Real-Time and Spatially Resolved Epigenetic Dynamics Tracking Beyond DNA Methylation via Live-Cell Epigenetic Sensors in 3D Systems

Modulation of gene expression is influenced by environmental exposures, genetic variation and stochastic processes. Epigenetic biomarkers that signal the outcome of their combined impact could be important early predictors of susceptibility to chronic noncommunicable diseases. Can epigenetic epidemiology shed light on causality and mechanism? Common complex traits arise over time and are the end result of lifestyle choices and a combination of environmental exposures during periods of developmental vulnerability and epigenetic plasticity in the presence of a combination of susceptible genetic variants. Genome-wide association studies to identify the genetic contributions to complex traits have identified many high probability allelic associations, but almost invariably each with a small effect, explaining only a modest percentage of the heritability of the trait. Notably, many estimates of heritability are inflated and do not adequately take into account-shared environment and other familial factors. It is not new to suggest that tissue-specific epigenetic modulation plays a major role in the development of complex traits, yet few have managed to support this using a robust experimental approach. Whereas the genome remains constant, gene expression is labile, demonstrates cell type and developmental specificity, age and sex differences, and is variably responsive to changes in the environment. What are the relationships between genetic variation and different environments that influence epigenetic responses? How do we begin to understand causal relationships that influence gene expression? From an experimental perspective, it is challenging to design studies that can address these questions, yet present a feasible and affordable approach. Large international and interdisciplinary studies are beginning to provide a good framework to better understand gene expression regulation in humans. Though open to criticism, their value should not be underestimated. They include ENCODE that examined 147 cell lines to search for functional elements in the genome [1]; the Epigenomics Roadmap [2] that examined 127 tissue biopsies from embryos and adults, both diseased and healthy, with a focus on identifying functional regulatory elements; the International Human Epigenome Consortium [3]; and the Genome Tissue Expression project (GTEx) that explores the relationship between genetic variation and tissue-specific gene expression and alternate splicing by sequencing whole genomes and examining gene expression in over 20,000 tissue samples from 900 individuals [4]. These data are beginning to give insight into cell specific, developmental stage, sex, age and ethnicity influenced, and stochastic gene expression differences at an individual and population level. The layered annotations in genome browsers highlight the importance of integrating transcriptome data (mRNA, miRNA and lncRNA) with DNA methylation, histone modification and chromatin interaction data to more fully understand function. Epigenome-wide association studies (EWASs) to identify regulatory mechanisms Epigenetic epidemiology: is there cause for optimism?

