Keywords

1 Introduction

The realization that DNA sequence is not the sole determinant of clinical phenotype was a result of the observation that identical twins, who carry the same DNA, are often disease discordant, whereby one of the twins is sick and the other is healthy (Fraga et al. 2005; Javierre et al. 2010; Costello et al. 2000). The term “epigenetic” was first proposed by Waddington (1939), who introduced the term “epigenetic landscape” to describe the molecular and biologic mechanisms that transform a genetic trait into a visualized phenotype. This encompasses the idea of the genotype and the phenotype (Esteller 2008; Rideout et al. 2001). Modulation of gene expression is a way that the DNA sequence can be regulated leading to stimulation or suppression of pathways or molecules that may lead to health or disease.

Currently, DNA methylation is a well-characterized and intensely studied epigenetic modification tracing back to the research done by Mahler and Griffith in 1969, who showed that DNA methylation may play an important role in the function of long-term memory (Bird 2002). Besides DNA methylation, other epigenetic modifications include histone modifications, miRNA and nucleosome accessibility (Fig. 1.1). The continuous interest in epigenetics has resulted in discoveries of a role for epigenetics in diseases ranging from autoimmune diseases to cancer, congenital disease, mental retardation, endocrine diseases, pediatric diseases, neuropsychiatric disorders, and many others (Fraga et al. 2005; Javierre et al. 2010).

Fig. 1.1
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Primary mechanisms of epigenetic modifications include DNA methylation, histone modifications, miRNA, and nucleosome accessibility

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2 The Science Behind Epigenetics

The central dogma in genetics is the doctrine that information in our cells flows only in one direction—from DNA to RNA to proteins (Portela and Esteller 2010). It was an absolute dogma that has now been essentially debunked, due to the role of the environment in modulating the expression of genes. A new term, “epigenetic”, literally meaning outside of or above the gene, has become one of the hottest and newest emerging fields in the scientific world. Epigenetics does not involve the biochemical alteration in DNA sequences, rather, it turns on or off different genes that can make us susceptible to developing disease.

Epigenetic research aims to unearth how environment, social condition, psychosocial factors, and nutrition affect an individual’s expression of genetic information (Fig. 1.2). In multicellular organisms, variable phenotypes may result from the same genotype because of the potential ability of epigenetic markers appearing during development to be passed on to offspring (Kaminsky et al. 2009). Researchers have already found that the phenomenon of division and differentiation of single cell during embryogenesis is tightly associated with epigenetics (Meissner et al. 2008). The result is that monozygotic twins have the same genetic information, but may have a different epigenetic profile, determined by the environment in which they live and grow, leading to differences in health and disease phenotype (Kaminsky et al. 2009). Theoretically, a cloned animal, with genetic material from the same donor, can potentially develop a different disease from the donor (Costello et al. 2000). Epigenetics can be part of the answer to variable phenotypes and plays a crucial role in cell division and differentiation.

Fig. 1.2
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The interaction among epigenetics, genetics, and the environment

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3 Epigenetics and Human Disease

The three main mechanisms of epigenetics are DNA methylation, histone modification, and miRNA. These mechanisms are responsible for the initiation and maintenance of epigenetic silencing and regulation of the gene expression profile and are cornerstones of a series of cellular processes, including cell differentiation, gene expression, X chromosome inactivation, embryogenesis, and genomic imprinting (Holliday and Pugh 1975; Riggs 1975). Unveiling the relationship among these components has rapidly and surprisingly resulted an improved understanding of regulation of gene expression. Furthermore, disruption of an epigenetic profile can have a significant impact on cellular function, which can lead to dysregulation of gene expression and can potentially lead to the occurrence and development of “epigenetic disease”.

An aberrancy in DNA methylation is a common manifestation of epigenetic disease. Methylation occurs at the 5′-position of a cytosine residue, which is regarded as a fundamental gene silencing mark (Holliday and Pugh 1975). This cytosine residue can be methylated and maintained by numerous DNA methyltransferases (DNMTs), which play an important role in the silencing of transcription factors, as well as defense against expression of endogenous retrovirus genes and repression of transposable elements (Roulois et al. 2015). The addition of a methyl group to the untouched C-5 position of a cytosine by DNMTs during DNA replication contributes to the occurrence of de novo DNA methylation (Jones and Baylin 2002). The methylation occurring in 5′-CG-3′(CpG) can easily be deaminated spontaneously to the thymine, while the unmethylated CpGs can be converted to uracil. The expected number of CG pairs in the human genome is about 20% (Jones 2012). The observed number is often lower than the expected number, due to a high mutation rate for methylated CpG sites.

Some promoter regions enriched with CG, named CpG islands, are at least 200 bp and are greater than 55% conserved throughout evolution. Maintaining the primary epigenetic status is fundamental to maintaining normal development. Disruption of this balance can lead to an aberrant epigenetic landscape on the basis of time and place. The DNA located at some promoter regions, when methylated, may cause heritable transcriptional silencing. The hypermethylation occurring at some important genes, such as p16INK4A, CDH1, DAPK, p14ARF, can contribute to the tumorigenesis (Esteller 2007).

Histone modification is another key mechanism of epigenetics (Fig. 1.3). Histone complexes are composed of two unstable dimers H2A, H2B and a tetramer of H3 and H4, wrapped by 147 bp of DNA to form the nucleosome (Schotta et al. 2004). The histone complex facilitates the condensation of genomic DNA and has an impact on post-transcriptional modification. Several modifications, such as acetylation, methylation, ubiquitination, phosphorylation, and sumoylation, occur on the conserved lysine at the histone tails (Nakayama et al. 2001; Yuen and Knoepfler 2013).

Fig. 1.3
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Histone modifications include acetylation, methylation, ubiquitination, phosphorylation, and sumoylation

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Histone acetylation and deacetylation are essential for gene regulation. Acetylation generally leads to active transcription, whereas hypoacetylation is an indicator of inactive transcriptionally. Histone methylation can indicate both active and inactive transcription, and the state of mono-, di-, and trimethylation has different effects. Methylation is facilitated by the enzymes known as histone methyltransferases (HMT).

Histone modification to H3 is the most well studied and characterized. The di- and trimeric forms of H3K4 and H3K36 are frequent targets of histone modification and lead to activation of transcription. In contrast, H3K92/3 and H3K27me2/3 modification lead to gene silencing. It should be noted that the histone component H3K9 is found primarily in a gene-poor region, such as telomeres and centromeres, and is a permanent marker for the formation of heterochromatin. This histone component is also associated with X chromosome inactivation and gene repression at promoter regions (Nakayama et al. 2001). Conversely, the H3K27 is generally found in gene-rich regions and acts as a temporary marker correlating with the development of regulators (Santenard et al. 2010). Studies show that histone H3 mutations are associated with giant bone cell tumor and chondroblastoma and have also been found to be a mutation of high frequency in high-grade gliomas in children (Schwartzentruber et al. 2012). Any mutation of histone-associated enzymes may contribute to the development of diseases, such as cancers, autoimmune diseases, endocrine diseases, and psychologic disorders.

Mature miRNA is another key player in epigenetics. miRNA is a class of non-coding small RNA, about 22 nucleotides in length. MicroRNA are complementary to single or a series of messenger RNA (mRNA). It cannot be translated into protein, rather their main function is to downregulate gene expression in different ways, including mRNA cleavage, translational inactivation, and deadenylation to produce a mitotically heritable result (Tufarelli et al. 2003). Emerging evidence indicates that miRNAs play significant roles in cell division, differentiation, and development. Abnormalities in miRNA are associated with a wide variety of human diseases, including cancer, autoimmune diseases, and cardiac diseases (Calin et al. 2002). Consequently, miRNAs are becoming extremely useful as clinical biomarkers, and diagnostic tools have been developed especially in the field of cancer. In addition, miRNA play a crucial role in many other biological systems. An example would be in cardiology. Through regulation of gene expression, miRNAs play a significant role in regulating cardiac function or dysfunction, including cardiac rhythm, ventricular wall integrity, contractility, and myocyte growth.

4 The Clinical Application of Epigenetics

One of the personal human challenges in health and disease has to do with uncertainty. In the vast majority of cases, there is no single test that will definitely provide an answer for patients. Patients often need multiple studies, which take a significant amount of time, and this adds to the anxiety of seeing a physician. The promise of epigenetic is that it can provide new insights into clinical development of diagnostic and therapeutic methods and bridge the gap between effects of the environment and host genetics. Epigenetics has the potential to be used as biomarkers for the detection and diagnosis of disease, disease monitoring, and response to treatment. In the past few decades, pharmacoepigenetics has attracted much interest and epigenetic drug (epidrug) development has achieved significant advancement.

4.1 Epigenetic Biomarkers

The discovery and utilization of biomarkers have the potential to impact patient management and clinical outcomes (Garcia-Gimenez et al. 2017). Biomarkers may be directly related to pathogenesis or may be surrogate markers or important for disease prognostication or monitoring. Some biomarkers may also be potential therapeutic targets or may indicate where the search for such targets should commence (Costa-Pinheiro et al. 2015; Dirks et al. 2016). The identification of potential markers is only the first step, as these markers must be validated and confirmed as a reliable and statistically acceptable reflection of the disease. Epigenetic markers have already been incorporated into clinical application and are being used in the prevention, diagnosis, and treatment of cancers, autoimmune diseases, as well as neurological and cardiac disorders.

