes that inhibit endothelial cell inflammation in the setting of atherosclerosis63 and to attenuate salt-induced hypertensive heart failure64 and agonist-induced cardiac hypertrophy65 .Histone acetyltransferases also constitute a large family of genes and have numerous nuclear and non-nuclear substrates  and have been characterized with varying degrees of specificity in cardiac and vascular cells.67 SET and MYND containing histone methyltransferase  family member Smyd1, which is restricted to striated muscle, has been shown to participate in cardiac phenotype through loss-of-function studies in the adult heart, which resulted in hypertrophy, dilation, and derepression of some cardiac disease genes.68 Although Smyd1 localizes to the nucleus and interacts with chromatin in the adult heart68 , a substantial portion of the protein is non-nuclear. This non-nuclear population of the protein has also been implicated in adult cardiac function  and heart development.74 A different SET family protein, G9a , has been shown by loss-of-function studies to play a key role in adult cardiac phenotype: inducible MerCreMer-dependent depletion resulted in cardiac hypertrophy, modestly depressed ejection fraction, and fibrosis through a mechanism that involves targeted derangement of multiple histone methylation marks (including H3K9me2/3 and H3K27me3) at genes involved in cardiac function.75 SET7 was shown in a human microvascular endothelial cell line to regulate target gene (IL-8 [interleukin 8] and HMOX1 [heme oxygenase 1]) expression in a H3K4me1-dependent and -independent manner.76 Intriguingly, this process is tightly linked to glucose levels, demonstrating a metabolic sensing mechanism on chromatin.77 On the removal side of methylation, genetic loss or augmentation of the histone demethylase JMJD2A, respectively, blocked or exacerbated cardiac hypertrophy in mice.78 A recent pharmacological study showed that inhibition of histone methylation at H3K9 with the compound chaetocin (which targets the enzyme [SU(VAR)3–9] responsible for conversion of H3K9me2 to H3K9me3) attenuates some aspects of salt-induced cardiac dysfunction —while not affecting pathological gene regulation and only modestly impacting hypertrophic growth—in part through diminished H3K9me3 at repetitive elements and mitochondrial genes.79The histone methyltransferase SET domain containing 2 was recently shown to be essential for myoblast differentiation in a process involving modification of histone H3K36 trimethylation.80 suggesting that features of myocyte proliferative/regenerative plasticity may be revived through histone-modifying enzymes. Ezh2 stabilizes forming blood vessels in the mouse embryo,81 and pharmacological inhibition of Ezh2 (and some H3K27me3 target loci) improved outcomes in hindlimb ischemia.82 Some naturally occurring compounds target histone-modulating enzymes (and nonhistone lysine residue-containing proteins) and have substantial in vivo benefit in conditions, such as cancer.84 Because many of these compounds are present in diets shown epidemiologically to promote cardiovascular health (such as cruciferous vegetables), part of this effect may be through actions to promote—at the subcellular level and across organs—favorable epigenomic health.The polycomb repressive complex (PRC) is one of the best-studied gene silencing complexes (responsible for H3K27me3 deposition) and has been implicated—usually through the actions and genetic disruption of one or more of its components—in a wide variety of higher phenotypes in mammals. In the cardiovascular system, the Ezh subunits 1 and 2 were shown to be differentially involved in cardiac development and regeneration: both were necessary for normal development, with Ezh1 but not 2 being required for neonatal heart regeneration and with Ezh1 but not 2 being capable, via overexpression, of promoting regeneration in the hearts of mice aged outside the established neonatal regenerative period,71 HDACs on myofilaments,85 and HDAC family members of the sirtuin class which regulate many substrates in mitochondria and cytosol57).Although the histone isoforms and even specific residues targeted by individual enzymes have often been worked out in reconstituted systems in vitro, such information is almost universally lacking from such studies in animal models of the cardiovascular system . It is also important to note that many of these histone-modifying enzymes target nonhistone substrates that have no direct relationship to gene expression or epigenetics but yet exhibit powerful effects on complex organ level phenotypes . And second, how do the cadre of expressed-at-any-given-time enzymes decide which nucleosomes to modify ? One approach to answer these questions would be to identify intermediate indices of epigenomic function like accessibility and structure, designing interventions that modulate these indices.88 Because presence of bivalent marks at a given locus are necessary but not sufficient to specify a bivalent locus, recent studies have focused on evaluating the role of ATP-dependent chromatin remodelers in this process.89 Studies in the cardiovascular system have examined the role of these complexes in tissue level phenotypes by genetic manipulation of the ATPase subunits of the brahma-associated factor complex, Brm (Brahma), or Brg1 . For example, genetic disruption of either of these molecules alone had no effect on retinal angiogenesis in neonates, exercise-induced angiogenesis in adult skeletal muscle, or tumor angiogenesis, whereas mice with disruption of both Brm and Brg1 after birth exhibited fatal vascular malfunction in the heart and gut during the early postnatal period90 . Brg1 has been shown to be involved in zebrafish myocyte proliferation and cardiac regeneration,92 mesoderm, and hence cardiomyocyte, differentiation in cell culture, in part by modulating enhancer activity,93 whereas both Brg1 and Smarca3  are required for normal heart development in mouse96 . Early formation of vasculature and erythropoiesis in mouse is dependent on Brg1 but not Brm in hematopoietic and endothelial cells,97 implying functional distinction between complexes seeded with these different ATPases during cardiovascular development (a similar conclusion was made from genetic disruption in smooth muscle cells98), a functional involvement that may extend into adulthood in the setting of endothelial injury and presumably disease.99ATP-dependent chromatin remodeling enzyme complexes use the energy from ATP to translocate DNA through the nucleosome. That is, the ATP-dependent chromatin remodelers reposition nucleosomes along the genome according, in part, to cues harbored in the spectrum of histone tail PTMs, thereby enabling fundamental genomic processes, like transcription, nucleosome assembly/disassembly, mitosis, meiosis, and chromosome segregation. One of the most studied of these complexes is the SWItch/sucrose nonfermentable complex, originally identified in yeast (and known to have >10 protein components) and its mammalian cousin the brahma-associated factor complex . Models for the actions of these remodelers are informed by protein crystallography studies, in vitro biochemical assays, and ChIP-seq–based genome-wide measurements and are thus highly developed and can help to explain the observed dynamism of chromatin in development, between cell types and in stimulus response.100 such as neurons [where it may account for 25% of methylation], although this has only begun to be explored in the cardiovascular system102). Also contrasting with the plethora of proteins controlling histone PTM, DNA methylation is directly added or removed by a narrow suite of enzymes: maintenance (DNMT1) and de novo  methyltransferases which establish methylation patterns after mitosis and replication and alter the pattern of methylation during organismal development and disease, respectively. Demethylation of DNA occurs in part via nonenzymatic means during replication, as well as during normal and pathological conditions in nondividing cells. Conversion of 5-methyl-cytosine to 5-hydroxymethylcytosine is catalyzed by the 10-11 translocation methylcytosine dioxygenase 1 family of enzymes—an active, selective process. The 5-hydroxymethylcytosine is a less stable modification, prone to nonenzymatic conversion to (unmodified) cytosine, and has thus been proposed as a molecular beacon of genes switching from off to on. DNA methylation patterns are erased and re-established transgenerationally, but this process seems to involve faithful perpetuation of methylation marks along the genetic lineage, that is, from parent to progeny .DNA methylation is dynamic during vertebrate development, where it reinforces cell fate decisions and controls