Epigenomic Reprogramming in Cardiovascular Disease

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Yang Zhou, Jiandong Liu, Li Qian Department of Pathology and Laboratory Medicine, Department of Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, United States

INTRODUCTION Cardiovascular diseases (CVDs) have emerged as one of the leading causes of illness and death in the world and have been producing numerous health and economic burdens worldwide. As reported by American Heart Association (AHA), around 92.1 million adults in the United States currently have at least one type of CVDs [1]. There are multiple types of CVDs that involve heart or blood vessels with different causes and pathological characteristics [2]. Despite advances in the prevention and treatment of CVDs, the death rate attributable to CVDs continues to rise mainly because the therapeutic strategies for CVDs, especially for heart failure, are limited and inadequate. Therefore, deeper understanding of molecular mechanisms underlying CVDs and new technologies are needed to discover more efficacious therapeutic approaches. Considering the inherent low proliferative and regenerative capacity of mammalian cardiomyocytes (CMs), the most straightforward strategy for treating heart failure is to replenish functional CMs or replace the malfunctioned CMs in order to recover heart function [3e5]. Recently, the revolutionary work in the field of stem cell biology and cardiac regenerative medicine has progressed rapidly to deepen our understanding of cardiac development and open the new path to cardiac regeneration. Generation of autologous CMs via induced pluripotent stem cell (iPSC) reprogramming followed by differentiation and direct reprogramming from fibroblasts holds great promise as an alternative strategy for heart regeneration and disease modeling [6,7]. In studies over the past decade, iPSC reprogramming has been successfully achieved through the ectopic expression of master pluripotent transcription factors in various types of somatic cells from both murine and human [8e10]. The efficient differentiation of iPSCs into functional CMs mimics cardiac differentiation during early embryonic stage, providing not only the platform to dissect underlying mechanisms of cardiac development and diseases in patients, but also the source of CMs for potential utility in cell therapy [11e16]. More recently, inspired by iPSC reprogramming, functional induced cardiomyocytes (iCMs) have been derived from cardiac fibroblasts via forced expression of key cardiac transcription factors both in vitro and in vivo [17e24]. Direct cardiac reprogramming offers an appealing approach as it could accomplish in situ cardiac regeneration for regenerative therapy for heart diseases.

Computational Epigenetics and Diseases. https://doi.org/10.1016/B978-0-12-814513-5.00010-6 Copyright © 2019 Elsevier Inc. All rights reserved.

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Epigenetics is typically defined as the regulatory mechanisms of gene activity that are not due to alterations in DNA sequence. Epigenome means genome-wide epigenetic regulations, including DNA methylation, posttranslational modification of histone, chromatin remodeling, higher-order DNA organization, and noncoding RNA alterations, all of which are heritable and sequence-independent. Epigenetic dynamics in the local and global organization of chromatin has been recognized as critical regulators to precisely determine the transcriptome of a cell [25]. The understanding of epigenomes will be able to explain how identical genomes in diverse types of cells within an individual organism produce such varied transcriptomes specific to each cell type. Moreover, results from studies in animal models clearly demonstrate that not only genetic factors, but also epigenomic variability leads to phenotypic variability and influence disease susceptibility [26,27]. Thus, the epigenomic regulations and misregulations underlying normal development and diseases hold promise to develop innovative biomarkers and therapies for CVDs. With the rapid accumulation of the large-scale mapping of epigenomic and related data in CVD study, it has been highlighted that the use of computational tools is the core for data collecting, cleaning, clustering, modeling, and predicting, which gives rise to complex and comprehensive epigenomic information that is inaccessible using traditional approaches [28]. In addition, to facilitate the data analysis, massive data sources for epigenetic research have been collected and classified as invaluable databases [29], such as NIH Roadmap Epigenomics database [30], DNA methylation databases MethDB [31] and MethPrimerDB [32], VISTA human enhancer database [33], 3D-genome Interaction Viewer and database (3DIV) [34], Human Enhancer Disease Database (HEDD) [35]. Such tools and information allow for the systematic study of relationship between epigenomics and diseases. Herein, we focus on the recent computational analyses-based studies on landscapes and dynamics of chromatin modifications and structures in normal cardiomyocyte differentiation, CVD development, and heart regeneration, in particular direct cardiac reprogramming.

