Epigenomics in Health and Disease

Spain

Preface

Agustin F. Fernandez, Hospital Universitario Central de Asturias, Institute of Oncology of Asturias, Universidad de Oviedo, Spain

Mario F. Fraga, Nanomaterials and Nanotechnology Research Center, Institute of Oncology of Asturias, Universidad de Oviedo, Spain

Both genetic and epigenetic mechanisms are involved in the regulation of gene expression, cellular differentiation, and developmental processes. An essential difference is that epigenetic alterations do not involve a change to the DNA sequence, and therefore they can be reverted by the use of therapeutic drugs, such as DNA methylation or histone deacetylases inhibitors.

Although many epigenetic modifications are maintained throughout a person’s lifetime, at certain genomic loci, epigenetic marks can change over time, and this is thought to depend both on intrinsic, or genetic, and extrinsic, or environmental factors. The impact of genetic variance on epigenetic regulation, especially in pathologic contexts, is covered in Chapter 1 of this volume.

In 1989, only a few years after the first human oncogene (H-RAS) was found to be activated by point mutation, an epigenetic alteration of a tumor suppressor gene (Rb) in human cancer was discovered. The first epigenetic studies analyzed alterations in DNA methylation in tumor suppressor genes by using candidate gene approaches. Later on, in addition to DNA methylation, other epigenetic mechanisms, such as post-translational modifications (PTM) of histones and noncoding RNAs (ncRNAs), have been found altered in association with several human diseases.

During the last few decades, methods and technologies aimed at studying changes in epigenetic marks have evolved from the analysis of a small number of specific candidate genes to analyzing large numbers of genes, but the true qualitative and quantitative leap occurred with the appearance of the techniques of ultra-sequencing complete epigenomes. These new technologies have made it possible to investigate in greater detail the epigenetic alterations associated with human disease. In Chapters 2 and 3, various methodologies for the study and analysis of complete epigenomes, and their usefulness in the identification of new biomarkers associated with diseases are described. The emergence of these new technologies has revolutionized the way of approaching research projects, as much as the way we manage all the information obtained. Chapter 4 explores how to analyze the huge amount of data obtained by these methods, and the software tools currently available for the analysis of epigenomic data are described. Chapters 5–11 of this volume summarize studies where these next-generation technologies have been applied to the search for genome-wide epigenetic changes related to aging, cancer, and other human diseases, including neurologic disorders and autoimmune and metabolic diseases. The focus of Chapter 12 is the importance of factors that should be taken into account in the design of epigenomic epidemiologic studies. And finally, Chapter 13 discusses how epigenomic data can be useful in the clinical management of human disease, particularly in cancer, and the potential applications of epigenetic and genetic biomarkers in optimizing epigenetic therapies are described.

A major future challenge in the field of epigenetics will be to identify epigenetic marks in single cells, since epigenetics, unlike genetics, is cell specific. This epigenomic cell information, along with the genetic information, will provide the basis for defining customized therapies in the treatment of human diseases.

Our most sincere thanks to all the authors for their outstanding contributions.

Chapter 1

The Role of the Genetic Code in the DNA Methylation Landscape Formation

Holger Heyn,    Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, Barcelona, Catalonia, Spain

Abstract

The genetic and epigenetic codes are highly interconnected, and the understanding of causalities within this cross talk guides our understanding of the biology of healthy life and diseases. Particularly, epigenetic regulation helps interpret genetic variants, and in return functional variance in the epigenetic landscape can be identified through its relation to the genetic background. Epigenetic mechanisms, particularly DNA methylation, shaped the genetic sequence during evolution, and the intrinsic properties of the DNA have impact on the formation of the DNA methylation landscape. Here it is the complex process of gene regulation that is actively controlled by differential DNA methylation patterns. In contrast, transcriptional regulation, such as transcription factors binding to genetic consensus sequences, leaves its marks in the DNA methylome. Elucidating consequences of genetic variance and particularly its impact on epigenetic regulation provides important insights into phenotype formation in natural variation and pathologic contexts, including disease risk.