imprinting, or the dependence of gene/protein expression (and associated phenotypes) on whether a given version of a gene is expressed from maternal or paternal allele. Unlike histone modifications, which can occur on any number of different amino acids apparently without heed to locale , DNA methylation targets a single residue, cytosine, usually in a single context . Broadly construed, DNA methylation in CpG islands  and shores (areas around said islands) tends to be associated with gene silencing.106 Conversely, the bodies of mRNA-encoding genes tend to be methylated, without an established correlation allowing prediction of expression. Other methods for large scale analysis of DNA methylation include methylation immunoprecipitation (which has been used to identify methylation-dependent regulation of atherosclerotic risk in humans107) and DNA methylation arrays (notably the Illumina 450 chips), the latter of which has been extensively deployed in humans to characterize methylation patterns associated with a host of pathophysiological conditions including cancer,108 high blood pressure,109 body mass index and obesity,111 atrial fibrillation,112 inflammation,113 and death.114 Perhaps, because of cost and technical demands, reduced representational bisulfite sequencing (or much more expensive whole genome bisulfite sequencing) has been applied to a smaller list of diseases. Such data from mice, however, show that DNA methylation plays a powerful role in heritable differences in response to metabolic syndrome103 and may contribute to catecholamine-induced cardiac pathology.104 DNA methylation and hydroxymethylation abnormalities have been found in animal116 and human118 heart failure, associated with changes in expression of pathological genes. Work from mouse cardiomyocytes suggests that DNA methylation largely obeys chromatin structural features of A/B compartmentalization , wherein dynamics of DNA methylation during lineage commitment are enriched in A (active) compartments and genetic disruption of DNA methylation (via DNMT3a and 3b knockout) does not alter compartmentalization.102 This observation supports a passive relationship between DNA methylation and chromatin structure, at least in the formation phase.Bisulfite sequencing is the method for unequivocal determination of methylation status ; another is to favor relaxing of chromatin and transcription (as in gene bodies). These opposing effects must require the intervention of discriminating factor(s), perhaps including methyl-CpG binding proteins, but this field currently wants for established rules and actors. One investigation121 demonstrated that MeCP2, a methyl-CpG binding protein, is reversibly downregulated in a mouse model of pressure overload . These findings suggest that the actions of DNA methylation may be modulated in the diseased heart at multiple levels, including methylation, demethylation, and reading of methylation. Another mode of action is through trans effects, whereby a methylation event can regulate the expression of a gene in a distal region of the genome . Studies from human cardiac development reveal an enrichment of regulatory elements, including DNA methylation sites, in regions of genetic variation associated with heart disease,122 supporting a molecular link between chromatin regulation and genetic variation in the context of pathological phenotypes. By regulate, it is meant here that the methylation event is shown to correlate—with genome-wide statistical significance—with the expression of a gene, a methylation quantitative trait locus. This regulation may take the form of enhancer element formation/modification (discussed below) or other as-yet uncharacterized chromatin structure effect.What is the import that methylation patterns are associated with complex human phenotypes? One method through which DNA methylation has its molecular effects is to reinforce prevailing chromatin landscapes by preventing accessibility and facilitating compaction . Furthermore, these complex diseases are often associated with global changes in DNA methylation. In some cases, the genetic or epigenetic lesion occurs in a gene whose aberrant function can exert a dominant role in disease pathogenesis. In other cases, these epigenomic changes may instead be general hallmarks of perturbed cellular function, whereby the normal parameter space for gene expression is expanded, facilitating dysfunction of multiple cellular processes. For many observations on histone PTMs and DNA methylation in cardiovascular disease, the train or snow leopard question remains unanswered.The prevalence of cancer128). Of particular interest to chromatin biology, however, is the concept that long noncoding RNAs (lncRNAs) may participate in gene regulation by modulating chromatin structure.It is now appreciated that most of the genome is transcribed, if only a small portion of that transcriptome encodes mRNA destined for translation, with intriguing differences in this noncoding transcriptome across cell types and after pathological insult. Noncoding RNA biology is a specialized discipline unto itself, with new species of RNA—ascribed really cool and sometimes bizarre functions—identified seemingly endlessly and will not be extensively reviewed herein (excellent reviews on the roles of various noncoding RNAs in cardiovascular biology have emerged130), is Xist, which is centrally involved in X chromosome inactivation. Xist is transcribed from and acts in cis to silence the X chromosome through a process that recruits, via direct binding of Xist to the proteins, the PRC2 complex, and YY1. A depositor of histone H3K37me3 silencing marks and transcriptional repressor, respectively, these proteins in turn compact the X chromosome and prevent further transcription.131 This model—lncRNA binding to a specific region of chromatin and recruiting histone-modifying enzymes—is appealing because it solves the problem of DNA sequence recognition, of which many histone modifiers are incapable. Another well-characterized lncRNA that binds PRC2 subunits is Hotair, involved in gene silencing in mammals and shown to regulate chromatin in trans outside of the context of X inactivation.132 These studies led to a gold rush on PRC2-interacting lncRNAs, which have been estimated to range in number from hundreds to thousands in mice133 and humans.134 The general properties, if they exist, through which these lncRNAs couple PRC2 to chromatin are the focus of continued investigation.136 It may be that the genes for these molecules are distributed across the genome and the lncRNAs in turn all act in a local manner to recruit and modulate chromatin machinery to a given gene expression environment.137 Yet there are clear limitations were the cell to attempt to repeat this process with other lncRNAs: physiological transcriptional profiles in adult cells do not involve turning on or off entire chromosomes, with genes temporally coregulated often residing on different chromosomes  and beset by numerous histone modifications. Reflecting this fact, the spectrum of lncRNA functions has expanded138 to include actions in trans  as well as cis to enhance transcription, to block it, to scaffold chromatin interactions, and to aggregate microRNAs (thereby making them unavailable to regulate mRNAs).lncRNAs have been proposed as a potential mechanism for how different chromatin marks are deposited—specifically and reproducibly—across the genome. One of the best-studied lncRNAs, a general definition of which is an RNA >200 nucleotides with no discernable open reading frames  and RNA pol II elongation.141 Other lncRNAs have been discovered to play a role in disease-associated gene regulation. Chaer binds the Ezh2 subunit of PRC2, and its genetic manipulation leads to alteration in H3K27me3 levels around pathological genes and cardiac hypertrophy in the mouse.142 An antisense transcript in the β-MHC (beta-myosin heavy chain) locus was found to associate with that locus in a manner independent of the PRC subunit EZH2 in the setting of pressure overload.143 Interestingly, that same paper showed the EZH2 interaction with chromatin was regulated by the noncoding RNA pri-miR-208b, hinting at a broader role for noncoding RNAs in regulating chromatin.128 Some lncRNAs seem to exert their effects on gene regulation through interaction with chromatin remodeling complexes, as is the case for Mantis, a lncRNA discovered in macaque and shown to regulate endothelial angiogenesis in a manner involving interaction with BRG1.144 Similarly, the cardiac-specific lncRNA Myheart binds and inhibits the actions of BRG1, thereby regulating expression of myosin heavy chain expression, along with other genes. Myheart is downregulated by pressure overload stress and its transgenic restoration protects against overload-induced hypertrophy.145 Interestingly, lncRNAs like Malat1 in vascular tissues146 and Chast in cardiac tissues147 are nuclear localized and regulate expression of nearby genes although it remains to be tested whether they accomplish these actions through recruitment of chromatin complexes. In the field of cholesterol metabolism, 2 recent lncRNAs have been discovered that exert powerful effects of lipid levels and atherosclerosis in vivo: LeXis,148 expressed in the liver, directly controls genes involved in cholesterol biosynthesis, consequently modulating plasma cholesterol levels, and MeXis,149 expressed in macrophages, regulates genes involved in cholesterol efflux .Initial investigations of lncRNAs in the heart revealed involvement in developmental growth and maturation. 