Decision letter: Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

Background: The neonatal mammalian heart has a transient capacity for regeneration, which is lost shortly after birth. A series of critical developmental transitions including a switch from hyperplastic to hypertrophic growth occur during this postnatal regenerative window, preparing the heart for the increased contractile demands of postnatal life. Postnatal cardiomyocyte maturation and loss of regenerative capacity are associated with expression alterations of thousands of genes embedded within tightly controlled transcriptional networks, which remain poorly understood. Interestingly, although mitogenic stimulation of neonatal cardiomyocytes results in proliferation, the same stimuli induce hypertrophy in adult cardiomyocytes by activating different transcriptional pathways, indicating that cardiomyocyte maturation may result from epigenetic modifications during development. Notably, DNA methylation and chromatin compaction are both important epigenetic modifications associated with a decrease in transcription factor (TF) accessibility to DNA. However, the role of both DNA methylation and chromatin compaction during postnatal cardiac maturation remain largely unknown.Hypothesis: Postnatal changes in DNA methylation and chromatin compaction silence transcriptional networks required for cardiomyocyte proliferation during postnatal heart development.Aims: This PhD Thesis focuses on characterising the role of DNA methylation and chromatin dynamics during postnatal cardiac development and maturation using in vitro and in vivo model systems. Three major Aims are addressed:• Aim 1: To characterize transcription and DNA methylation dynamics during postnatal rodent cardiac development (Chapter 3).• Aim 2: To determine the role of DNA methylation in regulating the expression of cell cycle related genes in mouse and human cardiomyocytes (Chapter 4).• Aim 3: To understand the relationship between chromatin accessibility and transcription during mouse and human cardiomyocyte development (Chapter 5).Results: In Aim 1, global DNA methylation changes were characterized during postnatal development. Marked changes in the expression levels of key DNA methylation enzymes were observed during the first month of postnatal heart development. Postnatal inhibition of DNA methylation using 5-aza-2’-deoxycytidine (5aza-dC; 1mg/kg/day) reduced global DNA methylation levels in the heart in vivo and was associated with a marked increase in cardiomyocyte proliferation (~3-fold) and ~50% reduction in binucleated cardiomyocytes compared to saline-treated controls, suggesting DNA methylation is required for postnatal cardiomyocyte cell cycle arrest. Nextgeneration mRNA sequencing (RNA-seq) and genome-wide sequencing of methylated DNA (methyl-CpG binding domain enrichment and sequencing (MBD-seq)) identified dynamic changes in the cardiac methylome during postnatal development (2545 differentially methylated regions (DMRs) from postnatal day (P) 1 to P14 in the mouse). ~80% of DMRs were hypermethylated and these DMRs were associated with transcriptional shut down of important developmental signalling pathways.In Aim 2, a specific subset of cell cycle genes displaying differentially regulated gene expression and DNA methylation patterns were profiled to understand how DNA methylation is involved in regulating cardiomyocyte proliferation. Gene expression profiling of these cell cycle genes during development and following 5aza-dC treatment in mouse hearts, as well as 3D human cardiac organoids (hCOs), verified that DNA methylation was required for transcriptional regulation of a small group of cell cycle genes. Interestingly, 5aza-dC (10µM) also induced ~2-fold increase in cardiomyocyte proliferation in hCOs. These findings suggest that DNA methylation has a direct role in the regulation of a subset of genes required for cardiomyocyte proliferation in both mice and humans.Chromatin accessibility results from the integrated action of multiple epigenetic marks. In Aim 3, cardiomyocyte nuclei were isolated from both human and mouse developmental heart samples for RNA-seq and for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile changes in transcription and chromatin accessibility during cardiomyocyte development in mice (P1 to P56) and humans (foetal (14-19 weeks (wks)), 0-10 years (yrs), 10-30 yrs and 30+ yrs). RNA-seq confirmed that cardiomyocyte cell cycle arrest occurs between P1 and P14 in the mouse. Moreover, ATAC-seq identified open chromatin signatures at various genomic features, including CG islands, transcription start sites (TSSs), enhancers, TF binding sites and protein coding regions, while intergenic regions were associated with compact chromatin. Integration of RNA-seq and ATAC-seq data demonstrated a strong correlation between transcription and chromatin accessibility at the different stages of cardiomyocyte development. TF motif analysis identified E2F transcription factor 4 (E2f4) and Forkhead box M1 (Foxm1) sites as being transcriptionally repressed and undergoing chromatin compaction from P1 to P56. E2f4 and Foxm1 are well known TFs that regulate cell cycle, thus suggesting that cell cycle genes are epigenetically silenced during cardiomyocyte maturation.Conclusion: This PhD Thesis provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation and chromatin compaction. Together, this PhD Thesis provides new insights into the role of DNA methylation and chromatin dynamics during cardiac development. This Thesis provides a new framework for the epigenetic regulation of transcription during postnatal cardiomyocyte maturation and points towards an epigenetic mechanism for cardiomyocyte terminal differentiation during the neonatal period.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