There are several advantages of epigenetic biomarkers. First, the biomarkers indicate a new direction in which molecular markers correlate with genetic and the environmental factors which contribute to the development of diseases (Lorincz 2011). What epigenetics does is to provide a functional biomarker which does not depend on DNA sequence alone. The epigenetic biomarker, especially those related to DNA methylation, falls outside of the DNA and RNA sequence based testing and may provide an alternate stability profile (Garcia-Gimenez et al. 2017). Epigenetic biomarkers can be checked in blood, tissue, body fluid as well as secretions which are commonly sampled during procedures and surgeries. Furthermore, any disruption of epigenetics can be checked in the context of the genome and even prior to or at the very early stage of the disease. This property is unique compared to the RNA and protein-based tests because RNA and protein abnormalities appear at relatively late stages and often in low quantities or concentration.

4.2 Epigenetic Therapy

Epigenetic therapy is a new treatment option utilizing epigenetic drugs (epidrug) or less obviously, non-pharmacological techniques of clinical management (Fig. 1.4). Recent research in epigenetics now offers an attractive way to target the epigenetic mechanism caused by cancers, autoimmune diseases, cardiac disorders, and mental illness.

Fig. 1.4
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Epidrugs for the treatment of human diseases. Several epidrugs have been approved for clinical application, and many epidrugs are undergoing clinical trials

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Large numbers of molecular inhibitors have been developed over the past several decades. The United States Federal Drug Administration (FDA) approved the first epidrugs azacytidine (5-AZA) and decitabine (5-AZA-CdR) in 2004 for the treatment of leukemia (Egger et al. 2004). These drugs are in fact DNA methyltransferase inhibitors, thereby categorized as epigenetic modifiers, which can reprogram the epigenetic profile and potentially reverse the disease. They are now indicated in the treatment of hematologic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia (CMML). Reprogramming and reshaping the epigenetic profile by reducing DNA methylation levels, especially on tumor suppressor genes, may lead to normalization of the gene expression profile (Table 1.1).

Table 1.1 A variety of epidrugs are already approved for clinical use or are undergoing clinical trials
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Histone acetylase inhibitors are another compelling class of epidrugs (Nagaraja et al. 2017). Panobinostat has been approved for the treatment of multiple myeloma (MM), Belinostat has been used in the treatment of refractory or relapsed peripheral T cell lymphoma (PTCL), and several clinical trials using sodium phenylbutyrate for Huntington disease and Vorinostat for HIV-1 are undergoing (Vojinovic et al. 2011). Other oncology research is focusing on the combination treatment of HDAC and DNMT inhibitors for AML, glioma, breast cancer, and CMML, thereby demonstrating a synergistic effect (Brocks et al. 2017) between two forms of epigenetic mechanisms.

Drug resistance remains a problem (Grasso et al. 2015). EZH2, one of the components of polycom-repressive complex 2 (PRC2), is a histone K27 methyltransferase. Recent studies on the EZH2 inhibitor have shown an arrest of cell growth by its ability to remove residual PRC2 (Mohammad et al. 2017). In a recent trial, an EZH2 inhibitor, tazemetostat, has been tested to treat refractory B cell lymphoma (NHL) (Morera et al. 2016). Another team is using MAK683, an inhibitor of embryonic ectoderm development protein (EED), to treat nasopharyngeal carcinoma and diffuse large B cell lymphoma (DLBCL) (Chiappella et al. 2017).

Epidrugs have showed promising effects in both clinical practice and preclinical and clinical trials. Scientists are not only focusing on the enzymes that generate or create epigenetic markers (readers), but also on the enzymes that wipe out epigenetic markers (erasers) and proteins that edit epigenetic markers (writers).

5 Epigenetic in Diseases

5.1 The Role of Epigenetics in Cancer

Epigenetic aberration is a common feature of cancer, characterized by hypermethylation at specific promoter regions and global DNA hypomethylation, and/or either loss or gain of acetylation or methylation of histone proteins (Liang and Weisenberger 2017). Disruption of chromatin is crucial for nucleosome positioning, DNA wrapping, accessibility of chromatin to transcription factors, and regulation of gene expression. Silencing of tumor suppressor genes and activation of oncogenes are the hallmarks of epigenetic aberrancy (Fig. 1.5). The dysregulation of the epigenetic profile plays a key role in carcinogenesis. Likewise, the dynamic and reversible character of epigenetic modulation is an attractive feature for novel clinical treatment modalities.

Fig. 1.5
figure 5

Epigenetic mechanism in cancers which are induced by both DNA methylation and histone modifications can be reversed by inhibitors of epigenetic modifiers

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5.1.1 AML

AML is a malignant tumor that arises from abnormal hematopoietic stem cells, characterized by massive proliferation of neoplastic precursor tumor cells, which causes hematopoietic aberrancies and alters bone marrow homeostasis. Current clinical protocols have limited options, involving intensive chemotherapy (Lavallee et al. 2016), and/or using poorly sourced stem cell transplantation (Powles et al. 1980). Despite promising results over the last few decades, AML is still a devastating disease in over half of young patients and almost 80% of elderly patients due to relapse and drug resistance, leading to increased morbidity and mortality (Adelman et al. 2019; Burnett et al. 2011). Aberration of the epigenetic landscape contributes to the development and the regulation of AML. Recurrent somatic mutations occur in specific genes that play a crucial role in epigenetic modulation, but epigenetic dysregulation is a more likely mechanism compared to the recurrent mutations alone. Consequently, there is an urgent need to unearth the epigenetic mechanisms and pathogenesis of AML in order to achieve better patient management.

DNA Methylation

Aberrant DNA methylation is a hallmark of cancer, with global hypomethylation at repetitive elements, and hypermethylation occurring in the promoter regions that are enriched with CpG islands. Emerging evidence shows that for leukemogenesis, the disruption of DNA methylation occurring outside of CpG island is equally important as those within CpG island regions, and that hypomethylation and hypermethylation contribute equally to oncogenesis. Recent studies show that myeloid malignancies are accompanied by recurrent mutations occurring at epigenetic modifiers including Tet2 and DNMT3A, which are associated with hypomethylation and hypermethylation. Unearthing the interaction of these DNA methylation modifiers can help to elucidate the mechanism of AML.

DNMT3A is a DNA methylation enzyme that can produce de novo methylation at CpG loci, and it is a common target of somatic mutations. These mutations occur in almost 40% of cytogenetic AML patients and about 20% of T-AML patients (Shlush et al. 2014). DNMT3A is viewed as a marker of early stage leukemia and may be beneficial in monitoring early events in AML. Increasing evidence has shown that DNMT3A mutations which occur in AML may appear in the T lymphocytes from the same individual.

Recent research has also demonstrated that elderly people can carry the DNMT3A mutation without evidence of hematologic malignancy, showing that these mutations may be involved in clonal hematopoiesis and can contribute to leukemogenesis (Bond et al. 2019). The mutation occurring in DNMT3A leads to an arginine substitution, which results in a reduction in enzyme activity (Russler-Germain et al. 2014). DNMT3A mutated mice showed dynamic DNA methylation patterns occurring in both regions of hypomethylation and hypermethylation. Normal DNMT3A is critical for HSC self-renewal and cell differentiation in adult wild-type recipient mice (Liu et al. 2005). DNMT1 is a key player in the fate of leukemia cells, and a deficiency of DNMT1 in a mouse model was associated with reduced DNA methylation with reduction of the capacity of tumor suppressor genes to reactivate (Mizuno et al. 2001).

The ten-eleven translocation (TET) family is another important target of DNA methylation through the transformation of 5-methyl-cytosine (5-mc) to 5-hydroxymethylcytosine (5-hmc). About 30% of AML patients have mutations in TET2, which leads to reduced 5-hmc levels (Cimmino et al. 2017). The association between TET mutations and poor prognosis of AML patients has been reported. Increased capacity of self-renewal, over-proliferation of hematopoietic stem and progenitor cells (HSPC), and increased cell differentiation have been observed in TET2 deleted mice. In about 30% of AML patients, mutations frequently occur in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2). Mutated TET and mutated IDH are mutually exclusive in adult AML. IDH can convert isocitrate into α-ketoglutarate naturally, but 2-hydroxyglutarate (2-HG) results from mutated IDH. This metabolite can compete with α-ketoglutarate and function as an inhibitor of TET2. Furthermore, the global hypermethylation signature in AML is associated with mutated IDH1/IDH2, and the overlapping hypermethylation signature has been observed in patients with mutated TET2. Murine models show that the level of 2-HG is increased in mutated IDH and the population of HSPC is expanded. Mouse findings were also consistent with those in AML patients (Cimmino et al. 2017).

Histone Modification

Histone acetyltransferases (HAT) and histone deacetylases (HDACs) are the primary mediators of histone acetylation and deacetylation, respectively. HAT is involved in sporadic translocation in AML patients (Izutsu et al. 2001). The myeloid oncoproteins, including PML-RARA and EVI-1, can interplay with either major complexes or scaffold proteins which aberrantly recruit HDACs. This results in abnormal chromatin condensation and remodeling, with the sites near transcription factors being more affected. HMTs are involved in translocation in AML, and HMT mutations occur in components of PRC2 and mixed-lineage leukemia (MLL) proteins (Basheer et al. 2019). The most common MLL protein, KMT2A, can modify H3K4 and transcriptionally activate targeted genes. MLL-induced translocations occur in about 10% of AML patients.