Keywords

Epigenetics; genetics; DNA methylation; transcription factor; genome-wide association studies; methylation quantitative trait loci; cancer risk; meQTL; GWAS

Chapter Outline

1.1 Bringing the Genetic Code to Life 1

1.2 Intrinsic Properties of DNA 3

1.3 Sequence-Pattern-Dependent DNA Methylation Profiles 5

1.4 DNA-Binding Factors Shaping the Epigenetic Landscape 6

1.5 The Dynamics of Transcription-Factor-Mediated DNA Hypomethylation 8

1.6 Translational Potential of Demethylation Dynamics 9

1.7 DNA Methylation Quantitative Trait Loci 9

1.8 Genotype-Driven Variance in Human DNA Methylation Profiles 10

1.9 Epigenetic Mediator Function for Human Risk Phenotype Formation 11

1.10 Epitype Association Guiding the Interpretation of Cancer Risk Polymorphisms 12

1.11 Closing Remarks 13

References 14

1.1 Bringing the Genetic Code to Life

At the beginning of this millennium, the human genome was decoded [1,2]. The sequencing of the major fraction of the genetic sequence and the resulting first version of an assembled human reference genome displayed an important step toward the understanding of cellular biology and human life. Using the then-current knowledge of genomic organization and sequence characteristics, the genetic sequence was estimated to be roughly 1% coding and the majority of the genome to be noncoding, from which a major part was considered junk DNA [3]. However, realizing that the human genome differs only marginally between individuals and is even conserved to around 99% between humans and chimpanzees [4], the noncoding part of the genome was quickly recognized to contribute to phenotype formation and interindividual and interspecies differences.

To illustrate the impact of gene regulation on phenotype formation, the human body presents the best paradigm. Considering the fact that every single cell in the human body carries exactly the same genetic information, it appears remarkable that cells can differ to such a large extent in appearance and function. The difference between a pancreatic cell that produces insulin and a hematopoietic stem cell, giving rise to numerous specialized blood cell types, does not lie within their genetic sequence but is grounded in the activity of the genome. A distinct set of active and repressed genomic regions defines intraindividual differences between cell phenotypes but is also responsible for variation between human individuals (interindividual) and across species (interspecies). Following the sequencing of more than 1000 human genomes [5] and the analyses of variable genetic loci in thousands of individuals in genome-wide association studies (GWAS) [6], it also became apparent that the majority of human phenotypes (traits), ranging from the color of the hair to the susceptibility to develop breast cancer, is encoded in the noncoding fraction of the genome.

Although the protein-coding fraction of the genome, including transcription and translation start sites, was well annotated, the knowledge of the noncoding genome was grounded in annotations based on distance to known genes (promoter regions) or single examples of cis-acting regulatory regions (enhancers). As the function of the genome outside the coding context could not be inferred by the sequence itself, efforts were focused on the comprehensive annotation of the noncoding part of the genome that was no longer considered silent or junk, but rather as important driver for phenotype formation. Here seminal contribution was made by the work performed within the ENCyclopedia Of DNA Elements (ENCODE) project, which, after annotating selected parts of the genome in a pilot phase (1% of the genome) [7], presented a comprehensive catalog of regulatory elements within the human genome and led to different estimates as to the extent of active regions in the human genome [8]. However, although the features tested to determine functionality (ranging from transcription activity to DNA factor binding occupancy and chromosomal conformation) covered a wide range of mechanisms involved in known gene regulatory processes, the tissue specificity of the regulome remained a major challenge to drawing a general conclusion. Also, the studies used cell lines from single individuals and hence could not address interindividual differences, and results could have been influenced by biases introduced by cell culture. However, due to its extensive number of performed experiments and width of analyzed features, the resulting annotation of the human genome presented a highly valuable insight into the actual activity of the genetic sequence. Importantly, integrated analysis of the wealth of data sets produced by ENCODE enabled the segmentation of the genome into distinct regulatory elements [9–11]. Specifically, active regulatory regions were annotated as promoter, enhancer, or insulator regions, wherein transcriptional activity was determined as a measure for actively transcribed regions that mark putative genic loci or those for which transcription contributes to the regulatory process (enhancers). In addition, transcriptional silent regions or in those carrying actively repressing marks were annotated in repressed or silent segments.