EpigenomicsVol. 7, No. 8 CommentaryMendelian randomization: applications and limitations in epigenetic studiesCaroline L Relton & George Davey SmithCaroline L Relton*Author for correspondence: E-mail Address: caroline.relton@bristol.ac.uk MRC Integrative Epidemiology Unit, University of Bristol, Bristol, BS8 2BN, UK & George Davey Smith MRC Integrative Epidemiology Unit, University of Bristol, Bristol, BS8 2BN, UKPublished Online:7 Dec 2015https://doi.org/10.2217/epi.15.88AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: causal inferenceDNA methylationepidemiologyepigeneticsmediationSNPPapers of special note have been highlighted as: • of interest; •• of considerable interestReferences1 Mathers JC, Strathdee G, Relton CL. Induction of epigenetic alterations by dietary and other environmental factors. Adv. 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Epidemiol. 44(2), 512–525 (2015).Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited ByEpigenome-wide epidemiologic studies of human immunodeficiency virus infection, treatment, and disease progression11 January 2022 | Clinical Epigenetics, Vol. 14, No. 1A comparison of the genes and genesets identified by GWAS and EWAS of fifteen complex traits19 December 2022 | Nature Communications, Vol. 13, No. 1Prenatal medication exposure and epigenetic outcomes: a systematic literature review and recommendations for prenatal pharmacoepigenetic studies29 April 2021 | Epigenetics, Vol. 17, No. 4Shared Genetic Basis and Causal Relationship Between Television Watching, Breakfast Skipping and Type 2 Diabetes: Evidence From a Comprehensive Genetic Analysis24 March 2022 | Frontiers in Endocrinology, Vol. 13Epigenetic Clocks14 April 2022EpigeneticsImpact of In Utero Folate Exposure on DNA Methylation and Its Potential Relevance for Later‐Life Health—Evidence from Mouse Models Translated to Human Cohorts13 December 2021 | Molecular Nutrition & Food Research, Vol. 261A meta-analysis of epigenome-wide association studies in Alzheimer's disease highlights novel differentially methylated loci across cortex10 June 2021 | Nature Communications, Vol. 12, No. 1Genetic impacts on DNA methylation: research findings and future perspectives30 April 2021 | Genome Biology, Vol. 22, No. 1Causal Inference Methods to Integrate Omics and Complex Traits17 August 2020 | Cold Spring Harbor Perspectives in Medicine, Vol. 11, No. 5Tobacco Smoking and the Association With First Incidence of Mood, Anxiety, and Substance Use Disorders: A 3-Year Prospective Population-Based Study15 March 2021 | Clinical Psychological Science, Vol. 9, No. 3Time trends in epigenetic signatures and population health risksInvited Review – A 5‐year update on epigenome‐wide association studies of DNA modifications in Alzheimer's disease: progress, practicalities and promise30 August 2020 | Neuropathology and Applied Neurobiology, Vol. 46, No. 7Understanding the Interplay Between Health Disparities and Epigenomics20 August 2020 | Frontiers in Genetics, Vol. 11Lifetime Smoking and Asthma: A Mendelian Randomization Study4 August 2020 | Frontiers in Genetics, Vol. 11Genetically, Dietary Sodium Intake Is Causally Associated with Salt-Sensitive Hypertension Risk in a Community-Based Cohort Study: a Mendelian Randomization Approach26 June 2020 | Current Hypertension Reports, Vol. 22, No. 7Antipsychotic Medications and DNA Methylation in Schizophrenia and Bipolar Disorder: A Systematic ReviewPharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, Vol. 40, No. 4Epigenetics and gestational diabetes: a review of epigenetic epidemiology studies and their use to explore epigenetic mediation and improve prediction17 October 2019 | Diabetologia, Vol. 62, No. 12DNA methylation aging clocks: challenges and recommendations25 November 2019 | Genome Biology, Vol. 20, No. 1Mendelian randomization provides support for obesity as a risk factor for meningioma22 January 2019 | Scientific Reports, Vol. 9, No. 1Appraising the causal relevance of DNA methylation for risk of lung cancer24 September 2019 | International Journal of Epidemiology, Vol. 48, No. 5Mendelian randomization studies on atherosclerotic cardiovascular disease: evidence and limitations17 May 2019 | Science China Life Sciences, Vol. 62, No. 6A brief tour of epidemiologic epigenetics and mental healthCurrent Opinion in Psychology, Vol. 27An integrative approach to detect epigenetic mechanisms that putatively mediate the influence of lifestyle exposures on disease susceptibility29 June 2019 | International Journal of Epidemiology, Vol. 48, No. 3Causal Modeling in Environmental HealthAnnual Review of Public Health, Vol. 40, No. 1Mendelian Randomization and the Environmental Epigenetics of Health: a Systematic Review18 February 2019 | Current Environmental Health Reports, Vol. 6, No. 1Postnatal Social Factors: The Epigenome and the Skeleton29 June 2019Epigenomics in tobacco risk assessment: Opportunities for integrated new approachesCurrent Opinion in Toxicology, Vol. 11-12Unravelling the Roles of Susceptibility Loci for Autoimmune Diseases in the Post-GWAS Era27 July 2018 | Genes, Vol. 9, No. 8DNA methylation in human lipid metabolism and related diseasesCurrent Opinion in Lipidology, Vol. 29, No. 2Protocol for the EMPHASIS study; 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The REGICOR study15 September 2016 | Human Molecular Genetics, Vol. 25, No. 20How to make DNA methylome wide association studies more powerfulXinyi Lin, Sheila Barton & Joanna D Holbrook7 April 2016 | Epigenomics, Vol. 8, No. 8 Vol. 7, No. 8 Follow us on social media for the latest updates Metrics Downloaded 297 times History Published online 7 December 2015 Published in print December 2015 Information© Future Medicine LtdKeywordscausal inferenceDNA methylationepidemiologyepigeneticsmediationSNPAcknowledgementsThe authors would like to thank members of the MRC Integrative Epidemiology Unit for their contribution in developing this area of research.Financial & competing interests disclosuresThe authors' work related to the application of Mendelian randomization to epigenetics is supported by the Medical Research Council Integrative Epidemiology Unit at the University of Bristol (MC_UU_12013/1 and MC_UU_12013/2). This work was also supported by CRUK (grant number C18281/A19169). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