DECIPHER HISTONE CODES OF CM TRANSCRIPTION In eukaryotic nucleus, the genomic DNA is wrapped around histone proteins in nucleosomes, which are the fundamental repeating structural units of chromatin. Each nucleosome is composed of about 146 base pairs of DNA wrapped around eight histones, called histone octamer, which contains two copies each of the histone proteins H2A, H2B, H3, and H4. The histones possess a diverse array of posttranslational modifications on specific residues along their N-terminal “tails”. To date, the histone modifications include acetylation, methylation, ubiquitination, and SUMOylation of lysine (K) residues, phosphorylation of serine (S) and threonine (T) residues, methylation of arginine (R) residues, ADP ribosylation, deamination, and isomerization of proline (P) [36,37]. The different histone modifications maintained and altered via corresponding enzymatic systems have been proposed to act sequentially or in combination to form a “histone code” that is read by other histone binding proteins or readers to bring about distinct genome activities and gene regulation [38]. Nowadays, numerous posttranslational modifications of histones have been documented and revealed with critical roles in mediating the genome function and gene activity in response to upstream signaling pathways [39]. For instance, H3K4me3 at promoters and transcription start sites, H3K27ac at enhancers and promoters, H3K36me3 at transcribed gene bodies are associated with transcription activation [40]. In contrast, H3K27me and H3K9me2/3 are related to gene repression and heterochromatin formation [40].

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In addition, new methods harnessing the power of next-generation sequencing technology have been developed to interrogate chromatin dynamics at the genome-wide scale to reveal the link between epigenomic status and gene regulation, such as Chromatin ImmunoPrecipitation assay (ChIP-seq) and Assay for Transposase Accessible Chromatin (ATAC-seq), following with the generation of computational tools to interpret, visualize, and annotate this genome-wide information [41e44]. Thus, increasing enthusiasm of deciphering histone codes in the epigenome has been triggered.

IDENTIFY CHROMATIN MODIFICATION LANDSCAPES AND DYNAMICS DURING HEART DEVELOPMENT Both gene expression and epigenetic signatures are highly cell type specific. A map of tissue- or cell type-specific promoter and enhancer regions has been drawn in the mouse genome to demonstrate and utilize the tight link of chromatin state and transcriptional activity [45,46]. In mouse heart, cardiacspecific promoters are identified by enrichment of H3K4me3 or Pol II binding, while cardiac-specific enhancers are defined based on the presence of H3K4me1 or H3K27ac outside promoter [46]. In pericentriolar material 1 (PCM1) positive mouse CM nuclei, the activity of mRNA is highly correlated with occupancy of H3K4me1, H3K4me3, H3K27ac, and H3K36me3, but inversely correlated with H3K27me3. Among these histone modifications, H3K27ac is the most predictive one for deducing transcriptional activity [45]. Furthermore, epigenome-wide analysis of H3K36me3 patterns could facilitate the identification of cardiac gene isoforms expressed in CMs [45]. In contrast to the relatively stable genome, the epigenome is very dynamic during development and differentiation in order to establish and maintain cell type-specific gene expression based on cellular identity and function. During cardiac differentiation, cell morphology and function are changed sequentially, as a result of alterations in gene expression as well as dramatic changes in the epigenetic landscape, which is required for appropriate cell fate differentiation [47,48]. In particular, during mouse embryonic stem cell (ESC) differentiation into CMs, a pattern of chromatin state transition, in which H3K4me1 enrichment is prior to enrichment of H3K4me3 and RNA Pol II on the promoter region of genes, is associated with later gene activation [48]. Also, analysis of occupancy of H3K4me1 and/or H3K27ac at distal enhancer regions has led to the identification of numerous putative cardiac fate-specific enhancers, which are undergoing rapid transitions between poised and active states at each stage of differentiation [48]. Similarly, genome-wide analysis of chromatin modifications along the time course of cardiac differentiation from human ESCs showed stage-specific changes in H3K4me3 and H3K27me3 levels [47]. Notably, a cardiac-specific chromatin signature has been identified to discriminate master regulatory factors of CM differentiation from CM structure proteins involved in muscle contraction and energy production [47]. A majority of cardiac transcription factors and members of key signaling pathways had increased active chromatin modifications (H3K4me3 and H3K36me3) and decreased repressive chromatin modification (H3K27me3), while genes encoding cardiac structure proteins showed a similar increase in active chromatin modification but no H3K27me3 deposition at any time [47]. This cardiac specific chromatin signature is also able to predict new regulators for appropriate human cardiac development [47]. Interestingly, a recent paper took advantage of iPSC reprogramming to study the epigenetic remodeling of cis-regulatory elements in cardiac development and diseases [49]. They performed massive ChIP-seq experiments for histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3) in different types of somatic cells derived from the same human fetal heart and their respective iPSCs. Defining with enrichment of H3K27ac