Abnormal patterns of H3K79 methylation have been observed in MLL-transformed AML patients, and several related genes are also overexpressed. EZH2 can stimulate the di- and trimethylation of H3K27 and is generally considered to be a repressive modifier in numerous types of malignancies. Intriguingly, EZH2 mutations result in functional silencing in ALL, but the mechanism remains unclear. Other components, including SUZ12 and EED, play an important role in the leukemogenesis. Mutations of these components can lead to loss of function in ALL (Sinha et al. 2015).

miRNA

Aberrant activity of certain miRNAs can disrupt hematopoiesis and trigger leukemogenesis (Liu et al. 2019). miRNA can perform as either tumor suppressor or oncogenic agents, and while many new miRNAs are being found to have epigenetic activity each year, there are many that probably have not even been identified.

The most studied miRNA in AML is Let-7, which is known for its tumor-suppressive property in various types of cancers. It functions by targeting a number of different oncogenes, including KRAS, HMGA2, MYC, and IMP1, and plays a role in adult AML. Let-7b is also downregulated in pediatric AML patients, and it is associated with dysregulation of the oncogene c-Myc, indicating that both solid tumors and hematologic neoplasia may have abnormal expression of Let-7 (Pelosi et al. 2013). Another well-known miRNA is miR-29, which is downregulated in MLL-mediated AML. The main function of miR-29 is induction of apoptosis and regulation of the cell cycle. Studies in mice support that miR-29 can induce apoptosis and inhibit tumor cell growth (Garzon et al. 2009).

Oncogenic miRNAs such as miR-126 play a significant role in cancer. Overexpression of miR-126 in stable leukemia cell lines demonstrates inhibition of apoptosis and improved cell survival. AML patients also may show high levels of miR-24, and overexpression of miR-24 blocks the synthesis of mitogen-activated protein kinase phosphatase 7, promoting cell growth in AML (Organista-Nava et al. 2015).

5.1.2 Lung Cancer

Lung cancer is the leading cause of tumor-related death worldwide and is responsible for nearly 30% of cancer-related deaths, which is significantly more than the other top five cancers, including colorectal carcinoma, breast and prostate cancer. Despite medical advances, lung cancer continues to have a high mortality and low survival rate. Environmental tobacco smoke continues to be an important risk factor. Lung cancer can be clustered into two groups, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The latter group can be further categorized into two types, squamous cell lung cancer and adenocarcinoma. The abnormal initiation and progression of lung cancer may be caused by the interplay of genetic disruption and dynamic epigenetic aberrancies. Epigenetics plays a key role and is an attractive target for the study of lung cancer and the development of new treatments.

DNA Methylation

Studies of the abnormal epigenetic profile seen in lung cancers can improve our understanding of lung tumorigenesis. Aberrations in DNA methylation is a key factor that contributes to the initiation and development of lung cancer by silencing tumor suppressor genes. DNA hypermethylation at promoter genes is considered an early event, and about 3% of functional genes carrying CpG-rich regions are deactivated in advanced stages of lung cancer (Teixeira et al. 2019). Three DNMTs are widely studied, all of which are overexpressed in lung cancer. The expression of DNMT1 is increased at the early stage of lung cancer, and it can silence the expression of P16INK4A and RASSF1A. DNMT3B is also a key participant in the pathogenesis of lung cancer and is associated with poor prognosis. The interaction of these DNMTs establishes abnormal DNA methylation patterns and represses the expression of tumor suppressor genes. Numerous tumor suppressor genes are affected and silenced, including MGMT, CDH13, DAPK, and APC genes. The inactivation of RASSF1A occurs in about 40% of squamous cell carcinomas and almost 80% of small cell lung cancers (Giri and Aittokallio 2019).

Histone Modification

Global H3 methylation and H2A acetylation have been observed in both SCLC and NSCLC. Gene activation caused by the H3K9 methyltransferase SETDB1 has been observed in lung carcinogenesis and increases invasiveness of cancer cells and cell proliferation by regulation of the WNT pathway. ERK1/2 activation, induced by upregulation of the H3K36 modifier KDM2A, promotes invasiveness and cell growth and is associated with a poor prognosis (Gardner et al. 2017). There are other metastasis-associated genes activated through upregulation of H2F3A, increasing tumor invasiveness resulting in poor outcomes in early stages of lung cancer. Likewise, upregulation of H4K8 acetylation and hypoacetylation of H3K12/H4K16 have been reported to occur in squamous cell carcinoma (Roper and Sheng 2019).

miRNA

Downregulation of dicer has been observed in NSCLC and correlates with poor prognosis. A mouse lung cancer model showed that dicer deletion led to tumor development and lower survivor rates (Szczyrek et al. 2019). miR199, miRNA101, and miRNA 126 are dysregulated in rat models in the early stage of lung cancer and are downregulated in human NSCLC patients as well. miRNA-let plays a crucial role in cell apoptosis and the cell cycle; downregulation of let-7 promotes cell division in lung cell lines and upregulation inhibits cell growth. Xenograft mouse model studies show similar results.

A study of smokers who suffered from NSCLC found that polymorphisms in let-7 correlate with increased death risk (Del Vescovo and Denti 2015). miR-17-92 regulates cell apoptosis. Deactivation of let-7 and overexpression of MYC are common findings in lung cancer, leading to the upregulation of miR-17-92, overexpression of E2F protein, and increased lung carcinogenesis. Furthermore, miR-17-92 increases genomic instability and RB repression in SCLC cell lines. RB repression results in DNA damage by inducing the expression of y-H2AX (Zhang et al. 2018).

5.1.3 Pediatric High-Grade Glioma

In the past few decades, medical advances have greatly improved the survival rates of a number of pediatric cancers. This is not true for pediatric high-grade glioma (pHGG). Neurogenic tumors remain the leading cause of pediatric cancer, and pHGG is the leading cause of neurogenic tumors. It primarily affects the pons, brain stem, and cerebellum, with a survival rate lower than 1% (Donaldson et al. 2006). About 80% of pHGG is associated with histone H3 mutations, where K27 lysine is substituted by methionine (H3K27M) (Schwartzentruber et al. 2012). Treatment of pHGG is limited to surgery and radiation, with effective chemotherapy currently unavailable. Research targeting epigenetics provides a potential novel alternative to current treatment modalities.

DNA Methylation

DNA methylation patterns of pHGG show hypermethylation at CpG-rich promoter regions, leading to deactivation of tumor suppressor genes. H3K27M mutations commonly occur in pHGG affecting the midline structures and are associated with few O6-methylguanine-DNA methyltransferase (MGMT) methylation changes, dismal results, and poor prognosis (Wu et al. 2012; Taylor et al. 2014). Similarly, H3G34R/V mutations are associated with a poor prognosis, whereas IDH1 mutations have no significant impact on survival rates compared to the wild type. H3G34R/V mutations are commonly associated with hemispheric pHGG with MGMT methylation enrichment (Fang et al. 2018). The methylation profile of three proliferative oncogenes, pedGBM-RTK1, pedGBM-RTK2, and pedGBM-MYCN, are associated with poor prognosis. DNA methylation profiles present important information that can be used to determine treatment and prognosis.

Histone Modification

An H3 mutation is identified as an oncohistone. Nearly 80% pHGGs harbor the H3.3K27M mutation, which has already been introduced (Schwartzentruber et al. 2012). H3K27I is another mutation that occurs in H3.3, where a lysine is substituted by an isoleucine. Furthermore, H3G34R/V, another variant of pHGG, involves substitution of glycine 34 to either arginine or valine. Mutations of H3.1 and H3.2, including HIST1H3B and HIST1H3C, are common events leading to histone variations named H3.1K27M and H3.2K27M, respectively. Mutations of H3.3 K27M and G34R/V occur exclusively in pHGG and show unique DNA methylation patterns and gene expression profiles.

Interestingly, these two types of mutations in H3.3 often occur simultaneously with mutations in other genes. For example, nearly 30% of the K27M mutations are associated with an ATRX mutation and approximately 60% of mutations occur in conjunction with a TP53 mutation. Likewise, G34R/V mutations have ATRX and TP53 mutation as well, but the PDGFRA mutation has been found in the same individuals (Yuen and Knoepfler 2013). Together, this evidence suggests that mutations in H3 are an important risk factor and contribute to the pathogenesis of pHGG. Scientists have found that HDAC inhibitors have a promising effect on the treatment of pHGG (Grasso et al. 2015; Pang et al. 2009). According to this study, a combination of HDAC inhibitors, BRD4 and CDK7 blockers are a better strategy to overcome drug resistance (Nagaraja et al. 2017). EZh2 inhibitor, another inhibitor of histone modification, can remove PRC2 and inhibit cell growth and may be a promising therapeutic method for the treatment of pHGG (Mohammad et al. 2017).

miRNA

Studies of miRNA in pHGG are lacking compared to adult gliomas. The expression of miR-34 and miR-21 are upregulated, while miR-129 and miR-124 are downregulated in pHGG. Several miRNAs are involved in the regulation of gene expression through the MAPK pathway, involving regulation of BRAF-KIAA1549 (Pang et al. 2009). A xenograft mouse model of miR-487 showed inhibition of colony formation and downregulation of nestin and PROM1 genes. Other miRNA, including miR-204, miR-1296, miR-1224, miR-10a, and miR-34c, are downregulated, and miR-527, miR-769-3, and miR-200A are upregulated in pHGG (Riddick and Fine 2011).