150 determining their extent of enrichment in this tissue when compared with tissues of distinct developmental origin (liver and skin) and determining changes between embryonic, adult, and diseased noncoding transcriptomes . Likewise in myocardial infarction, the recovery/injury/remodeling period was found, in mice, to be associated with changes in lncRNA expression , some of which (the lncRNAs) were subsequently shown to modulate expression of mRNA-encoding genes known to participate in basic cardiac function.152 Studies from humans have charted differences in lncRNA expression between fetal and adult cardiomyocytes, linking their expression with known enhancer marks associated with protein-coding RNA transcription .153Discovery analyses in a mouse model of pressure overload revealed the expression profile of cardiac lncRNAs,cis), and can correlate with histone PTMs, binding some of the histone-modifying complexes, PRC2 in particular. It is unknown to what extent lncRNAs can act at a distance , the role of chromatin structure to coordinate such actions , if they bind directly to chromatin and DNA (perhaps involving triple helix formation155), and whether they are sufficient to coordinate the locus-specific activities of chromatin modifiers through a model in which multiple lncRNA genes, by virtue of their evolutionary distribution at key sites across the genome, establish local neighborhoods of regulation at which they recruit—or repel, according to wont—histone modifiers and polymerase machinery.What is known about lncRNAs in the cardiovascular system is that they can be cell type specific, often lack extensive sequence conservation across species , can regulate transcription  decorate regions of DNA that need not be—although may be (see below)—themselves transcribed, which in turn recruit binding of enhancer-associated proteins  and interact in 3 dimensions with the genes whose expression they enhance. This region of DNA, the appropriately demarcated histones, and any associated proteins together constitute the enhancer which is often validated as such by showing that either (1) its genetic disruption interferes with expression of its target gene or (2) that the enhancer DNA sequence can drive developmental and lineage appropriate transcription through a cell- or organism-based reporter assay. In the absence of chromatin conformation data, enhancers are usually assumed (and tested) to regulate the nearest downstream gene. Somewhat counterintuitively, then, enhancers tend to reside in areas of relative nucleosome depletion , such that enhancers can be identified by open chromatin assays  followed by DNA sequencing. A subgroup of enhancers, called super enhancers,159 has been classified based on the observation that the aforementioned enhancer features at times occur multiple times in close proximity to each other. Super enhancers can exhibit augmented transcriptional activation potential and thus may represent a distinct structural property of cell type–specific chromatin.160 Further specification of enhancer behavior includes delineation of poised (those ready for promoting transcription of their targets) versus active  enhancers, which can be distinguished by the presence of silencing histone marks (and the enzymes that deposit them) at poised enhancers and their absence (concomitant with the presence of greater levels of RNA pol II) at active enhancers.158Enhancers are regions of DNA that promote the transcription of other regions of DNA.161 and may thus operate in the RNA form. It could be that this transcription is a goal-directed process in the normal way we think about RNA doing things in the cell: the enhancer RNAs may have gene regulatory or other functions. It may also be that the enhancer RNA synthesis is a by-product of enhancer DNA in close apposition to churning transcription factories and serves no subsequent end and that its transcription serves the end of keeping a transcription factory churning and poised for ready enlistment in production of other RNAs that do serve subsequent ends (as RNAs). This fascinating concept of chromatin biology is an active area of investigation.162 Studies have begun to emerge examining the role of enhancer transcription in select cardiovascular processes, such as cardiac conduction163 and endothelial cell stress response.164 Active endothelial cell enhancers, defined by H3K4me2 and H3K27ac binding (plus some enhancer RNA transcription), exhibited altered transcription factor binding in human aortic endothelial cells after exposure to oxidized phospholipids.164 Intriguingly, SNPs (single nucleotide polymorphisms) associated with cardiovascular disease were over represented in these enhancers, suggesting a molecular scale explanation for how the former influences transcription and phenotype, a property that may be a common feature of enhancers across cell types and species.165It has more recently become apparent that some enhancers may themselves be transcribed166 A similar approach was used to characterize enhancers in fetal and adult human heart tissue ,167 revealing functional elements that may participate in human cardiac gene regulation. Angiotensin II–induced vascular growth, a key component of atherosclerosis, was found to proceed via dynamic utilization of super enhancers in human cells through a process that involves complex interplay among noncoding RNAs, chromatin readers, and transcriptional machinery.168 A theme of developmental processes being redeployed—not wholesale, but in a selective manner—in the disease setting may also play out for enhancers: regulatory elements marked by H3K27ac are specified in part by the master cardiac transcription factor GATA4 during development and some of these regions, devoid of GATA4 in healthy adult heart, are revisited by the protein on pathological stimulation, contributing to disease-associated gene expression.169 Pursuing this concept more directly in a complementary model, it has also been shown that cardiac enhancers undergo altered regulation by disease-associated transcription factors after pathological stress.170 The histone modification reader BRD4 binds super enhancers that are associated with cardiac disease genes. Interestingly, this process is finely tuned to differentially modulate association of BRD4 with these disease genes while leaving housekeeping genes unaffected, a process controlled in part by microRNA-dependent titration of BRD4 levels.171p300 occupancy has been used to identify enhancers in the developing mouse heart (embryonic day 11.5), many of which were found to exhibit tissue-specific activity.172: lineages of cells outwardly genetically identical that differ based on semistable, transmissible chromatin features. All cardiac myocytes and vascular smooth muscle cells are not the same, which means that although these cells do not proliferate and differentiate like cancerous cells do, it is reasonable to hypothesize that developmentally endowed epigenetic clones exist and contribute to organ level phenotypes in the adult. Indeed, distinct clonal populations  of cardiac cells contribute to different anatomic and functional features of the adult organ178 —epigenetic dissection of these populations may well reveal epigenetic clonality to be an underlying process contributing to this observation. The chromatin accessibility assay ATAC-seq, which reveals areas of open chromatin, has been applied in a single-cell format to a lymphoblastoid cell line, identifying subpopulations of cells based on chromatin accessibility.181 That such variability exists in postmitotic, healthy adult cells in the cardiovascular system remains to be demonstrated, but the observation of transcriptome variability in these cells, and the presence of chromatin accessibility variability in cells otherwise phenotypically similar, makes such a conjecture not unwarranted.The rapidly dividing phenotype