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and H3K4me1, cell type-specific enhancer elements were identified in human iPSCs and heart cells, and functionally validated in transgenic mouse embryos and human cells. Taken together, epigenome-wide analyses of histone modifications in combination with the transcriptome data not only provide new insights into the role of histone modifications and transcription factors in regulating cardiac-specific gene expression, but also identify stage-specific enhancers and promoters along heart development.

DYNAMICS OF REGULATORY CIS-ELEMENTS IN HEART DISEASE In addition to chromatin dynamics in normal heart development, a large number of regulatory cis-elements defined by a distinguished pattern of histone modifications have been identified in heart diseases [50e52]. Comparison of massive ChIP-seq and RNA-seq data between adult CMs isolated from hypertrophic hearts induced by transverse aortic constriction (TAC) and normal hearts demonstrated that pressure-overload hypertrophy is associated with changes in multiple types of histone modifications on a wide array of genes involved in cardiac functions [52]. Consistently, distinct global distribution of H3K36me3 and H3K4me3 enrichment correlated with mRNA abundance in cardiac cis-regulatory elements has been profiled in cardiomyopathic and normal hearts from rat models and human tissues [50,51]. Moreover, the epigenome study in mouse revealed that during cardiac hypertrophy, the epigenomic reprogramming occurs both on enhancers associated with normal heart development and on cis-elements specifically active in pathogenesis-wide [52]. It is also of great importance to understand whether and how the epigenomic dynamics contributes to the development of cardiomyopathy. The effect of histone modifications on cardiac diseases has been determined when they were removed or activated by manipulation of histone modification enzymes and administration of small molecules (see review in Ref. [53]). Such studies support the notion that epigenome-wide histone dynamics plays critical roles in heart failure pathogenesis (see review in Ref. [54]). Recently, epigenomic analyses showed that hyperacetylated chromatin induced by excessive activation of a histone acetyltransferase p300 is involved in pathological cardiac hypertrophy [55], while inhibition of p300 attenuates hypotrophic phenotype [56]. Overexpression of JMJD2, a demethylase of histone H3K9me3 and H3K36me3, exaggerated TAC-treated cardiac hypertrophy in CMs. Conversely, mice with JMJD2 loss were partially protected from pressure overload [57]. JMJD2 was shown to be associated with removal of H3K9me3 in specific loci of prohypertrophic genes [57]. In addition, a major component of polycomb repressive complex 2 (PRC2), EZH2, which catalyzes histone mark H3K27me3 for chromatin silencing, plays an important role during heart development and in the adult CMs [58,59]. Accumulating evidence implicates a potential mechanism by which altered chromatin signatures are recognized by specific DNA binding factors, or readers, to further regulate corresponding gene activity [60]. For instance, BET bromodomain reader protein was found to be critical transcriptional coactivator in activating pathologic genes that drive CM hypertrophy and heart failure progression. BET proteins recognize acetyllysine, which marks disease-specific genes in the genome, to increase the binding of Pol II complexes promoting chromatin remodeling, transcriptional initiation, and elongation [60]. Therefore, early administration of BET bromodomain selective inhibitor JQ1 blocks pathological hypertrophy in mice during pressure overload [60,61]. Collectively, dynamic changes in histone marks at the cis-regulatory regions of a genome allow DNA binding factors to activate and maintain specific gene expression with spatiotemporal precision during normal and pathological development.