5.2 The Role of Epigenetics in Immune Diseases

The etiology of autoimmune diseases remains unclear. Although genome-wide association studies (GWAS) are now widely available, they have failed to reveal a clear genetic pathophysiology of autoimmune diseases. Scientists now appreciate that the epigenome plays a critical role to initializing and stimulating autoimmune diseases (Fig. 1.6). Disruption of DNA methylation and histone modification leads to abnormal epigenetic profiles. Epigenetic aberration in autoimmune disease results in breaking self-tolerance, thus triggering autoimmunity. The epigenetics of autoimmune diseases is discussed in Chaps. 7–13 of this book.

Fig. 1.6
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Epigenetic modifications dysregulate gene expression on different types of immune cells and trigger autoimmune diseases

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5.2.1 Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by overactivation of the immune system and production of excessive autoantibodies. These antibodies may attack self-tissues or organs and are a potential cause of SLE. However, even with the medical advances of the past decades, the mechanism of SLE remains unclear. Epigenetics may provide new insights to elucidating some of the factors leading to the breakdown of tolerance.

DNA Methylation

DNA methylation is a key player in the pathogenesis of SLE. Altered DNA methylation in SLE is characterized by global hypomethylation on CD4+ T cells involving the extracellular signal-regulated kinase (ERK) signaling pathway. In murine models, studies have shown dysregulation of this pathway leading to downregulation of DNMT1 and overexpression of methylation-associated autoimmune genes. Hypomethylation in CD4+ T cells results in upregulation of relevant genes. Studies have found that CD11a (ITGAL), CD40LG, CD70 (TNFSF7), and perforin are all upregulated in lupus patients and are positively correlated with disease activity. It has been found that the promoter region of these genes is significantly hypomethylated in lupus T cells, compared to controls and patients with inactive lupus (Lu et al. 2002, 2007; Oelke et al. 2004). Similarly, lupus models were successfully induced after CD4+ T cell from healthy individuals were stimulated with phytohemagglutinin (PHA), followed by treatment with any of the DNA methylation inhibitors, including 5-aza, procainamide, hydralazine (Deng et al. 2003). The promoter regions of these genes are hypomethylated and the corresponding genes are greatly upregulated. Mice injected with either procainamide- or hydralazine-treated CD4+ T cells develop a lupus-like disease, which shows loss of methylation at promoter regions of some targeted genes and produces autoreactive antibodies.

Histone Modification

Abnormal histone modification is a key contributor to the pathogenesis of SLE. Active SLE patients show downregulation of global H3 and H4 acetylation in CD4+ T cells, with the acetylation level being negatively associated with disease activity (Coit et al. 2013). SLE murine models demonstrate that spleen cells have a low level of acetylation in H3. The use of HDAC inhibitors, such as suberoylanilide hydroxamic acid or trichostatin A, successfully treated splenomegaly and glomerulonephritis in SLE. In vitro, the acetylation level of histones H3 and H4 greatly improved after the use of HDAC inhibitors, with a decrease in the level of a number of cytokines, including IFN-γ, IL-6, IL-10, and IL-12. HAT mutations in a mouse model led to a propensity to develop lupus-like diseases, with positive anti-dsDNA autoantibodies, glomerulonephritis, and low survivor rates (Mishra et al. 2003).

miRNA

miRNAs function by regulating the transcription of mRNAs. Peripheral blood mononuclear cells (PBMCs) in SLE patients show an abnormal expression of miRNA. Disruption of miRNAs involving the ISG and TLR pathway is considered a key risk and contributes to the pathogenesis of SLE (Yan et al. 2014). Previous work demonstrated that miR-146a, which negatively regulates the IFN pathway, contributes to disease pathogenesis. Recent studies have demonstrated that an abnormal expression of LYN induced by miR-30a in B cells has an important role in SLE. In MRL/lpr murine models, DNMT1 was decreased by upregulation of miR-148a and miR-21, leading to a DNA hypomethylation pattern. Overexpression of miR-17-92 has been shown to lead to lupus in animal models. Mice knockdown (Wang et al. 2018) studies demonstrated a slow progression of lupus and better prognosis. miRNAs can also be used as biomarkers in clinical application, are associated with SLE disease activity index (SLEDAI), and are a good predictor of disease activity (Khoshmirsafa et al. 2019).

5.2.2 Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic relapsing autoimmune disease, characterized by aberrant targeting of joint linings by the immune system. RA leads to bilateral joint erosion, including knees, hands, or shoulders. The symmetric characteristic is useful to distinguish RA from other forms of arthritis. In RA, disruption of a normal epigenetic profile by DNA methylation, histone modifications, and miRNA is commonly detected in stromal and immune cells. Epigenome aberration affects multiple inflammatory and matrix-associated pathways and contributes to the pathogenesis of RA.

DNA Methylation

RA has a similar DNA methylation pattern as SLE, showing global hypomethylation of T cells and monocytes. Demethylation occurs in the promoter region of CD40L on silenced X chromosomes, leading to overexpression of CD40L, which contributes to the development of RA (Lu et al. 2007). CTLA-4 is hypermethylated at specific sites in Treg cells in RA patients, leading to a decrease in the expression of CTLA-4. Subsequent studies found that methotrexate decreases FoxP3 DNA methylation levels in Treg cells. Upregulation of CTLA-4 results from Foxp3 reactivation, which leads to normalization of Treg cell function. Aberrant methylation patterns also occur in PBMCs. Demethylation of IL-10-related genes greatly increases expression of IL-10 in PBMCs (Khoshmirsafa et al. 2019), whereas abnormal methylation of IL-6-related genes in PBMCs results in downregulation of IL-6. Loss of global methylation also occurs in synovial fibroblasts, CXCL12 and TBX5 in RA (Cecchinato et al. 2018).

Histone Modification

Dysregulation of several histone modifiers is a common feature of RA. Conflicting results have been found between expression of HATs and HDACs in synovial fibrocytes and activity of RA patients, probably caused by different criteria of recruited RA patients. Recent studies discovered that the expression of HDAC1 and HDAC2 is associated with and regulates TNF in synovial fibrocytes (Angiolilli et al. 2017). However, the expression of HDAC5 is negatively associated with IL-6 expression as well as disease activity. Recent work shows that SIRT1 is upregulated in RA synovial fibrocytes, leading to overexpression of IL-6 and IL-8 and reduction of apoptosis. Scientists studied the histone methylation status at the promoter regions of matrix metalloproteinases (MMP) in RA synovial fibroblasts and found that the active marker H3K4me3 was upregulated and the inactive marker H3K27me3 was downregulated in the promoter regions of MMP-1 and MMP-9. An arthritis mouse model using the HDAC inhibitor Givinostat demonstrated a reduction in cytokine levels and improvement in the prognosis of RA (Angiolilli et al. 2018).

miRNA

Early studies of miRNAs in RA found that expression of miR-155 and miR-146a is upregulated in RA synovial fibroblasts. Subsequently, larger studies showed that miRNA-155 is overexpressed in various tissue and immune cells, such as CD14+ cells derived from synovial fluid, B cells, macrophages, and PBMCs. Likewise, miRNA-146a is upregulated in PBMCs and CD4+ T cells. The association between miR-155 expression in PBMCs and swollen joints indicates that miR-155 promotes the progression of RA by triggering production and recruitment of cytokines. MiR-124a has been observed to be downregulated in RA patients. MiR-223 was found to be overexpressed in CD4+ T cells, synovial fibroblasts, and synovial fluid (Dunaeva et al. 2018) in RA patients.

5.2.3 Multiple Sclerosis

Multiple sclerosis (MS) is a chronic relapsing autoimmune disease involving the nerves of brain and spinal cord, leading to axonal degeneration and demyelination. The mechanism of MS remains unclear. Multiple studies discovered disrupted epigenetic profiles in immune system components which lead to demyelination and recurrent inflammation that contributes to the pathogenesis of MS.

DNA Methylation

Previous studies using a murine model for MS, namely, experimental autoimmune encephalomyelitis (EAE), found that T-bet/H2.0-like homeobox (Hlx), which is regulated by MBD2, plays an important role in the pathogenesis of MS. IFN-γ, expressed Th1 and natural killer (NK) cells, is in turn regulated by T-bet. The Hlx gene coordinates with T-bet to regulate the expression of IFN-γ. MeCP2, a member of the MBD family, is capable of myelin repair and remyelination when combined with brain-derived neurotrophic factor (BDNF) (KhorshidAhmad et al. 2016). Expression of Dnmt1, TET2, and 5hmC is greatly depressed in MS, and abnormal DNA methylation profiles at the promoter region of specific genes has been observed in PBMCs from patients with MS. Studies have also shown that the DNA methylation levels of HLADRB1 in CD4+ T cells correlate with the risk of developing MS (Tang et al. 2019).