Segmentation algorithm are extremely powerful to summarize the data produced by multidimensional experiments related to gene regulation and enable an estimate of the activity and function of a given region of interest. However, the diversity of regulatory processes and their individual particularities can only be assessed using in-depth analysis and, importantly, large sample cohorts and particular tailored study designs. This chapter focuses on a regulatory mechanism directly affecting the genetic sequence: DNA methylation. DNA methylation involves the covalent modification of DNA, converting cytosine in 5-methyl-cytosine (5-mC). As part of epigenetic regulatory processes, DNA methylation was shown to be stably inherited throughout cell division and to be crucial in developmental and differentiation processes [12]. However, DNA methylation represents the oldest described epigenetic mechanism and its role in gene regulation is still subject of discussion. In general, high levels of 5-mC at proximal promoters are associated to transcriptional silencing; however, outside these regions, its role is highly dependent on the genomic context [13]. For example, increased 5-mC levels within the gene body were related to an elevated transcriptional activity [14].

Similar to the controversial function of DNA methylation, the question of which elements determine the temporal and spatial distribution of 5-mC within the genome remains unresolved. Although the mechanisms covalently modifying the genetic code are clearly defined, with DNA methyltransferases catalyzing the addition of the methyl-group to cytosine, the driving forces guiding the activity of such enzymes is under intense discussion, and putative causalities are summarized in this chapter.

1.2 Intrinsic Properties of DNA

Taking into account that DNA methylation varies among different cell types within the same individual but is relatively stable in the same tissue of different individuals, the genetic code appears to play an inferior role in the variable nature of DNA methylation profiles. However, there are several examples clearly displaying how the genetic sequence is necessary and sufficient for the formation of the DNA methylation landscape [15]. Here, the density of CpG dinucleotides presents the most prominent example of a genetic feature directly associated to the DNA methylation status of the respective regions [16]. In particular, an elevated CpG density was related to an unmethylated (hypomethylated) status of DNA, whereas CpG-poor regions are mainly highly methylated (hypermethylated). Intriguingly, it was the DNA methylation itself that was shaping the distribution of CpG dinucleotides in the genome throughout evolution. Methylated cytosines tend to be mutated by deamination, which results in the formation of uracil and the incorporation of thymine after cell division (without DNA repair). As these transition mutations mainly affect methylated cytosine and almost exclusively occur in a CpG context, this specific dinucleotide was depleted in the genome during evolution. Consequently, the proportion of CpG dinucleotide within the human genome is far less than would be expected if all dinucleotides would underlie equal evolutionary pressures. Moreover, because hypomethylated CpG sites, a frequent feature of promoter regions, were protected from deamination processes, it led to their enrichment in promoters of constitutively transcribed genes and the formation of CpG clusters or CpG islands. However, CpG islands being located outside proximal promoter regions and a specific set of islands being mainly hypermethylated suggest that additional features shape the human DNA methylome [17].

One of these features was discovered as an intrinsic property of the DNA molecule itself, and such parameters as DNA stiffness or flexibility enlarged our understanding of the code encoded by the one-dimensional succession of nucleotides. Sophisticated analysis and computational modeling of DNA molecule properties resulted in the redefinition of the promoter signature and supported a hypothesis of an ancient regulatory mechanism encoded by the intrinsic physical properties of DNA [18]. Computational strategies, linking the DNA sequence to epigenetic mechanisms, consistently support an intrinsic association between the genome and epigenome. Here the cumulative effects of many weak interactions are thought to guide the epigenetic code and to present the blueprint for subsequent formation processes [19]. In this regard, the epigenetic landscapes could be partially predicted computational models and remarkably could be assembled in vitro, suggesting DNA methylation profiles to be partially encoded in the genetic code itself [20].

Interestingly, DNA methylation was described to be capable of altering physical DNA properties and hence modify downstream regulatory processes by altering its flexibility [21]. Specifically, molecular dynamics simulations observed that the high flexibility of the CpG sites is not extrapolated in CpG-dense regions. In contrast, poly-CpG fragments are hardly distinguishable from equally sized dinucleotides in terms of global unwinding and isotropic bending. Importantly, it is the covalent modification of cytosine in CpG-dense regions that dramatically alters the winding properties of DNA, which are particularly important, when DNA is wrapped around nucleosomes, an additional layer in the epigenetic code. Here, DNA methylation changes the winding properties of the DNA by making it stiffer. This was shown to be particularly important in CpG islands, wherein the altered flexibility of the DNA molecule changed the affinity of DNA to assemble into nucleosomes. As nucleosome positioning presents an important feature of transcriptional regulation at transcription start sites, differential DNA methylation can indirectly influence epigenetic regulation through its impact of the physical properties of the genetic sequence.