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DNA METHYLATION DURING HEART DEVELOPMENT AND IN DISEASE The major form of DNA methylation refers to the addition of a methyl group to the 5th position on the cytosine ring of DNA (5-methylcytosine, 5mC). Mammalian DNA methylation at CpG dinucleotides is mainly catalyzed by three known DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) [62]. DNMT1 mainly involves in methylation maintenance, while DNMT3A and DNMT3B are de novo DNMTs that primarily methylate unmethylated DNA independent of DNA replication [63]. Reversely, removal of DNA methylation is carried out by the ten eleven translocation (TET) family catalyzing the conversion of 5mC to DNA to 5-hydroxymethylcytosine (5hmC) [64,65]. DNA methylation provides a critical epigenetic means for defining and maintaining cellular identity by regulating gene regulatory elements such as promoters and enhancers [66,67]. In general, DNA methylation is associated with gene repression, since DNA methylation is able to prevent the binding of transcriptional machinery and of transcription factors directly or influence chromatin structure [68,69]. There are a growing number of DNA methylation profiling technologies, which based on how 5mC is distinguished from cytosine, including commonly used reduced representation bisulfite sequencing (RRBS) [70], whole-genome bisulfite sequencing (WGBS) [71], methylated DNA immunoprecipitation sequencing (MeDIP-Seq) [72], and hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-Seq) [73].

DNA METHYLATION IS ORCHESTRATED IN NORMAL HEART The study of DNA methylation in heart development and disease is at its early stage. DNA methylation has been increasingly recognized as a highly dynamic process [74,75], and highly associated with cell type-specific gene expression during development [76]. Whole-genome bisulfite sequencing of adult mouse heart tissues at base-pair resolution firstly provides a map of heart-specific differentially methylated regions (DMR), which are predominantly regulatory elements enriched with transcription factor binding motifs [76]. Furthermore, the DNA methylomes from nuclei of PCM-1 positive CMs in neonatal and adult mouse hearts were generated [74] and compared with those from ESCs [77] and whole heart tissues [76]. Interestingly, a highly dynamic pattern of DNA methylation at cardiac enhancers and gene bodies occurs during CM development and maturation [74]. In particular, fetal genes gain methylation and adult genes lose methylation on their gene bodies during postnatal CM maturation until adulthood, resulting in postnatal isoform switch of sarcomeric genes. Of note, postnatal methylation of fetal genes was dependent on the presence of de novo DNA methyltransferases Dnmt3a/b [74]. Most recently, high-coverage DNA methylomes have been generated by WGBS in FACS-sorted CM nuclei isolated from fetal, infant, adult, and end-stage failing human hearts [78]. Distinct mCpG patterns were identified in distal regulatory and genic regions and grouped into partially methylated regions (PMR), low methylated regions (LMR), and unmethylated regions (UMR), which are characterized with low level of active histone marks, enhancer histone mark H3K4me1, and promoter mark H3K4me3, respectively. They also found that during normal prenatal development and postnatal maturation, CM transcriptome is shaped by a highly dynamic interplay between mCpG on gene body and histone modifications. These results highlight the involvement of dynamic DNA methylation on gene bodies in CM maturation at postnatal stage. Investigation of DNA methylome in postnatal CMs in the absence of DNMT3a/b will address the issues whether DNA methylation is required for heart maturation. The underlying molecular mechanisms need further investigation.