of cancer has allowed researchers in this field to identify epigenetic clones182 What if this observation is evidence of a more generalized, myocyte-specific sensory apparatus on chromatin, that detects local calcium signaling, such as that involved in pathological gene activation, from calcium involved in contraction and nonetheless critical to influence gene expression ? Various pathological cell states, including cardiovascular disease and cancer, have been characterized by global changes  in histone modification. One possible reason for this unexpected observation was found to include regulation of cellular acidity183: global histone acetylation responds to perturbation of cellular pH (lower pH leads to less histone acetylation) and cells respond to modulation of histone acetylation by modulating pH, a sort of acetate capacitance system on chromatin to attenuate large swings in cellular acidity. Combined metabolomic and proteomic studies reveal that abundance of short chain acyl-CoA donors directly, although not indiscriminately, influences the modification of histone tails in human cells in culture.184 Indeed metabolic sensing by chromatin has been increasingly recognized to underpin cardiovascular physiology and disease.77Chromatin can act like a stress sensor complex, wherein there is no single factor controlling changes in disease-associated gene expression. Some investigators have described excitation–transcription coupling, with the term specifically applied to local calcium signals around the nucleus  inducing local CaMKII activation and HDAC mobilization.185 Association of chromatin with the lamina seems to be essential for nuclear structure, and disrupting this interaction has detrimental effects on nuclear integrity,186 particularly in cells subject to mechanical force. These actions are coupled to the mechanisms known to regulate chromatin’s role in gene expression, as supported by the observation that histone PTM influences nuclear rigidity and membrane integrity.187Aberrations in nuclear rigidity and structural integrity are associated with diseases like cancer and progeria, some of which are driven by so-called laminopathies, arising from malfunction of nuclear lamina proteins. Cardiomyopathies resulting from mutations in lamin A/C are one of the best-studied group of genetic diseases in clinical cardiology and have led to clinical trials, although in this context the effects on chromatin structure are unclear. Aberrant nuclear morphology, the blebbing of the nuclear membrane because of impaired laminar network architecture, is a hallmark of these diseases.188: cytoplasmic chromatin fragments—evaginated nuclear membrane containing DNA and nucleosomes decorated with heterochromatin marks—can induce inflammation and cell death through cytoplasmic signaling and circle-back transcriptional regulation. An even weirder story: rod and cone cells are terminally differentiated, specialized components of the retina, the light-sensitive component of the vertebrate eye. Evolution has hijacked chromatin in rods (but not cones) to serve the transcriptionally unrelated function of focusing light in the retinas of nocturnal but not diurnal mammals ,189 thereby providing a meta-function in service of that specific cell’s raison d’être.An unexpected non-nuclear, signaling behavior of not just chromatin-modifying proteins but actual intact multimolecular slabs of chromatin has been observed in cancer190 or areas designated to different forms of transcription, such as rRNAs and house-keeping sorts of protein-coding mRNAs, separated from stimulus responsive genes and furthermore from transcriptionally silent regions. This happens on a nuclear scale as reflected by the observation that transcription tends to happen toward the center of the nucleus whereas the periphery is an area of gene silencing. Another type of transcription factor-like activity is subchromosomal, in the form of chromatin looping, the formation of short- and long-range interactions to facilitate gene activation  or repression . Chromatin capture data have revealed that long range looping within the epigenome is dynamic and can bring together transcription start and end sites in 3 dimensions, perhaps to facilitate efficient cycling of machinery like polymerases and transcription factors  have been described in the setting of pressure overload hypertrophy, along with accumulation of RNA pol II in transcription end site, perhaps reflective of gene looping.Different structural units of chromatin establish distinct transcriptional environments. It can be helpful to think of chromatin itself as a transcription factor or transcriptional processor. Transcription does not happen willy-nilly throughout the nucleus but rather is localized to transcription factories,2) and below the level of the chromosome ? The goal of chromatin structural studies is to determine: what are the structural features between these scales and at what scale(s) are structural features functionally important? Next consider the pattern of chromatin observed in various cells of the cardiovascular lineage with a quotidian method, such as DAPI  labeling: while the pattern of staining is not random, there is no obvious reproducible pattern within a class of cells (and not shared between 2 classes) to which a functional consequence can be intuited , in contrast to chromosome patterns in mitosis/meiosis which definitively exhibit such tell-tale architecture. Fluorescence in situ hybridization experiments clearly demonstrated spatial segregation of chromosomes into territories193 while not revealing evidence of hierarchical arrangement. Recent higher resolution electron microscopy–based imaging of chromatin shows that its structure, in both interphase and mitotic cells, rarely achieves a scale >24 nanometers in diameter , with distinctions in arrangement between such cells coming from the density of compaction, which the authors interpret to be evidence of an absence of repeating, stable, hierarchical structure.194 How do these observations hold up in analyses of individual genes and with respect to histone post-translational modifications? In the cardiovascular arena, combination of Dam-ID and LaminB ChIP-seq (to identify loci associated with the nuclear periphery) and fluorescence in situ hybridization was used to demonstrate that differentiation in the myocyte lineage involves precise reorganization of expressed genes away from the myocyte nuclear membrane, itself found to be decorated with the silencing mark H3K9me2, and to a lesser degree by H3K9me3 .195Based on insights from chromatin capture and other epigenomic techniques, the organization of the epigenome is thought to involve key structural intermediates Figure . What is191). The vast majority of loci interact with a median number of other loci, and no privileged structural behavior can be assigned to the regions with large interactions .A prediction of a nonhierarchical model of chromatin is that the size of structural elements should be normally distributed. For chromatin interactions detected by Hi-C, for example in cardiac myocytes, this prediction has been shown to be true: the number of interactions plotted per locus follows a normal distribution, where most locus bins (bin size=5kb) have the same number of interactions (≈2500) and a small number have very few or very many interactions : topologically associated domains (TADs) are regions of chromatin with privileged local interaction; TAD boundaries, as nominally implied, demarcate regions of the genome where intrachromosomal interactions switch from interacting in 1 direction (say 5′ biased) to the opposite; distinct regions are insulated against expression, in part by chromatin structural proteins and histone modifications; short and long range interactions reproducibly form . The boundaries represent epigenomic cornerstones, directing interactions of nearby DNA and proteins in alternating directions, in part through the binding of chromatin structural proteins like CTCF. Cohesin and CTCF knockout animals and cells indicate these proteins are involved in TAD maintenance,199 but these proteins alone are not the whole story: embryonic stem cells198 with only 4% normal CTCF protein levels still exhibited TADs and cardiomyocytes191 with 20% normal CTCF protein levels exhibited sparse, minor changes in TAD boundaries and strength. These studies also show that TAD formation/maintenance and A/B compartmentalization can be decoupled experimentally as loss of cohesin or CTCF did not affect A/B compartmentalization. A compartments have more genes and are defined by having less interactions than would be expected for a given distance (B has more), indicating less compact chromatin. Eu- and