In conclusion, there is evidence of a direct influence of the DNA sequence on the spatial formation of DNA methylation profiles. These intrinsic properties of the genome on the epigenome present the blueprint for downstream regulatory events, and their understanding will guide our knowledge about cause and consequences in epigenetic regulatory mechanisms. However, physical properties are not sufficient to explain the flexible nature of DNA methylation, which, undoubtedly, involves additional mechanisms. Taking into account that the DNA methylation machinery is embedded in a complex network of regulatory processes, it is likely their interplay that guides the temporal variation of methyl-cytosine distribution in the genome.

1.3 Sequence-Pattern-Dependent DNA Methylation Profiles

Initial experiments aiming to determine the driving forces behind distinct DNA methylation states identified an important impact of the surrounding genomic environment on the methylation state [22]. However, earlier observations that the binding of transcription factors, such as SP1, can actively contribute to establishing an unmethylated state of targeted region suggested an effect of local regulatory activity and underlying DNA-binding factor sequence motifs on DNA methylation levels [23,24].

Years later, to which extent the genetic sequence acts as determent for the DNA methylation state was still unclear, and crucial evidence that intrinsic properties of the DNA act as major factor for the hypo- and hypermethylated state of DNA came from experiments inserting numerous sequences at a unique genomic locus with subsequent DNA methylation profiling efforts [25]. Here promoter sequences correctly recapitulated the original DNA methylation profiles, even being correctly altered in the transition from pluripotent to differentiated cells. Intriguingly, small methylation-determining regions (MDRs) were shown to control the methylation state of larger regions in cis and were both necessary and sufficient for regulating DNA methylation. Critical features, besides CpG density, that define MDR activity consisted of developmental state and transcription factor-binding motifs, suggesting that a concerted function of the regulatory network mediates the correct establishment of DNA methylation profiles. Subsequent studies further underlined the role of DNA-binding factor for DNA methylation levels [26]. High-throughput engineering of DNA sequence and comprehensive data modeling suggested a principal role for transcription factor binding sites and that CpG density alone is a minor determinant of an unmethylated state. Consistently, sequence variations among individuals at regulatory loci can lead to the differential binding of regulatory factor, with direct impact on the DNA methylation profile in the respective regions [27]. However, it is of note that some transcription factors are sensitive for CpG methylation, also pointing to an active regulatory role of the epigenetic mark in gene regulation [28,29].

In line with the definition of MDRs, a specific set of CpG-dense DNA regions was identified to be exclusively hypermethylated in somatic tissue types and proposed as cis-regulatory elements to facilitate methylation at CpG islands containing promoters and to act as the hypermethylation driver sequence [17]. Remarkably, these cis-regulatory motifs were utilized to engineer an in vivo model system that confirmed the disease-driving potential of epimutations (alteration in epigenetic state) in oncogenesis [30]. Specifically, an MDR that mediates hypermethylation was placed in the promoter region of the tumor suppressor p16Ink4a, resulting in epigenetic silencing of the gene. Moreover, the transgenic mice carrying the induced epimutation presented increased incidence of spontaneous cancers. This model perfectly illustrates the impact of genetic sequence on DNA methylation levels and its subsequent mediator function in phenotype formation.

The genetic sequence also provided crucial clues about the widely observed phenomenon of partially methylated domains (PMDs) in cancer samples [31–33]. PMDs span 20–40% of the genome and are thought to contribute to increased regulatory variability within their affected regions [33]. However, the intermediate DNA methylation state was observed by averaging individual CpG levels, and a more detailed analysis at the base-pair resolution level identified an extensive variability along the epigenome of these regions, with DNA methylation levels ranging from 0% to 100% [34]. The heterogeneity of the epigenetic landscape could be traced back to the underlying genetic sequence, wherein sequence features, such as CpG density, presented a major driving feature. Surprisingly, CpG-dense region within PMDs were associated with increased methylation levels, an opposite trend to the previous described hypomethylation in CpG islands. Conclusively, CpG density and sequence motifs present a major factor defining DNA methylation levels. However, as variable epigenetic states were observed for single genetic features, the actual DNA methylation profile highly depends on the underlying context.