BackgroundSmall RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.ResultsPaternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.ConclusionsDifferential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.

Update on epigenetics in allergic disease

DNA methylation and chromatin accessibility play important roles in gene expression, but their function in subgenome expression dominance remains largely unknown. We conducted comprehensive analyses of the transcriptome, DNA methylation, and chromatin accessibility in liver and muscle tissues of allotetraploid common carp, aiming to reveal the function of epigenetic modifications in subgenome expression dominance. A noteworthy overlap in differential expressed genes (DEGs) as well as their functions was observed across the two subgenomes. In the promoter and gene body, the DNA methylation level of the B subgenome was significantly different than that of the A subgenome. Nevertheless, differences in DNA methylation did not align with changes in homoeologous biased expression across liver and muscle tissues. Moreover, the B subgenome exhibited a higher prevalence of open chromatin regions and greater chromatin accessibility, in comparison to the A subgenome. The expression levels of genes located proximally to open chromatin regions were significantly higher than others. Genes with higher chromatin accessibility in the B subgenome exhibited significantly elevated expression levels compared to the A subgenome. Contrastingly, genes without accessibility exhibited similar expression levels in both subgenomes. This study contributes to understanding the regulation of subgenome expression dominance in allotetraploid common carp.

Simple SummaryTo understand the effect of metformin on epigenetic regulation, we analyzed histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin showed little effect on DNA methylation or chromatin accessibility but significantly reduced H3K4me3 levels at the promoters of positive cell cycle regulatory genes. Metformin downregulated H3K4 methyltransferase MLL2 expression and knockdown of MLL2 resulted in suppression of H3K4me3 expression and lung cancer cell proliferation. We further evaluated the clinicopathological significance of MLL2 in tumor and matched normal tissues from 42 non-small cell lung cancer patients. MLL2 overexpression was significantly associated with poor recurrence-free survival in lung adenocarcinoma. Our study facilitates the understanding of the effect of metformin on the regulation of histone H3K4me3 at promoter regions of cell cycle regulatory genes in lung cancer cells, and MLL2 may be a potential therapeutic target for lung cancer therapy.This study aimed at understanding the effect of metformin on histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin significantly reduced H3K4me3 level at the promoters of positive cell cycle regulatory genes such as CCNB2, CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. Metformin suppressed the expression of H3K4 methyltransferases MLL1, MLL2, and WDR82. The siRNA-mediated knockdown of MLL2 significantly downregulated global H3K4me3 level and inhibited lung cancer cell proliferation. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, and a Cox proportional hazards analysis showed that recurrence-free survival of lung adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08–4.72; p = 0.02) times poorer than in those without it. Metformin showed little effect on DNA methylation and chromatin accessibility at the promoter regions of cell cycle regulatory genes. The present study suggests that metformin reduces H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. Additionally, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.

Ectopic NSD2 Remodels H3K36me2 and DNA Methylation to Promote Oncogenic Gene Expression in Multiple Myeloma