heterochromatin marks dominate in A and B compartments, respectively. Prevailing evidence suggests that at the level of TADs, chromatin architecture is quite similar between cell types. Sub-TAD interactions, and less abundant—yet reproducibly detected—long-range interactions that span multiple TADs , may be the scales at which cell type–specific chromatin interactions are observed.Additional insights from the explosion of chromatin capture techniques to determine endogenous interactions have been informative.200 Mitotic chromosomes lack TADs and chromatin neighborhoods, instead exhibiting a uniform, homogenous pattern of hierarchical interactions.201 A similar observation was made for oocytes in metaphase II: an absence of TADs and chromatin neighborhoods in these cells persisted in the zygote, with long range chromatin interactions manifesting at the 8-cell and inner cell mass stages.202 Interestingly, physical segregation of alleles was seen to persist until the 8-cell stage as well,202 even after the formation of long range chromatin contacts, suggesting that chromatin structure is an emergent property of an allele, can vary between alleles, and thus may participate in allelic inheritance.Chromatin structure is dynamic during the cell cycle. The predominance of longer range interactions present in postmitotic cells is rapidly lost upon entrance into G1, followed by further depletion throughout S and G2, in favor of shorter range, local interactions. This process abruptly reverses itself, with a return of TADs and long-range interactions after nuclear division.203 Using data sets from human lymphoblastoid cells, this approach was used to schematize genome structure, revealing interchromosomal surfaces of apposition and detecting new anatomic properties of the nucleus, such as the physical clustering of centromeres of different chromosomes and the anatomic positioning of euchromatin and heterochromatin pockets with respect to other nuclear landmarks. Unlike traditional fluorescence in situ hybridization or chromosome painting, in such an exercise one can know which loci are responsible for a given anatomic feature and in what part of the nucleus this feature tends to occur relative to other features.Probabilistic modeling has been used to reveal 3-dimensional organizational principles from Hi-C data sets, the goal here being not a structure per se, but a population-based representation of the structural features of the chromosomes as they associate in the nucleus.17 has become a ready-to-hand tool for chromatin interrogation, shaping how studies are designed and interpreted. However, as discussed elsewhere in this essay, hundreds of histone PTMs are now known to exist. Also, the histone code and related ideas204 lack an underpinning in mathematical rules, a limitation addressed by investigators who have used dry laboratory approaches to characterize chromatin states205 or rules of nucleosome positioning206 that reconcile ChIP-seq and chromatin accessibility data with genome sequence and transcription. Apart from the accordant histone and chromatin-binding proteins associated with different flavors of chromatin, how distinct chromatin domains form, in a physical sense, is not completely understood. Recent evidence from Drosophila and human chromatin suggests that heterochromatin domains comprised H3K9me3-marked nucleosomes, heterochromatin protein 1, and DNA can exhibit phase separation behavior, which may be an explanation to link domain-scale and molecular-scale properties of heterochromatin foci which can display both liquid and stable phase properties,207 a phenomenon supported on a broader scale by contemporaneous studies.160 The way forward is to integrate structural studies from imaging and sequencings techniques with genome occupancy studies to build a new model governed by principles that incorporate all these sets of data.The histone code209 must do so by wholesale reprogramming of chromatin . iPS-derived cells coaxed toward a cardiovascular lineage acquire regulatory elements  reminiscent of their endogenous counterparts,211 and yet studies from noncardiovascular tissues have shown that iPS-derived cells retain some epigenetic memories from their cells of origin, which, perhaps not surprisingly, is also the case for cardiovascular cells derived in cell culture from developmental precursors .212 Cardiac cells exhibiting progenitor-like behavior isolated from adult hearts indeed exhibit, commensurate with transcriptome changes, DNA methylation changes in genes associated with the mature cardiomyocyte lineage, vis-à-vis adult cardiomyocytes lacking such progenitor-like behavior.213Histones were originally identified as inhibitors of transcription. This concept remains a kernel of chromatin theory: heterochromatin increases during differentiation and loss of pluripotency, and some diseases, notably cancer, have been found to be associated with a more euchromatic environment. Because they change concomitant with gene expression and phenotype, chromatin modifications are ispo facto taken as responsible for the unidirectional progression of cell fate commitment in the cardiovascular system. Another hypothesis cached therein is that histone PTM and other chromatin marks stabilize cell identity. Regarding this conclusion, here is a premise that should be rejected: identification of a chromatin-modifying enzyme in the heart or vasculature whose genetic manipulation impairs or reverses developmental state is a necessary and sufficient condition to prove a role for chromatin in deciding and stabilizing cell fate. Here is another such premise of tenuous utility: if chromatin modifications stabilize cell phenotype, then reprogramming strategies that restore pluripotency  or directly convert one cell type to another215 General features of heterochromatic mark (H3K27me3) loss were observed around genes that were expressed , and genes that would be expressed in subsequent stages of development were sometimes  enriched with H3K4me1  before acquisition of H3K4me3 and RNA pol II concomitant with expression. If one were so inclined, the following observations may be taken as evidence that chromatin becomes more plastic in the setting of cardiac pathology , as well as a decrease in the linker histone H1 to core (measured by H4) ratio8; association across genetically variable mouse strains between select chromatin structural proteins HMGB2 and CTCF and cardiac phenotype and the ability of HMGB2 to modulate cardiomyocyte hypertrophy and chromatin accessibility in cardiac myocytes217; and loss of CTCF (which induces cardiac dysfunction) or pressure overload hypertrophy is accompanied by a global decrease in genomic interactions detected by Hi-C.191Cell culture studies of distinct stages of cardiac lineage commitment explored the changes in chromatin marks associated with this process.218 Interestingly, DNA methylation signatures at this locus in blood were correlated with allergic sensitization, potentially hinting at a gene–environment interaction leaving an epigenetic signature. A similar observation of epigenetic risk conferred in a cell type–specific manner by marks present across different tissues was recently reported in the context of dilated cardiomyopathy.219 The durability of these marks in a temporal sense remains an open question. Unequivocal determination of transgenerational inheritance of chromatin features like DNA methylation or histone modifications (discussion of which often conflates the 2 distinct concepts of epigenetics and Lamarckian evolution or the inheritance of acquired traits) is tricky,220 and many examples are hotly debated. Epigenome-wide association analyses in liver demonstrate103 DNA methylation–dependent—and sequence variation independent—associations with clinical traits important for cardiovascular disease, such as insulin level, as well as other omics end points, providing proof of concept for population level epigenomic regulation of complex disease traits through the actions of DNA methylation variation to control phenotype, presumably through effects on chromatin structure or accessibility. Something ostensibly heritable through cell division or meiosis may masquerade as epigenetic, and may even be of chromatic origin, while in fact proceeding via genetic means.221Meta-analysis of heart failure GWAS (Genome-Wide Association Study) studies recently uncovered a novel risk allele associated with mortality and located in a noncoding enhancer region.222). Maternal smoking, for example, is known to induce widespread DNA methylation differences in newborns, some of which persist into the offspring’s adulthood, including in genes known to be associated with smoking-related birth defects,223 although