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Furthermore, the effect of DNA methylation on heart development can be suggested in studies of mice lacking DNMT. A general knockout of Dnmt3b leads to embryonic lethality between E13.5 and E16.5 with ventricular septal defects [63]. Dnmt3a homozygous knockout mice die at postnatal 3 weeks [63]. Whereas, CM-specific Dnmt3a and/or Dnmt3b knockout mice were generated in independent laboratories and showed complicated results [79,80]. aMHC-driven CM-specific loss of Dnmt3b resulted in compromised systolic function, widespread interstitial fibrosis, and myo-sarcomeric disarray [80]. In contrast, Myl7-driven CM-specific deletion of catalytic domains of both Dnmt3a and Dnmt3b showed no significant difference in CM function and CM response to pressure overload induced by TAC [79]. Moreover, in vitro RNAi-induced knockdown of Dnmt3a but not Dnmt3b via siRNA in cultured mouse embryonic CMs leads to sarcomere disassembly, and decrease in contractility and cytosolic calcium signaling [81]. Since these studies only showed restricted expression changes and methylation ablation in knockout or knockdown cells, it is still elusive whether DNA methyltransferases play pivotal roles for normal heart function. Combinational analyses of transcriptome and DNA methylome upon depletion of DNMTs will be valuable for a better understanding of DNA methylation in CMs.

DNA METHYLATION IS POTENTIAL THERAPEUTIC TARGET IN HEART DISEASE Based on comparative analyses of DNA methylation in normal and diseased hearts, DNA methylation is increasingly recognized as a fundamental epigenetic modification associated with cardiac diseases. As compared with postnatal CMs isolated from normal hearts, failing CMs partially resemble DNA methylation patterns in neonatal mouse CMs rather than those in adult CMs [74]. Moreover, two recent studies have reported differential DNA methylation signatures in hearts from patients with cardiomyopathy [51,82]. For the first time, genome-wide cardiac DNA methylation on human dilated cardiomyopathy in patients was generated by methylation chip and compared with controls from healthy patients [82]. The authors found that genes with differential DNA methylation were significantly enriched in pathways related to cardiac disease. In addition, they identified novel candidate regulators LY75 and ADORA2A via alteration of DNA methylation and mRNA expression for dilated cardiomyopathy, and evaluated the function of these genes in zebrafish model [82]. In the other study, compared with normal human hearts, DNA methylation maps generated by methylated DNA immunoprecipitation sequencing (MeDIP-seq) in end-stage failing hearts revealed that methylation changes in promoter CpG islands and gene bodies of genes that play critical roles in myocardial stress response, but not in intergenic CpG islands and enhancer CpG islands [51]. Intriguingly, loss of methylation in promoter regions is only correlated with upregulated genes in cardiomyopathic hearts, while no significant changes of methylation at the promoter of downregulated genes. However, different results were found in the comparative analysis of DNA methylome in nonfailing and heart failing human CMs, purified with specific CM marker phospholamban (PLN) [78]. Differentially expressed pathological genes show minimal alterations in mCpG of genic and cis-regulatory regions. Instead, they found that single-nucleotide polymorphisms (SNPs) associated with cardiac disease traits are highly enriched in low methylated cis-regulatory regions of human CMs. Collectively, apart from the identification of a specific DNA methylation dynamics of heart disease, we learned two more lessons from these studies. First, since different CVDs have disease-specific DNA methylation signatures [51,74,82], it is reasonable to hypothesize that DNA methylation signatures in CVDs can serve as potential diagnostic biomarkers and therapeutic targets. Most recently, in a multiomics study,

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several epigenetic loci have been identified to be significantly associated with dilated cardiomyopathy and might be potential epigenetic biomarkers for heart failure [83]. Second, through epigenome-wide analysis of DNA methylation combined with maps of histone marks, novel disease-related genes have been identified with functional relevance and will be potentially druggable targets in CVDs.