1.4 DNA-Binding Factors Shaping the Epigenetic Landscape

The binding of transcription factors, such as SP1, to MDRs was established as driving events toward a hypomethylated state of the DNA and confirmed by mutation experiments of respective consensus-binding motifs [25]. It is noteworthy that the study also showed that transcriptional activity is not necessary to maintain hypomethylated states as promoter regions and fragments not related to genic sequence recapitulated the original methylation state when placed in a transcriptional silence context. The scenario that the actual binding of factors to the DNA, and not the sequence itself, determines the methylation state of the region was supported by the fact that bacterial DNA, with comparable sequence composition, did not cause an hypomethylated state of the respective DNA [25]. This closely links DNA methylation to underlying DNA sequence features and suggests that a substantial fraction of methylation changes occur downstream of gene regulation [35].

Whole-genome DNA methylation maps further underpinned the relationship between DNA factor binding and hypomethylation [36]. Here, base-pair resolution profiling of mouse embryonic stem cell (ESC) and derived neuronal progenitor cells revealed the presence of a particular epigenomic feature, represented by small lowly methylated regions (LMRs), which were mostly located distal to promoter regions. LMRs resembled features of cis-regulatory elements, such as enhancer regions, being enriched in marker histone modifications (H3K4me1 and H3K27ac) and DNA sensitive to DNaseI digestion, a marker for open chromatin formation. Most notably, LMRs were enriched in DNA-binding factors, such as pluripotency-related factors; in specific cases, they outranged the enrichment observed at promoter regions (e.g., Nanog binding). A specific case relates to the enrichment of the chromatin organization factor CTCF (CCCTC-binding factor), whose binding can not only act to regulate transcription activity but also controls in the spatial organization of chromosomes [37]. Taking into account DNA methylation profiles, the prediction of CTCF binding to the genome could be significantly improved compared with the analysis of sequence motifs alone [36]. Moreover, CTCF was used to confirm the direct relationship between factor binding and differential DNA methylation. The study altered CTCF binding site sequences (in a transgenic approach) to reduce CTCF affinities to the DNA. Consistent with prior results, the absence of DNA occupancy of CTCF conferred a gain of CpG methylation within these regions and directly confirming the active impact of CTCF binding on the DNA methylation signature. Most remarkably, CTCF binding could actively promote a hypomethylated state, as hypermethylated reporter constructs lost methylation following CTCF binding. Interestingly, this effect could even be assessed for genetically variable loci (polymorphisms), wherein a polymorphic genetic sequence can alter CTCF binding affinity. Here, genotypes unfavorable for CTCF binding were associated with increased DNA methylation levels at these loci. However, these results are in conflict with those of previous studies reporting a methylation sensitivity of CTCF binding in imprinted regions [38,39]. Thus, CTCF binding is likely to be able to actively participate in demethylation, however, with DNA methylation preventing CTCF binding in specific regions.

The relationship between transcription factor binding and DNA hypomethylation was further underlined by using genetically engineered knockout mice for the DNA-binding factor REST (REST−/−) [36]. In line with previously observed associations, the absence of REST was accompanied by a gain of CpG methylation at loci that were previously hypomethylated and occupied by the transcription factor. Consistently, reintroduction of REST resulted in a de novo hypomethylation of the respective loci. These results support the role of transcription factors in the formation of the DNA methylation landscape and predict that the dynamic change of DNA methylation during differentiation is, at least partially, mediated by the function of tissue-specific transcription factors.

1.5 The Dynamics of Transcription-Factor-Mediated DNA hypomethylation

Further insights into the dynamics of DNA binding and DNA methylation turnover came from ChIP (chromatin immunoprecipitation) experiments enriching for CTCF-bound regions coupled with bisulfite sequencing [40,41]. Herein, the actual molecules occupied by CTCF could be interrogated for their methylation states, and surprisingly, CTCF binding could not be directly related to CpG hypomethylation. As the frequency (ChIP signal intensity) of CTCF binding was significantly associated with the likelihood of a demethylation state and as increased heterogeneity of methylation was detectable in low affinity bound CTCF regions, a dynamic DNA methylation turnover in these regions was hypothesized.