whether these effects are because of natal exposure remains unknown. There have been limited experimental studies directly testing the role of nongenetic inheritance of cardiovascular risk in animal models . In vitro fertilization experiments in mice using gametes from obese or normal weight parents (note: obesity was induced by high-fat diet) and surrogate, normal chow fed, mothers revealed that a propensity for increased body weight can be inherited by nongenetic means.225 Metabolic gene regulation has been shown to be a modifiable, and subsequently heritable feature, in that mice fed low-protein diets passed hepatic gene expression profiles transgenerationally through the paternal germ line, commensurate with heritable changes in DNA methylation .226 Genetic variability contributes to chromatin accessibility (measured via FAIRE-seq) in the basal state and following complex metabolic changes, such as those accompanying high-fat diet.227 Human studies in ethnically diverse populations have revealed DNA methylation variation associated with nicotine and alcohol dependence (and the codependency between these forms of addiction)228 although no evidence of inheritance or precedence of the phenotypes by the epigenetic features was demonstrated. In addition to the obvious prognostic and diagnostic potential of genomic and epigenomic measurements in the clinical arena , it is noteworthy that tools for reducing epigenomic treatment to practice have begun to emerge . Such approaches may enable targeting of entire transcriptomes, rather than individual molecules, in a gene therapy workflow that at once provides both specificity and temporal tuning.It has been increasingly recognized that epigenomic modifications are influenced by various diet and lifestyle factors that affect cardiovascular health  that are now experimentally mature can, when applied to chromatin, provide greater still molecular detail on how transcriptomes are specified and cell type–specific behavior governed in health and disease. Effective utilization of this wealth of knowledge to promote human health will require innovative thinking: some strategies will involve multimarker panels  whereas other strategies will use the intermediate end points that emerge from the collective actions of multiple classes of molecules as the readout for diagnoses or as target for treatment .Genomic and epigenomic technologies are providing high-resolution descriptions of susceptibilities and pathogenesis of cardiovascular diseases—that is, we now have the ability to acquire a far greater number of data points on patient populations, enabling identification of new biology but also, in the clinical setting, better stratification.It occurs to us that another definition of epigenomes would be the molecular features that make a living creature the same unit tomorrow that it is today. As described in the preceding sections, recent studies have identified the actions of discrete protein components and modifiers of chromatin in cardiovascular health and disease, providing potential targets for therapy. Epigenomic investigations have described chromatin landscapes, providing the data necessary to discover principles for the actions of the protein and RNA modifiers. These epigenomic investigations also enable discovery of intermediate properties, like chromatin accessibility and structure, which may be traits that contribute to higher level phenotypes. As the integrator of genetics and environment, and the substrate of cellular memory, chromatin features may provide the basis for understanding normal and pathological cardiovascular function.We regret that space limitations have forced us to omit reference to many important papers in the field of cardiovascular epigenomics. We thank Dr Emma Monte for critical feedback on this manuscript. Author contributions are as follows: concepts, all authors; text, T.M. Vondriska; figures, M. Rosa-Garrido and D.J. Chapski; and final version, all authors.Work in the Vondriska lab is supported by the National Institutes of Health, the American Heart Association, and the Cardiovascular Theme in the David Geffen School of Medicine at University of California, Los Angeles.None.


Telomeres are specialized structures that evolved to protect the end of linear chromosomes from the action of the cell DNA damage machinery. They are composed of tandem arrays of repeated DNA sequences with a specific heterochromatic organization. The length of telomeric repeats is dynamically regulated and can be affected by changes in the telomere chromatin structure. When telomeres are not properly controlled, the resulting chromosomal alterations can induce genomic instability and ultimately the development of human diseases, such as cancer. Therefore, proper establishment, regulation, and maintenance of the telomere chromatin structure are required for cell homeostasis. Here, we review the current knowledge on telomeric chromatin dynamics during cell division and early development in mammals, and how its proper regulation safeguards genome stability. In eukaryotes, the genome is organized in different compaction states. Schematically, euchromatin, which defines the open compaction state, contains active genes, whereas heterochromatin, which is the repressive compacted chromatin, includes gene-poor regions and different types of repeated elements with a specific chromatin structure  and proteins that are typically found at pericentric heterochromatin (PCH), such as trimethylation of lysine 9 of histone H3 (H3K9me3), trimethylation of lysine 20 of histone H4 (H4K20me3), hypoacetylation of histone H3 and H4, and HP1 proteins  polymerase tankyrase 1, thus preventing its binding to telomeres and the resolution of cohesion between sister chromatids. Persistent cohesion would then promote recombination by T-SCE  leads to ALT . Similarly, the epigenetic regulator structural maintenance of chromosomes (SMC) flexible hinge domain-containing 1 (SMCHD1) is involved in the silencing of subtelomeric DUX4 repeats   through a process called gametogenesis. Importantly, during this process, male and female germ cells undergo meiosis, a specialized cell division characterized by one round of DNA synthesis followed by two rounds of cell division, resulting in the production of haploid gametes. During and after meiosis, germ cells undergo chromatin remodeling, leading to specific DNA and histone methylation patterns, and to histone to protamine exchange in male germ cells  do not show any obvious phenotype and can be maintained for at least six generations, suggesting that the overly long telomeres of laboratory mice could compensate for the telomeric sequence reduction. Nevertheless, telomeric DNA is gradually lost at each generation with increasing incidence of chromosomal abnormalities  can be used for in vivo or in vitro fertilization, they are less efficient than wild-type gametes and lead to aberrant embryos with pronucleus formation defects, cytofragmentation, and impaired preimplantation development . This could lead to SUV39H1 recruitment to mediate H3K9me3 and heterochromatinization  is among the genes that are specifically expressed during ZGA  are characterized by rapid and telomerase-independent telomere lengthening. Telomeres are bound by recombination proteins and undergo T-SCE ), marking the maternal-to-zygotic transition   results in Zscan4 decrease and telomere shortening, while Tet TKO in ES cells cultured in 2i conditions (“ground state”) induces increased Zscan4 expression and telomere elongation  Chaf1a is downregulated and in myoblasts from patients with FSHD  could participate in Dux and Zscan4 repression and allow the establishment of a canonical heterochromatin state at later developmental stages  in somatic cells to induce pluripotency  in which a donor nucleus  is injected into an enucleated oocyte. The oocyte will reprogram the donor nucleus in a similar way as the reprogramming of the maternal and paternal genomes after natural fertilization (Sepulveda-Rincon et al. The discovery that after the histone to protamine exchange, the fraction of histones retained in mature sperm carries PTM led to the idea that male germ cells could transmit epigenetic information to the progeny (Gill et al. The parental origin influence, if any, on telomeric chromatin regulation is also quite enigmatic. A parental-specific chromatin structure is visible at PCH domains in the zygote. H3K9me3 and HP1 are absent from paternal PCH, while they are highly enriched at maternal PCH. Conversely, Polycomb complexes bind to these domains and repress expression of major satellites from the paternal pronucleus (Puschendorf et al. Telomeres are essential structure to ensure genome stability, tissue homeostasis, and embryo development. Telomeric chromatin plasticity is a striking feature that allows the dynamic regulation of chromosome ends following developmental cues, cell cycle, or metabolism. These plasticity features are crucial during natural development or experimental reprogramming of somatic nuclei. Thus, understanding the mechanisms that operate at telomeric DNA in these conditions will greatly help the development and optimization of assisted reproductive technologies or therapeutic reprogramming techniques (Sepulveda-Rincon et al.