DNA HYDROXYMETHYLATION REGULATES GENE EXPRESSION IN CARDIAC DEVELOPMENT AND HYPERTROPHY Although the role of DNA hydroxymethylation is not fully understood, studies have suggested that 5hmC is not only an intermediate product in the active DNA demethylation of 5mC, but also recognized as another stable epigenetic mark on DNA to regulate gene expression in several cell types [84e88]. 5hmC is found to be located on gene body and associated with active transcription in multiple cells [84e88]. Whereas, enrichment of 5hmC was also found on the transcription start sites of repressed but poised genes, whose promoters carry bivalent histone methylation marks, H3K4me3 and H3K27me3 in ESCs [86,87]. Moreover, it is widely reported that the occupancy of 5mC and 5hmC in the genome is highly correlated [84,89]. Recently, the base-resolution analysis of 5hmC in the CM genome dissects the role of DNA hydroxymethylation in cardiac development and hypertrophy [90]. The genome-wide 5hmC distribution was determined by hydroxymethylated DNA immunoprecipitation (hMeD-IP) coupled with high-throughput sequencing in CMs isolated from embryonic, neonatal, adult, and TAC-induced hypertrophic hearts. The majority of 5hmC was located at introns and intergenic regions. However, accumulation of 5hmC on the gene body is strongly correlative with active cardiac gene expression during development, while accompanied with loss of 5hmC on intergenic regions. Interestingly, in line with the finding in DNA methylome during heart development [74], cardiac hypertrophy leads to a shift of 5hmC modification towards a neonatal-like pattern. Furthermore, appropriate 5hmC distribution and gene expression in CM require the presence of TET2. Taken together, DNA hydroxymethylation is largely reprogrammed in heart development, as well as cardiac hypertrophy. Further studies need to elucidate the balance between 5mC and 5hmC for gene regulation in cardiac physiology and disease.

CHROMATIN CONFORMATION IN CARDIOMYOCYTES As mentioned above, besides modifications on histone proteins and DNA, epigenomic regulation also includes changes of chromatin structures, which reflects dynamic accessibility of genetic information and inter- and intrachromosomal communication [91]. Using newly developed chromosome conformation capture technology (such as 3C, 4C, 5C, Hi-C) and related computational tools [92], the 3-dimensional organization of chromatin has been investigated at high resolution in the whole genome [91,93]. Vast amounts of genome-wide interaction data discovered that chromosomes consist of discrete topologically associating domains (TADs), genome compartments, chromatin looping and interactions between cis-elements [94], which are defined by boundary binding of CTCF [95]. The loss of CTCF, which is the critical insulator for proper chromatin structures, disrupts morphogenesis and maturation of embryonic hearts before death at embryonic day 12.5 [96]. Interestingly, another aMHC-driven CM-specific loss of CTCF leads to cardiomyopathy even worse than TCA-induced phenotype [97]. Then Hi-C and high-throughput sequencing were applied to adult CMs from normal, TCA-treated, and CTCF-CKO hearts to investigate the contribution

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of chromatin structure in healthy and diseased CMs [97]. In heart failure models, the large-scale alterations in chromatin structure have been identified. Notably, pressure overload and CTCF depletion selectively altered chromatin looping near genes associated with disease, especially influenced the interaction between enhancers and genes [97]. This study provides a valuable resource for further investigation of epigenome dynamics when combined with many other data sets such as DNA methylome, histone mark ChIP-seq and ATAC-seq in cardiac development and diseases.