Histone Modification

Disruption of histone modification is a key factor in the initiation and progression of MS. Studies have shown that histone methylation is critical for the development of the M2-macrophage phenotype. The upregulation of H3K4me3 and downregulation of H3K27me2/3 have been observed at the promoter region of M2 marker genes. Overexpression of Jmjd3 removes H3K27me2/3 and affects the M2 phenotype from macrophages. HDACs are associated with differentiation of CD4+ T cells and secretion of cytokines by regulating histone patterns (Sun et al. 2018a). Furthermore, HDAC inhibitors suppress the immune system and inhibit the inflammatory process.

HDAC inhibitors play a role in inhibition of cell growth of CD4+ T cells and blocking of the production of IFN-y. HDAC inhibitors decrease T helper (Th) cell associated pro-demyelinating cytokine formation in macrophages, such as TNF-α, IL-12, and IL-6. Consequently, HDAC inhibitors affect Th1–Th2 conversion and Treg proliferation and can help alleviate symptoms of MS by suppressing systemic immune responses (Faraco et al. 2011). Upregulated histone acetylation in oligodendrocytes is associated with inhibition of cell differentiation, resulting in damaged remyelination in MS. In addition, histone deacetylation upregulates the transcriptional profile of oligodendrocytes in the context of myelination and remyelination.

miRNA

The expression of miR-326, correlating with the production of IL-17 in Th17 cells, plays an important role in the initiation and development of MS. MiR-326 is upregulated in Th17 cells and can decrease the expression of Ets-1, leading to an increase in Th17 differentiation (Du et al. 2009). Furthermore, miR-155 has been shown to be able to regulate Th17 cells by controlling the suppressive effects of Jarid2, which functions as a recruiter of PRC2. EAE murine models show upregulation of let-7e in CD4+ T cells, amplifying the function of Th1 and Th17 cell and increasing IL10 activity, leading to an increase in the severity of EAE. Scientists have shown that overexpressed miR-128 and miR-27b exist in naïve cells and miR-340 overexpression occurs in CD4+ memory T cell. These miRNAs favor differentiation of Th1 and abrogate differentiation of Th2 cells. Decreased expression of miR-320a has been found in B cells, leading to overexpression of MMP-9 (Yang et al. 2018).

5.3 Role of Epigenetics in Endocrine Diseases

The endocrine system comprises endocrine glands that generate the fundamental hormones that act on various organ systems, regulating cell proliferation, differentiation, metabolism, and tissue function. Studies have found that epigenetic modifications bridge the gap between genetic and environmental factors in modifying endocrine function and have a great impact on the endocrine system by regulating gene expression of endocrine networks. Disruption of epigenetics contributes to the pathogenesis of endocrine diseases.

5.3.1 Type 2 Diabetic Mellitus

Type 2 diabetes mellitus (T2DM) is a chronic endocrine disease which results from inactive responses of insulin to high blood glucose caused by damaged capacity in insulin-responsive tissues or organs and is associated with deficient β-cell compensation. Epigenetics play a key role in differentiation of endocrine cell and performance of islet cells. Epigenetic modifications affect the proliferation and development of β-cells and contribute to the pathogenesis of T2DM.

DNA Methylation

Abnormal insulin secretion of pancreatic islets contributes to the pathogenesis of T2DM. Hypermethylation at the promoters of transcription factor Pdx1, Ppargc1a as well as insulin-associated genes negatively controls the expression of mRNA. Genome-wide Infinium450K array has identified over 1000 CpG sites and genes, including Tcf7l2, Pde7b, Cdkn1a, and Kcnq1, that have a different DNA methylation pattern in T2DM (Kodama et al. 2018). In T2DM, the promoter of Cdkn1a and Pde7b is hypermethylated, resulting in downregulation of these two genes and reduction of transcription activity of β-cells and abnormal secretion of insulin.

Another study demonstrated that there is a significantly higher number of hypomethylated CpGs in T2DM compared to normal controls. Aberrant DNA methylation patterns in islets of T2DM have been observed in both PBMC of T2DM and β-cell lines during hyperglycemia, suggesting that changes in DNA methylation is a key factor in the pathogenesis of T2DM. Researchers have found that the promoter of insulin-like growth factor-binding protein 1 IGFBP1 and IGFBP7 is hypermethylated in PBMCs (Drogan et al. 2016). In addition, global hypermethylation has been observed in B cells and natural killer cells in T2DM. Metabolic abnormalities also arise from liver, adipose tissue, and skeletal muscle, possibly resulting from aberrant DNA methylation patterns in T2DM. Previous T2MD murine models revealed that the promoter of hepatic glucokinase (Gck) and L-type pyruvate kinase (LPK) is hypermethylated and associated with disease initiation and progression. Recent studies indicate that hypermethylation of ubiquinone oxidoreductase subunit B6 (NDUFB6) decreases the expression of NDUFB6, leading to changes in insulin sensitivity (Kacerovsky-Bielesz et al. 2009).

Histone Modification

Histone tail modification is a critical regulator for the pathogenesis of T2DM. Recent studies indicate that histone hyperacetylation plays an important role in glucose pathway regulation and insulin secretion, implicating HAT and HDAC as key players that modulate T2DM gene expression (Bocchi et al. 2019). HNF-4 acetylation regulates DNA binding and is associated with gene activation. T2DM murine models show that CBP enhances insulin responses and increases glucose tolerance in heterozygous mice.

The transcription factor PDX1 regulates gene expression of proinsulin. Several studies show that HDAC inhibitor treatment leads to downregulation of pro-inflammatory cytokines, such as IL-6, IL-8, ICAM-1, and MIF. In T2DM cell lines or murine models, the methyltransferase Set7 is associated with diabetic complications. Set7 methyltransferase combines with H3 tails to promote H3K4me1 binding to the promoter of RELA. Polymorphisms in SUV39H2, one of the H3K9 methyltransferases, alter inflammatory cytokine expression and have been found to play a role in T2DM (Syreeni et al. 2011).

miRNA

miRNAs play a role in the regulation of β cell proliferation, cell differentiation, insulin secretion, and insulin resistance in T2DM. miRNAs function as pivotal components in glucose homeostasis. Disruption of miRNA leads to interference of electrical excitation of the β cell membrane, affecting insulin production, development of pancreatic cancer, and regulation of glucose metabolism (Wu and Miller 2017). Repression of miR-124a is important for β cell development and secretion pathway regulation through upregulation of Foxa 2 and GTPase Rab 27a, leading to reduction of insulin secretion in islet cells of T2DM patients. Overexpression of miR-34a regulates survival of β cells and apoptosis of MIN-6 cells in both mouse and human T2DM (Wu et al. 2019; Li 2014). Animal models show that miR-375 is upregulated in T2DM and is responsible for β cell proliferation and development by regulating several growth-associated genes in pancreatic β cells (Li 2014).

5.3.2 Graves’ Disease

Graves’ disease (GD) is an autoimmune disease caused by excessive autoantibody binding to thyrotropin receptors. Hyperthyroidism is the most common clinical manifestation. Increasing evidence demonstrates that epigenetic modifications occurring as a result of environmental exposures lead to dysregulation of gene expression, contributing to the pathogenesis of GD.

DNA Methylation

Studies have shown that global DNA hypomethylation occurs in GD, leading to an immune response against thyroid tissue. Analysis of the DNA methylation profile in patients with GD revealed hundreds of genetic regions, including the genes DNMT1, ICAM1, CTLA4, and MECP2, with altered DNA methylation patterns (Guo et al. 2018). There are over 300 methylated regions in CD4+ T cells and over thousands of methylated regions in CD8+ T cells, with many genes involved in T cell immune signaling pathways. Previous studies have shown that hypermethylation of TSHR correlates with GD. Polymorphisms in methionine synthase reductase (MTRR), as well as DNMT1, are associated with DNA hypomethylation and increased susceptibility to GD. Polymorphisms of 5,10-Methylenetetrahydrofolate reductase C677T contribute to the development of GD (Mao et al. 2010).

Histone Modification

Lower levels of histone H4 acetylation and higher levels of HDAC1 and HDAC2 in the PBMCs of patients with GD have been observed (Sarumaru et al. 2016). Genome-wide studies reveal that H3K4me3 and H3K27ac are downregulated at T cell signaling associated genes in GD. Furthermore, H2A.X, a histone phosphorylated protein, was found to be dysregulated in T cells and thyrocytes in a GD murine mouse model. Dysregulation of TG caused by IFN-α enriches methylation of the Lys-4 residue in H3, which can trigger GD. IFN-α is a T cell secreted cytokine, which during viral infection leads to upregulation of H3K4me1 and H3K4me3 in thyrocytes (Coppede 2017).