Nutrition represents one of the greatest environmental determinants of an individual’s health. While nutrient quantity and quality impart direct effects, the interaction of nutrition with genetic and epigenetic modifications is often overlooked despite being shown to influence biological variation in mammals. Dissecting complex traits, such as those that are diet or nutrition related, to determine the genetic and epigenetic contributions toward a phenotype can be a formidable process. Epigenetic modifications add another layer of complexity as they do not change the DNA sequence itself but can affect transcription and are important mediators of gene expression and ensuing phenotypic variation. Altered carbohydrate metabolism and rates of fat and protein deposition resulting from diet-induced hypo- or hyper-methylation highlight the capability of nutritional epigenetics to influence livestock commodity quality and quantity. This interaction can yield either products tailored to consumer preference, such as marbling in meat cuts, or potentially increasing productivity and yield both in terms of carcass yield and/or offspring performance. Understanding how these and other desirable phenotypes result from epigenetic mechanisms will facilitate their inducible potential in livestock systems. Here, we discuss the establishment of the epigenome, examples of nutritional mediated alterations of epigenetics and epigenetic effects on livestock production. In order to meet the dietary needs of the world’s projected population of 9.1 billion by 2050, the Food and Agriculture Organization estimates that food production must increase by 70% . AttainiThe pathways governing nutritional regulation of epigenetic modifications in turn can be considered at each level of the substrate input, the molecular target, as well as the endpoint product. Many molecular forms of epigenetic modification have been described including changes in chromatin packaging due to histone acetylation and methylation, repression of gene expression by non-coding RNA, and repression of gene transcription by DNA methylation. These epigenetic mechanisms, in addition to others, have been recently described in detail . EpigeneFigure 1).To understand how epigenetic modifications mediate and affect gene expression it is important to understand how and when the epigenome is established. While we do not fully understand how all of the complex epigenetic genome changes occur, we do know that the genome is demethylated, or “erased,” and remethylated, yet some epigenetic information is retained and transmitted to the next generation. Furthermore, studies, primarily in mice, but also in cattle  have proAt fertilization genetic material from two haploid gametes must come together to create a new diploid organism. Since the mid 1970’s scientists have understood that one germ cell from each sex are required for the development of a viable embryo. Pathological analyses of human germ cell tumors have provided important biological information regarding the difference of parental genetic origins. Through histopathological analyses of ovarian teratomas and hydatidiform mole, functional differences of what the maternal and paternal genome each contribute to cell growth and differentiation was recognized. Ovarian teratomas occur in the ovary and consist of three germinative layers  whereas At fertilization the parental genomes start to go through global demethylation where prior epigenetic programming is erased so that new totipotent cells can be reset for specific cell linage determination in a manner which results in the correct cellular differentiation. This is one cycle or wave of epigenetic reprogramming which notably occurs of the developing zygote within the maternal environment. However, because each of the parental genomes is necessary to contribute specifically to the different and specialized manner of the development of the zygote the demethylation changes occur in a manner specific to the parent of origin. The male pronucleus, for example, is rapidly and actively demethylated involving Ten–Eleven Translocation (TET) dioxygenases proteins , whereasde novo methylation in a cell lineage specific fashion. DNA methylation increases in the cells which give rise to the embryo, a primitive ectoderm, but not the cells which give rise to the placenta and yolk sac, or primitive endoderm (de novo methylation via DNA methyl transferase (DNMT), DNMT3a, DNMT3b, and DNMT3L  induce the PGC to express B-lymphocyte-induced maturation protein-1 (BLIPM1) protein, and BLIMP1 suppresses homeotic genes (HOX) which activate somatic cell differentiation  become the founder cell population which will ultimately give rise to the germ cell population. Signaling events by bone morphogenetic protein 4  of mitosis and all the prospermatogonia will undergo remethylation before meiosis and birth. As such the maternal uterine environment is the direct influential environment of the developing male embryo and male primary germs cells remethylation. Subsequent to the establishment of the epigenome and birth, male germ cells will undergo multiple cell divisions, then meiosis prior to the development of mature sperm. Therefore, the methylation established in the early cell population needs to be maintained during cell replication post puberty and throughout the reproductive years. Finally, volume compacting necessary to produce the mature sperm is achieved by the replacement of approximately 85% of the histones with protamines in spermatozoa.Xist RNA level to maintain two active X chromosomes for subsequent gamete formation. The female germ cells enter meiosis, complete pachytene and arrest at diplotene stage of anaphase 1 at or before birth unmethylated. It is not until after birth, during the follicular growth phase of the oocyte that the arrested primary oocytes are remethylated. Therefore, the critical window when the female genome is remethylated and sensitive to changes is during the follicular growth phase of those eggs. At puberty and in response to endocrine signals the germinal vesicle oocyte resumes meiosis and completes the first meiotic division. Subsequent to the extrusion of the first polar body and upon fertilization the second meiotic division (MII) will take place.Conversely, in the female embryo, the PGCs remain in an unmethylated state. In the PGCs, and only the PGCs of the female embryo, there is a reduction in While mechanistically naïve to the epigenetic process, the oft considered “father of epigenetics”; vy locus and are fed a ration with sufficient methyl donating constituents; such as folate and betaine then the expression of the agouti gene is diminished and imprinting decreases both the yellow coat and the obesity. Agouti coat color in mice was the first experiment to show that maternal diet had the ability to affect epigenetic modifications and gene expression in offspring. Whereas, honeybees utilize differential nutrition with genetically identical larvae to generate their worker caste system . Nutrients such as folic acid and betaine have been shown to influence the function of enzymes that participate in the methylation process  and sterol regulatory element binding protein 1(SREBP1) while decreased methylation levels were found in the promoter region of ATP binding cassette sub-family A member 1 (ABCA1). In addition to studying the effects of betaine supplementation on methylation, PEPCK1) gene promoter was found to be hypermethylated but promoter regions of phosphoenolpyruvate carboxykinase 2 (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1) were hypomethylated in livers of piglets prenatally exposed to betaine. As expected the methylation levels were inversely proportional with expression levels. When examining histone enrichment, PEPCK1 was found to be enriched for H3K27me3 while PEPCK2 and FBP1 were found to be enriched for histone mark H3 lysine 4 trimethylation (H3K4me3). These results are not surprising, given that histone mark H3K4me3 tends to be associated with gene activation. Betaine has been utilized in livestock and chickens as a feed additive to improve a variety of economically important traits . FurtherS-methyltransferase (BMHT). BMHT catalyzes the transfer of methyl groups from betaine to homocysteine, a key component of one-carbon metabolism , have only recently been studied . Currenttabolism , and is  altered . The autene ZIP4 , a zinc FGFR4) and protein tyrosine phosphatase, receptor type, S (PTPRS) (PPARα) and glucocorticoid receptor (GR). IUGR piglets who were fed a control diet showed evidence of hypomethylation but IUGR piglets whose diet was supplemented with folic acid showed the same methylation levels as normal birthweight piglets (liver X receptorα is hypermethylated (HNF4a) in pancreatic cells (POMC) and GR in the sheep brain ” since maternal protein insufficiency generally manifests as reduced birth weight of offspring. Low birthweight associated with protein insufficiency is correlated with elevated risk for hypertension, hepatic steatosis, cardiovascular disease and leptin and insulin resistance . When me (PTPRS) . Subsequ (PTPRS) . Key fin piglets . More tathylated , as is hic cells . Though,ic cells  and prooep brain . The essRXR-alpha) gene explains as much as 26% of childhood obesity. Furthermore, there is limited evidence that suggests that the maternal dietary lipid profile impacts RXR-alpha gene and PPAR gene methylation , and a mitochondrial protein of the electron transport chain (NDUFB6) (Il13ra3) and hepatic cyclin-dependent kinase inhibitor 1A (cdkn1a) both are decreased in methylation and are associated with elevated risks for metabolic syndrome (Many studies have examined the variation in propensity for obesity associated with intake of a high-fat diet (HFD). A comparison of isocaloric high-fat and low fat rations demonstrated that the expression of several genes associated with lipid synthesis are altered in the HFD treated group, namely there is an increase in methylation in the promoter domains of fatty acid synthase ((NDUFB6) . In the (NDUFB6) , 2011 an(NDUFB6) . Periphesyndrome . There iIGF2) that was initially linked to the Dutch famine  experiments, suggesting that a mutation to this region recruits different transcription protein complexes (Sus scrofa microRNAs 136 and 500 (ssc-miR-136 and ssc-miR-500)  . Therefocallipyge phenotype in sheep is only expressed in heterozygotes that inherit the mutation from their paternal parent. callipyge mutation and implicated histone modifications as the epigenetic factor responsible for the phenotype  were reported. In this context, a cis-meQTL is defined as a genomic region that includes SNPs within 1 Mb of the CpG site associated with methylation state. It has been demonstrated that SNP variation strongly influences the level of DNA methylation at sites that contain intermediate levels of methylation. These studies demonstrate that elements of DNA sequence can affect methylation levels which in turn affect phenotypic variation. Now, the question exists as to how the livestock community can utilize epigenetic alterations for production of economically important phenotypes. While this subject has been discussed, there seems to be disparity regarding the matter  and higher protein mass (chicken) gains as well as altered expression of key gluconeogenic enzymes (pigs). In mammalian species, additional research to discern the role of environmental factors such as nutrients and genomic sequence effects on epigenetic mechanism is essential, especially at critical developmental time points. Generation of new tools and methodologies for interrogating the livestock epigenome will assist in disentangling genetic and epigenetic effects of complex traits. Ultimately, utilizing nutritional epigenetics for increasing and/or sustaining food production will require additional research.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer SK and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Ischemic cell death during a myocardial infarction leads to a multiphase reparative response in which the damaged tissue is replaced with a fibrotic scar produced by fibroblasts and myofibroblasts. This also induces geometrical, biomechanical, and biochemical changes in the uninjured ventricular wall eliciting a reactive remodeling process that includes interstitial and perivascular fibrosis. Although the initial reparative fibrosis is crucial for preventing rupture of the ventricular wall, an exaggerated fibrotic response and reactive fibrosis outside the injured area are detrimental as they lead to progressive impairment of cardiac function and eventually to heart failure. In this review, we summarize current knowledge of the mechanisms of both reparative and reactive cardiac fibrosis in response to myocardial infarction, discuss the potential of inducing cardiac regeneration through direct reprogramming of fibroblasts and myofibroblasts into cardiomyocytes, and review the currently available and potential future therapeutic strategies to inhibit cardiac fibrosis. Heart failure (HF) is a major public health issue that affects more than 23 million people globally , and endothelial cells (through EndoMT) have been described in cardiac tissue  whose expression and functions are strictly controlled in order to maintain the homeostasis of ECM degradation and synthesis  is expressed by multiple cell types and regulates adhesion and migration of cells , tenascins, periostin, osteopontin, and the CCN family of proteins , is a key mediator of ECM production under pathological fibrotic conditions  can cause narrowing of the vessel lumen and has been associated with impaired coronary blood flow  and through direct ECM-fibroblast connections mediated by transmembrane receptors called integrins , at the cell surface. Ligand binding to TβRII recruits the type I receptor  and induces its transphosphorylation. The intracellular signaling routes include the Smad-dependent regulation of gene expression and the Smad-independent activation of signaling cascades including mitogen-activated protein kinase (MAPK) signaling and signaling through the small GTPase Rho. In particular, signaling through TGF-β-activated kinase (TAK1) and p38 MAPK has been implicated in myofibroblast transdifferentiation, and pharmacological p38 MAPK inhibition is protective against cardiac fibrosis in a rat model of MI , it up-regulates the expression of TGF-β and IL-6 in cardiomyocytes, fibroblasts, and myofibroblasts. Both Ang II and TGF-β synthesized and secreted at the infarction site have been suggested to play a role in the development of reactive fibrosis in the non-infarcted myocardium  is the central signaling molecule of the renin-angiotensin system (RAS) with regard to cardiac fibrosis. Its immediate in vivo effects include vasoconstriction and increased blood pressure, but it also has direct remodeling-inducing effects on various cardiac cell types renin receptor  is predominantly produced by endothelial cells, but other cardiac cell types  can also synthesize and secrete it , and platelet-derived growth factor  that are thought either to promote  or to inhibit  cardiac fibrosis have been identified  with the help of cardiac transcription factor overexpression was first reported by Ieda et al. in 2010  and two miRNAs  combined with the pharmacological inhibition of TGF-β. Inhibition of fibrotic signaling with TGF-β or Rho-associated kinase (ROCK) inhibitors improves reprogramming efficiency, as does the overexpression of Akt/protein kinase B  results in significantly higher reported efficiency than classification by using a functional measure such as CaGata4, Tbx20, Tbx5, Nkx2-5, Hand2, and Mef2c at significantly higher levels than tail fibroblasts  in vitro Table . When grCurrent HF treatment recommendations are based on RAS inhibition and β adrenergic receptor antagonists, supplemented with mineralocorticoid/aldosterone receptor antagonists, ivabradine, and/or digoxin as necessary  inhibitors and AT1 receptor blockers (ARBs) unsurprisingly inhibit cardiac remodeling and fibrosis in various experimental models  reductase, namely statins, are effective and widely used for both primary and secondary prevention of ischemic cardiovascular events because of their cholesterol-lowering effects. Additionally, increasing evidence suggests that they exhibit anti-remodeling properties, which contribute to their beneficial clinical effects. Under in vitro conditions, statins have been shown to directly inhibit cardiac fibroblast proliferation and migration, fibroblast-myofibroblast transdifferentiation, and ECM turnover, all of which are expected to confer beneficial effects in the myocardial remodeling process  and B  have emerged as important candidates for the development of therapeutic agents for heart failure  in non-infarcted areas with the induction of the regeneration of the infarcted myocardium Fig. , for exa