RAPID CHROMATIN SWITCH DURING SOMATIC REPROGRAMMING Underlying mechanisms of direct cardiac reprogramming are not clear, yet we know that cardiac reprogramming process is associated with extensive epigenetic changes [98e101]. Recently, we investigated the occupancy of H3K4me3, H3K27me3 and DNA methylation on specific cardiac and fibroblast-related loci and showed temporal changes of epigenetic status during cardiac reprogramming, in which cardiac loci were rapidly bound with active marks, while loci associated with fibroblast fate were gradually labeled with repressive marks [102]. Moreover, decrease of repressive mark H3K27me3 by downregulating PRC2 complex components or pharmaceutical inhibitors appeared to be promotive for the initiation of iCM reprogramming mediated by a microRNA combination of miR-1, miR-133, miR-208, and miR-499 [98]. Besides, we and others identified major epigenetic barriers to iCM reprogramming through loss-of-function and gain-of-function screens of epigenetic factors [99,101]. As the key component of polycomb repressive complex 1 (PRC1), Bmi1 represses cardiac gene expression during Mef2c/Gata4/Tbx5-induced iCM reprogramming through histone repressive mark H2AK119ub on cardiac loci. Removal of Bmi1 deactivates cardiac gene expression, especially Gata4, leading to successful reprogramming with only two factors, Mef2c and Tbx5 [101]. Meanwhile, Liu et al. found that overexpression of Men1 reduced reprogramming efficiency. Men1 is the coactivator of methyltransferase Mll1, which is one of the “writer” proteins of H3K4 methylation. Consistently, inhibition of Mll1 by small molecules enhanced iCM generation [99]. However, the dynamic epigenomic landscape highly associated with cardiac fate conversion and maintenance remains not clarified. Nevertheless, during direct neuronal reprogramming, epigenomic changes mediated by pioneer transcription factors have been investigated and showed critical roles of epigenomic dynamics in the achievement of induced neuronal cells (iNs) [103]. The higher order chromatin architecture is relatively less clear, but loose and condensed chromatin structures reflecting DNA accessibility to regulatory factors and complexes are thought to have a pronounced impact on gene regulation [39] and control of cell fate [104]. Recently, the chromatin accessibility changes of direct reprogramming of fibroblasts into iNs have been studied in a genome-wide fashion and showed the rapid chromatin switch mediated by Ascl1 throughout the course of iN reprogramming [103]. The analysis of ATAC-seq data indicated that the majority of genomic loci are affected from as early as 12 hours to 5 days post-infection, while little chromatin remodeling occurs in sorted iN expressing neuronal reporter gene TauEGFP at day 5 and later stages. In combination with ChIP-seq data for Ascl1 and RNA-seq data for the corresponding time points during iN reprogramming [105], network analysis of ATAC-seq data identified several novel critical transcription factors Zfp238, Sox8, and Dlx3. Each of them is able to generate iNs in combination with another iN reprogramming factor Mytl1 [103]. Although it is still unknown if the epigenomic landscape change is a common feature between different direct reprogramming systems, these findings highlight the importance of chromatin state switch from donor cell type to the target one and might be valuable for future translation of direct reprogramming for regenerative medicine.

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CONCLUSION Epigenomic reprogramming is engaged during normal heart development and cardiac diseases. All of the above findings including altered histone modifications, global DNA methylation, and the plastic genome structures support the notion that epigenome offers a new perspective in the control of gene regulation, with a promising application to CVD therapy. With a rapidly growing number of epigenomes being determined in normal and diseased cells, one of the main challenges we will face is how to better integrate epigenomic data with other omics data, like transcriptome, proteome, and metabolome to gain useful biological insight. More effective bioinformatics and standardized computational tools are urgently needed. Further efforts to combinational omics analyses will provide new mechanisms of chromatin regulation at different chromatin levels and inspire new treatment strategies for heart failure therapy. Meanwhile, comprehensive decoding of epigenetic patterns in somatic cell reprogramming could facilitate understanding of the underlying mechanism of cell fate conversion and clinical translation, and ultimately pave the way for the development of personalized medicine for CVDs.

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