Recent studies have found that H2B has the capacity to identify DNA fragments in autoimmune thyroiditis, leading to upregulation of immune response genes. Polymorphisms of histone-associated genes contribute to the development of GD. SIRT1, an HDAC, has been associated with an overexpression of autoantibodies in GD.

miRNA

The most studied miRNA in GD is miR-146a (Zheng et al. 2018a). Repression of IL-1R-associated kinase 1 mediated by MiR-146a-5p triggers antigen presentation by dendritic cells (DC). MiR-155-5p mediates transcription factors and immune response molecules, by targeting SOCS1 and STAT3, leading to functional aberrancies in T helper or dendritic cells. Dysregulation of miR-155-5p and miRNA-146a-5p thereby erodes the immune microenvironment and breaks immune tolerance. In addition, MiR-346 negatively regulates the expression of Bcl-6 and activates CD4+ CXCR5+ T cells and is decreased in GD. Several overexpressed miRNAs, including miR-183-5p and miR-22-3p, and other repressed miRNAs, such as miR-660-5p, miR-101-3p, and miR-197-3p, have been observed in thyrocytes in patients with GD. miR-125a-5p is repressed in PBMCs of patients with GD, whereas miR-30a–5p and miR-519e-5p are significantly upregulated, and miR-19b-3p and miR-146a-5p are downregulated in Treg cells, which contribute to the development of GD (Qin et al. 2015).

5.3.3 Pituitary Adenomas

Pituitary adenomas (PA) are benign brain tumors, characterized by the release of high levels of hormones, including prolactin, growth hormone, and thyrotropin. Although genetic mutations are known to occur in PAs, recent studies demonstrate that altered epigenetic profiles may also contribute to the initiation and progression of tumorigenesis.

DNA Methylation

DNMT1 induces DNA methylation and alters the expression of the neuronatin (Nnat) gene (Yacqub-Usman et al. 2012). Overexpression of DNMT3b has been observed and can produce de novo DNA methylation. The promoter of DNMT3b is hypomethylated in PA compared to normal controls. In pituitary adenoma cell lines, repressed DNMT3B leads to activation of retinoblastoma protein (RB). Loss of RB in the mouse results in tumors arising from the intermediate lobe of the gland. Infinity HM450K array analysis revealed that p15(INK4b), p16(INK4a), RB1, and p27(Kip1) are hypermethylated and inactivated, and that about 90% of patients with PA showed altered DNA methylation patterns and dysregulated gene expression (Yao et al. 2017).

Histone Modification

Downregulation of BMP-4 caused by altered histone modifications has been demonstrated in PA (Yacqub-Usman et al. 2012). In PA patients, high levels of H3K9 acetylation positively regulate tumor immigration and invasiveness by targeting the MIB-1 (Ki-67) gene, as well as p53. Histone modification of melanoma-associated antigens (MAGE) and hematopoietic stem cell chromatin remodeler were found to be greatly overexpressed in PA. Upregulation of MAGE interacts with FGFR2, leading to hypomethylation of promoter regions and de-repression of targeted genes.

HDAC regulates pituitary tumor-transforming gene (PTTG) and leads to a high level of PTTG expression (Wierzbicka-Tutka et al. 2016). Increased acetylation of PTTG promoter has been observed, upregulating PTTG expression, resulting in an unfavorable outcome by modulating the signaling pathways of c-Myc and FGF2. Recent studies showed that dysregulation of Ikaros mediates the acetylation removal at the promoter of GH, restricting access to the Pit-1 activator. In PA patients, IK6, an isoform of Ikaros without a DNA binding domain, has a negative effect on gene expression, resulting in upregulation of H3 acetylation with overexpression of anti-apoptotic Bcl-XL, contributing to the development of PA (Ezzat et al. 2003).

miRNA

Overexpression of miR-493 and miR-122 has been observed in PA patients compared to normal controls. Decreased expression of let-7a, miR-15a, and miR-16, miR-21, and miR-141 has been found in PA as well (Nemeth et al. 2019). Upregulation of miR-26a modulates cell proliferation by targeting AtT20. Abnormal expression of miR-128 and miR-26b results in dysregulation of gene expression by targeting PTEN-AKT in GH-correlated PA. Furthermore, the interaction between downregulated let-7 and high-mobility group AT-hook2 (HMGA2) results in Ki-67-associated cell proliferation, invasiveness, immigration, and tumor growth, which contributes to the pathogenesis of PA. Decreased miR-23b and miR-130b have been shown to occur in PA. Upregulation of these two miRNAs arrests cell growth in PA cell lines (Leone et al. 2014).

5.4 The Role of Epigenetics in Respiratory System Diseases

5.4.1 Asthma

Asthma is a chronic and relapsed inflammatory lung disease, characterized by recurring and reversible airway constriction as well as bronchospasm. Despite recent advances in pharmaceuticals for asthma, the pathogenesis of asthma remains incompletely elucidated, and thousands of people still die from asthma every year. It is reasonable to believe that since asthma is a disease that is significantly impacted by environmental exposures, and since asthma is not a monogenic disease, epigenetics can help answer how regulation of gene expression may play a role in the risk of developing asthma as well as variable phenotypes of asthma (Fig. 1.7). The epigenetics of allergic diseases, including asthma, is discussed in Chaps. 4–6 of this volume.

Fig. 1.7
figure 7

Epigenetic modifications regulate gene expression and contribute to the pathogenesis of asthma

Full size image

DNA Methylation

Studies have shown that epigenetic modification plays an important role in T cell differentiation and have shown that altered DNA methylation contributes to the pathogenesis of asthma. Two arginase genes (Arg1 and Arg2) play a role in nitric oxide production, and altered DNA methylation in the promoter of these molecules has been associated with pediatric asthma (Li et al. 2006). DNA methylation at the promoter of Alox12 has been found to correlate with persistent bronchospasm. Abnormal DNA methylation patterns in Treg cells and altered DNA methylation of Foxp3 have been shown to occur in patients suffering from asthma with or without exposure to air pollution (Li et al. 2018). Polluted air contributes to decreased chemotaxis of Treg cells. Environmental tobacco smoke (ETS) also contributes to the development of asthma. Altered DNA methylation of Pcdh-20 has been found in sputum samples of asthma patients after comparing the asthma smoker to control smoker groups, and Pcdh-20 interacts with Pax-5α to increase the risk of asthma.

Diet is another factor that may also affect the epigenetic profile in asthmatics. Pregnant mice were fed with high and low methylation diets, and ovalbumin was used to stimulate the offspring. The offspring of the mothers fed with high methylation food showed higher IgE and more severe bronchial inflammation compared to the group on the low methylation diet. Abnormal DNA methylation patterns have been observed in CD11 DC cells of baby mice with vertical transmission from the mother. A murine asthma model showed hypermethylation occurring in the promoter region of IFN-γ and overproduction of IFN-γ in CD4+ T cells. Furthermore, loss of DNMT3A in CD4+ T cells overexpresses IL-13 and reduces DNA methylation at the promoter of IL-13, leading to aggravation of bronchial inflammation and overexpression of IgE (Yu et al. 2012).

Histone Modification

Asthma patients have higher expression of H4 acetylation compared to normal controls, which correlates with a reduction in HDAC activity and overexpression of inflammation-associated genes (Royce and Karagiannis 2014). Furthermore, airway inflammation in asthma patients can be controlled by glucocorticoids by regulating the expression of histone acetylation. Several studies have shown that modification of histone acetylation can be achieved in glucocorticoid-resistant asthma patients, and these modifications can reverse the glucocorticoid resistance. Recent studies have demonstrated that TGF-β2 can inhibit expression of ADAM33, a gene which has been cited as having a role in the pathogenesis of asthma, resulting in downregulation of histone acetylation of H3 and H4, as well as histone hypermethylation of H3K9 (Khaleva et al. 2019).

miRNA

Dysregulation of miRNA in CD8+ T cells of asthma patients has been observed, and expression levels correlate with disease severity. Downregulation of miR-146a and miR-146b in T cells, as well as decreased miR-28-5p, has been observed in CD8+ T cells in asthma patients (Comer et al. 2014). A number of animal studies have also shown that miRNAs play a crucial role in the pathogenesis of asthma. Animal models have shown that inhibition of miR-126 can block Th2 activity by modulating the expression of MyD88. In addition, repression of miR-145 can block inflammatory pathways, decrease mucous secretion, and downregulate Th2 cytokines. Multiple studies have shown that members of the let-7 family are upregulated after stimulation by allergens. Since let-7a stimulates the expression of IL-13, let-7a inhibitors may lead to remission of the inflammatory response (Chen et al. 2019).

5.4.2 Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow and irreversible impaired airway structure and globally is one of the leading causes of death. Epigenetic aberration plays a key role in the pathogenesis of COPD.

DNA Methylation

Environmental tobacco smoke (ETS) is an important factor contributing to the development of COPD, possibly as a result of its role in aberrant DNA methylation. A genome-wide DNA methylation study found that over 300 CpGs sites are associated with pathogenesis of COPD and have a great impact on the reduction of lung function (Bermingham et al. 2019). In addition, studies of innate immune cells in COPD patients showed that aberrant DNA methylation patterns and the severity of disease correlate with smoking status.

Murine COPD models sensitized to smoke also demonstrate abnormal DNA methylation patterns and altered gene expression profiles. DNA methylation at the promoter of p16 is positively associated with the severity of smoking (Sundar et al. 2018). Smoking can alter the expression level of NRF2, corresponding to the DNA methylation alteration in PBMCs inpatients with COPD. Loss of DNA methylation of A1AT is a risk factor for the development of COPD. Murine models sensitized to smoke have shown that overexpressed hypomethylation of HDAC occurs in COPD, resulting in cilium degeneration and dysregulation of mucus clearance.