Introduction: Subpopulations of highly tumorigenic, drug resistant cancer stem-like cells (CSCs) play a key role in cancer recurrence and progression. Surprisingly, these aggressive cells can arise repeatedly de novo from bulk tumor cells independently of mutational events. We investigated whether transition to and from a cancer stem-like state is associated with epigenetic alterations, such as DNA methylation and chromatin accessibility. Materials and methods: Using FACS and Hoechst dye staining, side population (SP) cells marked by high tumorigenicity and drug resistance vs. non-side population (NSP) cells lacking these properties were isolated from bladder cancer cell lines. Global DNA methylation and chromatin accessibility were mapped for SP vs. NSP cells using AcceSssIble assay on the Infinium EPIC platform. Differential gene expression for SP vs. NSP cells was assayed by HuEx array. AcceSssIble and HuEx results were overlapped to generate a subset of genes with differential accessible promoter regions and expression. One such gene, E2F3, was validated by qRT-PCR and analyzed clinically using the Oncomine and Basespace online data mining engines, as well as functionally by lentiviral shRNA depletion in bladder cancer cells for cell migration, invasion and drug resistance. 5-AZA was used to modulate global DNA methylation in bladder cancer cells. Results: SP and NSP subpopulations exhibited differential DNA methylation and chromatin accessibility at thousands of gene loci by AcceSssIble assay, and a majority of differential chromatin accessibility was independent of DNA methylation. When overlapped with HuEx data, 38 genes exhibited differential expression associated with differential promoter chromatin accessibility. One such gene validated by qRT-PCR was E2F3, a cell cycle regulator that was found to be associated with bladder cancer in the Oncomine and Basespace databases. In vitro E2F3 depletion by lentiviral shRNA resulted in smaller SP subpopulations and reduced cell migration, invasiveness, and drug resistance. Epigenetic interference with 5-AZA blocked the phenotypic transition, reduced SP subpopulations, and lowered E2F3 expression. Conclusion: Rapid cyclical transition between the SP and NSP subpopulations was associated with dramatic shifts in chromatin accessibility with concomitant changes in gene expression of clinically relevant genes that drove the SP phenotype, cell migration, invasiveness, and drug resistance. These observations implicate epigenetic plasticity as a driver of the switch between cancer stem-like and non-cancer stem-like states, suggesting new potential therapeutic avenues to control cancer resistance and progression. Citation Format: Tong Xu, Hongtao Li, Rong Xu, Jenny Wei, Meng Li, Gangning Liang, Amir Goldkorn. Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4335.

Vitamin B12, a co-factor in methyl-group transfer, is important in maintaining DNA (deoxycytidine) methylation. Using two independent assays we examined the effect of vitamin B12-deficiency (plasma vitamin B12<148 pmol/L) on DNA methylation in women of childbearing age. Coagulated blood clot DNA from vitamin B12-deficient women had significantly (p<0.001) lower percentage deoxycytidine methylation (3.23±0.66%; n = 248) and greater [3 H]methyl-acceptance (42,859±9,699 cpm; n = 17) than DNA from B12-replete women (4.44±0.18%; n = 128 and 26,049±2,814 cpm; n = 11) [correlation between assays: r = –0.8538; p<0.001; n = 28]. In contrast, uncoagulated EDTA-blood cell pellet DNA from vitamin B12-deficient and B12-replete women exhibited similar percentage methylation (4.45±0.15%; n = 77 vs. 4.47±0.15%; n = 47) and [3 H]methyl-acceptance (27,378±4,094 cpm; n = 17 vs. 26,610±2,292 cpm; n = 11). Therefore, in simultaneously collected paired blood samples, vitamin B12-deficiency was associated with decreased DNA methylation only in coagulated samples. These findings highlight the importance of sample collection methods in epigenetic studies, and the potential impact biological processes can have on DNA methylation during collection.

PML-RARα/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia

Comparison of Expression and Epigenetic Profiles in Human and Mouse Erythropoiesis and Megakaryopoiesis Using a Systems Biology Model

Genome-wide epigenetic modification dynamics, including DNA methylation and chromatin modification, are involved in biological processes such as development, aging, and disease. Quantitative identification of differential epigenetic modification regions (DEMRs) from various temporal and spatial epigenomes is a crucial step towards investigating the relationship between epigenotype and phenotype. Here, we describe EpiDiff (http://bioinfo.hrbmu.edu.cn/epidiff/), an integrated software platform that supports quantification of epigenetic difference and identification of DEMRs by Shannon entropy. Two main modules, quantitative differential chromatin modification region (QDCMR) and quantitative differentially methylated region (QDMR) are provided for bioinformatic analysis of chromatin modifications and DNA methylation data, respectively. The third module, quantitative differential expressed gene (QDEG), can be used to identify differentially expressed genes. The platform-free and species-free nature of EpiDiff makes it potentially applicable to a wide variety of epigenomes at an unprecedented scale and resolution. The graphical user interface provides biologists with a practicable and reliable way to analyze and visualize epigenetic difference.