And, indeed, the active involvement of transcription factor binding in DNA hypomethylation mechanisms was elucidated in the ESC and the genome-wide mapping of 5-hydroxy methyl-cytosine (5-hmC). 5-hmC represents a derivate of 5-mC and is considered an intermediate state toward hypomethylation of cytosine. This demethylation cascade is highly active in the ESC as well as neuronal cell types but was also reported, although to a lesser extent, in other somatic tissue types [42,43]. Functionally, the active demethylation is catalyzed by ten-eleven translocation (TET) enzymes, and interestingly, TET1 binding was observed to be enriched at the LMRs in the ESC, suggesting an involvement of the enzyme in the reversible DNA methylation state of these loci [36]. Indeed, profiling the distribution of 5-hmC in the ESC and derived neuronal progenitor cell, determined an enrichment of 5-hmC at the LMRs [41]. Furthermore, comparing both differentiation states revealed differential 5-hmC to be highly frequent at the LMRs. Supporting evidence that transcription factor binding is driving active DNA demethylation came from studies in knockout mice for the DNA-binding factor REST (REST−/−), wherein the absence of the transcription factor was associated with decreased levels of 5-hmC at region previously occupied by the factor [41].

In conclusion, these results suggest an active participation of transcription factors in shaping the DNA methylation landscape during differentiation processes. Moreover, the guided turnover of CpG methylation gives valuable mechanistic insights into the dynamics and flexibility of DNA methylation and further underpins a tight interplay between distinct features in the formations in gene regulatory processes that mediate distinct phenotypes, such as cell type states. Although, the intrinsic properties of the DNA sequence act as important determinant for epigenetic states, nongenetic trans-acting factors contribute to its final shape. Thus, external stimuli (environmental impacts) and genetic variability contribute to interindividual variability of the epigenetic landscapes.

1.6 Translational Potential of Demethylation Dynamics

Importantly, the knowledge of direct dependencies of DNA methylation and transcription factor binding is of direct translational value, since it can be applied to the identification of causal events that disrupt the epigenetic landscape in diseases. This strategy was applied on comprehensive epigenetic data sets, including several whole-genome DNA methylation landscapes in samples from patients with medulloblastoma [44]. Initially, the study confirmed the relationship between transcription factor binding and DNA hypomethylation, by integrating chromatin–immunoprecipitation data for the transcription factor OXT2 (resulting in a genome-wide map of OXT2 occupancy on DNA) and DNA methylation profiles. Following the validation of a direct relationship between both parameters in the investigated context, the study proceeded to use transcription factor binding motifs for the identification of driving forces for medulloblastoma subtype formation. Particularly, specific hypomethylated regions within previous defined subtypes of patient samples were analyzed for enriched binding motifs, and remarkably, transcription factors that putatively shaped the DNA methylome of the distinct subtypes were determined and were suggested as putative drivers of the different cancer phenotypes. Importantly, this strategy enabled the identification of novel candidates that participated in tumorigenesis as potential disease-driving factors, which could be of translational value for diagnostic or therapeutic approaches.

1.7 DNA Methylation Quantitative Trait Loci

In addition to the relationship between DNA methylation and the directly underlying genomic sequence, wherein changes in genotypes induce a dynamic change in methylation state, genetic variance is also capable of influencing DNA methylation levels over distance. In this regard, genotype variability was described to affect neighboring (cis-regulation) or far-reaching (trans-regulation) CpG methylation levels [45,46] and led to the definition of DNA methylation quantitative trait loci (meQTLs). As the majority of meQTL-related epigenetic variability occurs in proximity to their associated genotype [47,48], the local regulatory environment and spatial conformation of the chromatin was hypothesized to be the main contributor to the connection. The multidimensional scenario involved the direct alteration of the epigenetic state by genetic variability, which triggers traceable downstream events, including chromatin conformation differences or altered regulatory or transcriptional activity of gene loci [49]. Consistent with this model, sequence polymorphisms were described to influence the binding of transcription factors [50,51], to alter the local profiles of histone modifications [52], or to be associated with distinct chromatin states [53]. Thus, genetic variability confers variant epigenetic states, defines the regulatory potential of the respective regions, and enables a systematic association between genotypes and epitypes [54,55]. Consequently, genetic variability is altering the local regulatory environment and can be detected in downstream events. Besides the triggered variance in DNA methylation (meQTLs), genotypes were shown to directly determine the transcriptional activity of target genes and were defined as expression quantitative trait loci (eQTLs) [56]. Surprisingly, meQTL and eQTL rarely overlap, which suggests the independent nature of both features [57]. Modeling of the relationship between genotypes and epigenetic and gene expression states have consistently revealed a complex relationship among the three features and has questioned an exclusive cause–mediator–consequence relationship [58]. The absence of a direct association between genetically driven variances could also be explained by temporal differences of the events, wherein the expression levels display the status quo of the cell state, and DNA methylation provides information about their