DNMT expression is affected by smoke in COPD. Smoke exposure downregulates the expression of DNMT1 and upregulates DNMT3B in COPD (Qiu et al. 2018). Abnormal DNA methylation patterns after smoke exposure induce gene silencing by targeting PRC2. RUNX3, an anti-tumor gene, is regulated by PRC2 and is positively correlated with duration of smoking and methylation status.

Histone Modification

Upregulation of inflammation-associated genes, including AP-1 and NF-κB, induces the overexpression of cytokines by upregulating acetylation levels of histones in COPD patients (Sun et al. 2018b). Upregulation of NF-κB has been observed in COPD patients. A murine COPD model has shown that AP-1 and NF-κB are upregulated by ETS. TNF-α and other inflammatory cytokines can then trigger the inflammatory response by altering the expression of NF-κB, leading to massive production of IL-8.

Abnormal histone phosphorylation stimulated by smoke has been found to activate MSK1 in both humans and mice models. MSK1 interacts with p65, a member of NF-κB family, and is downregulated by post-transcriptional modification of phosphorylation and acetylation of H3 and H4. Studies have found that inflammatory cytokines and chemokines can be negatively controlled by HDACs (Lai et al. 2018). HDAC3 deacetylates p65 and negatively modulates expression of NF-κB. Furthermore, the expression of HDAC is reduced in PBMCs and airway tissue from smoking derived COPD patients. Impaired activity and reduced expression of HDACs are associated with disease status, although there is relative stable expression of HDAC5 and HDAC3. In smokers with COPD, HDAC2 activity decreases in small but not large airways. Downregulation of DJ-1 and NRF2 is correlated with disease status in COPD patients. Murine studies revealed that DJ-1 knockdown inhibits the activity of NRF2 and downregulates the expression of antioxidant genes (Malhotra et al. 2008).

miRNA

Recent studies have found that miRNAs can trigger lung inflammation, and abnormal miRNA activity can contribute to the development of COPD. Studies have found that smoking has a repressive effect on the expression of miRNA, and that miRNAs are significantly downregulated in smokers with COPD compared to normal controls (Szymczak et al. 2016). Abnormal expression of miR-452 is involved in the dysregulation of MMP12. Altered expression of miRNAs plays an important role in the TGF-β and Wnt pathways, which are crucial for the initiation and progression of COPD (Heijink et al. 2016). Studies have also demonstrated that miR-181d, miR-30c, and miR-638 expression may play a role in the pathogenesis of COPD by regulating oxidative stress.

5.4.3 Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder with dismal prognosis and few therapeutic options. IPF is characterized by progressive fibrosis and an irreversible decline in lung function. The pathogenesis and mechanism remain unknown. Epigenetic changes may shed light on the pathophysiology of IPF.

DNA Methylation

Studies have shown that hypermethylation at the promoter of Thy-1 downregulates Thy-1 in IPF, resulting in proliferation of fibroblasts and impaired lung function. The methylation status at the promoter of a-SMA in different cells, including myofibroblasts and fibroblasts, is associated with a-SMA gene expression (Tzilas et al. 2019). DNMT inhibitors have a repressive effect on DNMT activity and activate the expression of a-SMA. Recent studies show that a-SMA can bind to MeCP2, altering the expression of a-SMA in fibroblast cells in the lung (Coward et al. 2014). Fibroblast apoptosis is affected by the methylation levels of PTGER2 and ARF, and hypermethylation in the promoter decreased the expression level of these genes in IPF. PTGER2 can upregulate the activity of DNMT3a and increase the methylation level, resulting in upregulation of genes that arrests cell growth in fibroblasts. IPF patients have a loss of methylation and an increase in the level of TP53INP1 expression (Sanders et al. 2012). It has been shown that PDGF, NOTCH1, and CASZ1 alter DNA methylation and gene expression, contributing to the pathogenesis of IPF.

Histone Modification

Loss of acetylation of histone at the promoter of apoptosis and anti-fibrosis genes, such as CXCL-10 and COX-2, can reduce gene expression and decrease protein activity, leading to the proliferation of fibroblasts and impeding normal apoptosis in IPF. Studies have found that a histone deacetylase, Sirtuin, mediates the degradation of p21 proteasomal and abolishes the senescence mediated by TGF-β in bronchial epithelial cells (Korfei et al. 2018). The histone deacetylase inhibitor can be performed as an antagonist of TGF-β1 and inhibits fibroblasts’ differentiation into myofibroblasts, leading to decreased production of collagen. IPF animal models have shown that a histone deacetylase inhibitor can activate the expression of Thy-1 in fibroblasts. Furthermore, histone deacetylase inhibitors block differentiation pathways regulated by TGF-β1 and collagenation in pulmonic fibroblasts. A novel histone deacetylase inhibitor, SpA, can arrest cell growth and block the differentiation of cells that contribute to IPF (Davies et al. 2012).

miRNA

Mir-29, let-7d, miR21, and miR154 are upregulated in IPF patients compared to normal controls (Mizuno et al. 2017). A murine IPF model shows that let-7d has a protective function by blocking the pathway of epithelial to mesenchymal transition mediated by Smad-3. Let-7d can be blocked by an miRNA inhibitor, leading to a decrease in the expression of epithelial cells and an increase in the expression of mesenchymal markers in epithelial cells (Pandit et al. 2010). miR-154 has been found to be overexpressed in IPF patients (Milosevic et al. 2012). Introduction of miR-154 into fibroblasts increases cell growth by activating the WNT pathway. Human and animal studies also have shown that miR-21 and miR-29 are upregulated in fibroblasts, and that inhibition of these miRNAs can modulate TGF-β1 and reduce fibrosis.

5.5 The Role of Epigenetics in Dermatologic Disorders

Dermatologic diseases vary extensively in symptoms and physical appearance. Some manifestations may be benign, and others may be life-threatening. Genetic and environmental factors both play a role in the pathogenesis of dermatologic disorders. Recently, large-scale studies have shown that disrupted epigenetic profiles can trigger the initiation and progression of dermatologic diseases.

5.5.1 Psoriasis

Psoriasis is a dermatologic disorder involving the accelerated proliferation of keratinocytes. Clinically, skin presents red and rough patches covered with excessive silver scales. The etiology and mechanism of psoriasis remain unclear, but it is thought to be an autoimmune disease, although no autoantibody has been identified. The epigenetics of psoriasis is discussed in detail in Chap. 8.

DNA Methylation

Global hypomethylation in CD4+ T cells and hypermethylation in PBMCs have been observed in psoriasis patients. Studies have found that DNMT1 is overexpressed in PBMC in psoriasis patients compared to normal controls (Zhang et al. 2010). Downregulation of MeCP2 and MBD2 has been observed in PBMCs from patients with psoriasis. Loss of methylation has been detected at the promoter of SHP-1, which is important for the cell growth. An isoform of SHP-1 has been found to be overexpressed in psoriasis patients compared with normal controls (Ruchusatsawat et al. 2006).

The promoter of p16 has been found to be demethylated and the expression of p16 upregulated in mononuclear cells from psoriasis patients. Loss of methylation of p15 and p21 has occurred in psoriasis patients, resulting in arrest of hematopoietic cell growth. Studies have found that the promoter region of a tumor suppressor gene, p14ARF, is heavily hypermethylated and the expression level downregulated in psoriasis patients (Chaturvedi et al. 2003). Genome-wide studies have identified over hundreds of genes that are hypermethylated in psoriasis, of which about 100 are associated with the X chromosome. Immune-associated genes, such as TLR-7 and IRAK1 on the X chromosome, are hypermethylated in T cells. A recent study revealed that the methylation status of CpG sites can be categorized into different groups in CD4+ T cells of psoriasis patients, and many of these genes are associated with regulation or expression of cytokines and chemokines (Coit et al. 2019).

Histone Modification

Psoriasis patients have been demonstrated to have global H4 hypoacetylation in PBMCs, with the level of H4 acetylation associated with PASI scores as well as disease activity. Upregulation of HDAC1, EZH2, and SUV39H1 and downregulation of HATs, SIRT1, and CBP have been observed in psoriasis (Zhang et al. 2011). Studies have also found that SIRT1 can arrest cell growth and induce cell differentiation of keratinocytes by inhibiting the activity of E2F1 (Fan et al. 2019). E2F1, a member of the E2F family, can function as an activator or repressor of genes. The activated pathway of E2F is responsible for hyperproliferation and impaired apoptosis, while the repressed pathway of E2F correlates with a reduced cell growth rate. In psoriasis patients, downregulation of SIRT1 may be responsible for the overgrowth of keratinocytes. Studies have shown that HDAC inhibitors may be a potential therapeutic epidrug for the treatment of psoriasis.

miRNA

Recent studies have found that about 10 miRNAs are repressed and 40 miRNAs are overexpressed in psoriasis patients compared to normal controls, and almost all of these miRNAs are involved in the pathogenesis of psoriasis (Hawkes et al. 2016). Upregulation of miR-146a can mediate the expression of TNF, which may trigger initiation and progression of psoriasis. Repression of miR-125b can mediate the expression of FGFR2. MiR-21 is negatively correlated with T cells apoptosis and is overexpressed in psoriasis patients. MiR-31, which is regulated by TGF-1, is upregulated in keratinocytes of psoriasis patients.

Collagen IV is regulated by miR-135b. Inhibition of miR-135b inhibits keratinocyte proliferation. In psoriasis patients, miR-424 is significantly downregulated, leading to excessive proliferation of keratinocytes by activating cyclin E1 and MEK1 (Tsuru et al. 2014). Some psoriasis patients have been treated with anti-TNFα. Recent studies have shown that levels of miRNAs involved in the regulation of inflammation are altered in the serum of patients with psoriasis. Upregulation of miR-210 has been observed in CD4+ T cells in psoriasis, and it can inhibit the expression of FOXP3 and facilitate the development of autoimmunity (Wu et al. 2018).

5.5.2 Systemic Sclerosis

Systemic sclerosis (SSc) is an autoimmune disorder of connective tissue, characterized by an accumulation of collagen and persistent thickening and stiffening of the skin, caused by impairment of small blood vessels and accumulation of collagen.

DNA Methylation

Studies have already shown that SSc is characterized by global DNA hypomethylation in CD4+ T cells and upregulated autoimmune-disease-associated genes, such as NLRP1. It has also been found that DNA methylation modifiers, including DNMTs and MBDs, play a role in SSc. Hypomethylation in the promoter regions of CD40L and CD70 in CD4+ T cells and upregulation of the genes have been observed in patients with SSc (Yalcinkaya et al. 2016; Hedrich and Rauen 2012). A murine model demonstrated that loss of methylation occurs at the promoter of CD40L, resulting in overexpression of the gene and fibrosis. In addition, abnormal DNA methylation and overexpressed CD40L appear only in female SSc patients, mainly because CD40L is located on the X chromosome.

In contrast, DNA methylation levels of fibroblasts in patients with SSc are increased compared to normal controls, correlating with upregulation of the DNA methylation modifiers, DNMT1, MECP2, and MBD1 (Dees et al. 2014). Hypermethylation has been found to occur in the promoter of FLI1, which results in reduced synthesis of collagen. Downregulation of this gene by DMNT inhibitors can decrease the amount of fibrinogen in the fibroblasts of SSc patients. As in other diseases previously discussed, components of the Wnt pathway may be hypermethylated, leading to reduced expression of Wnt pathway antagonists SFRP1 and DKK1, thus triggering fibrosis. From a therapeutic standpoint, DNMT inhibitors may activate these genes and repair Wnt signaling, thus blocking fibrosis. Studies have also shown that the promoter of BMPR2 is hypermethylated and the gene is downregulated, contributing to the pathogenesis of pulmonary hypertension in SSc patients (Gilbane et al. 2015).

Histone Modification

HDAC inhibitors can induce acetylation and downregulate the expression of FN1 and COL1A1, both of which contribute to fibrosis. Besides inhibiting gene expression of collagen synthesis in SSc, HDAC inhibitors can also activate the expression of profibrotic genes, including CTCF and ICAM-1, which can impact the development of fibrosis in SSc (Pang and Zhuang 2010). The expression of p300, a histone acetyltransferase, is upregulated in fibroblasts of SSc patients, and it acts to increase fibrosis along with Egr1 and TGF-β in SSc. Studies have also demonstrated that inhibition of H3K27me3 can block collagen production and decrease fibrosis induced by TGF-β in SSc. Altered acetylation levels of H3 and H4 have been observed in B cells in patients with SSc. Acetylation levels of H4 are increased and there is a loss of methylation of H3K9. These alterations impact SUV39H2 and HDAC2, induce dermatologic fibrosis, and increase severity of disease (Wang et al. 2013).

miRNA

Upregulated miR-21 increases the expression of fibrosis-associated genes, including COL1A1 and FN1, by modulating the expression of SMAD7 in fibroblasts of SSc patients. Studies have also found that miR-21 is upregulated in other fibrotic diseases, including pulmonary fibrosis and dermatologic fibrosis. In contrast to miR-21, miR-29 has a negative impact on fibrosis (Bagnato et al. 2017). Downregulation of miR-29 has been found in SSc and other diseases in which fibrosis is a feature. Downregulated miR-29 leads to a decrease in the expression of COL1A2 and COL3A1, leading to an increase in the level of ECM proteins and triggering the development of fibrosis in SSc.

Decreased let-7a increases the level of collagen I in SSc, and a murine SSc model demonstrated that an increase in the expression of let-7a inhibits fibrosis. miR-196a functions as an inhibitor of collagen production under the regulation of TGF-β. Downregulation of miR-196a leads to excessive production of collagen and the development of fibrosis in SSc patients. The expression of miR-129-5p is mediated by IL-17A and downregulated by TGF-β signaling (Nakashima et al. 2012).

5.5.3 Melanoma

Melanoma results from aberrant proliferation and differentiation of melanin-producing cells called melanocytes and is one of the most malignant cancers with poor prognosis. The etiology of melanoma still remains unknown, but ultraviolet (UV) light exposure increases the risk of disease.

DNA Methylation

The DNA methylation pattern of melanoma is similar to other types of cancer, characterized by global hypomethylation and specific promoter hypermethylation, leading to tumor suppressor gene silencing and oncogene activation, resulting in tumorigenesis. One study found that melanoma patients can be classified into several groups based on different DNA methylation patterns, as in the case with colorectal cancers.

Colorectal cancer can be classified into several groups based on CpG island methylator phenotype (CIMP), and the same process can be applied to melanoma (Tanemura et al. 2009). CIMP status is dependent upon mRNA transcription and protein translation. A recent study assigned mutations in IDH1 and ARID2 into high CIMP groups. Another study found that genetic mutations correlate with an altered epigenetic profile, leading to dysregulation of gene expression. Hypermethylation at the promoter of RASSF1A results in the deactivation of encoded genes, which occurs in about 60% of melanoma cases. But this is only found in advanced stages of melanoma, suggesting that methylated RASSF1A may be a good marker for monitoring the progression of melanoma. Loss of p16 resulting from methylated CDKN2A has been observed in melanoma, and this leads to melanocyte hyperproliferation (Li et al. 2019).

DNA hydroxymethylation is another important mechanism which contributes to the pathogenesis of melanoma (Fig. 1.8). 5-hydroxymethylcytosine (5hmC) is produced through enzymatic action of TET family proteins. Melanoma has a low 5hmC level compared to normal controls, and the level is associated with tumor stage, suggesting a potential role as a biomarker for the prognosis of melanoma (Bonvin et al. 2019). Another study found that several TET genes as well as IDH2 are deactivated, contributing to the downregulation of 5hmc. A murine melanoma model showed that TET2 insertion can restore expression of 5hmc, indicating that it may be an attractive therapeutic target for the treatment of melanoma.

Fig. 1.8
figure 8

Global loss of 5hmc in melanoma can be a biomarker for the diagnosis of melanoma

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Histone Modification

Downregulation of H3K27ac and upregulation of H3K27me3 have been observed in melanoma, which leads to an increase in the expression of SOX10 (Cronin et al. 2018). A SOX10 knockout model demonstrated a slower melanoma growth rate compared to wild-type controls. SAHA, an HDAC inhibitor, induced acetylation of H3 and H4 at CDKN2A, leading to a depression of p14ARF. A murine melanoma model showed that in advanced stages of melanoma, mH2A is downregulated due to DNA hypermethylation and reduced expression of mH2A2, leading to an increase in migratory capacity. Abnormal expression of E2F and BRD2, which binds to H2A.Z.2, is associated with increased malignant potential of melanoma. Knockdown of this altered histone variant downregulates the expression of these encoded genes, including E2F and BRD2. HDAC6 regulates the expression of STAT3, which is responsible for the expression of PD-L1, suggesting that combination treatment of HDAC inhibitors and immunotherapy may be a treatment option (Lombard et al. 2019). Studies have found that Temozolomide, a chemotherapy drug commonly used in melanoma by targeting MGMT, often encounters drug resistance in melanoma patients. A study using HDAC inhibitors to treat chemo-resistance melanoma patients found that the acetylation level was upregulated at the promoter region of MGMT, although the expression level of this gene was not changed (Chen et al. 2016).

miRNA

miRNAs can function as either tumor suppressive or onco-miRNAs to regulate the development of melanoma. MiR-31, functioning as a tumor suppressor miRNA, represses tumor growth by inhibiting EZH2 expression. Downregulation of miR-31 has been observed in melanoma, leading to overexpression of EZH2 and contributing to hyperproliferation of the tumor (Zheng et al. 2018b). Previous studies identified that miR17 was overexpressed and miR-25 is underexpressed in metastatic samples of melanoma, and thus it can potentially be developed as a biomarker for melanoma. Dysregulated miRNAs, including miR-26a, miR-221, and let-7a, are involved in regulating cell apoptosis, cell growth, and tumor metastasis, all of which contribute to the initiation and progression of melanoma (Romano et al. 2017).

5.6 Role of Epigenetics in Cardiovascular Diseases

Cardiovascular diseases (CVD) that may be under the influence of epigenetic regulation include atherosclerosis, angina, myocardial infarction, cardiomyopathy, heart arrhythmia, and congenital heart disease. CVD is one of the leading causes of death worldwide.

5.6.1 Atherosclerosis

Atherosclerosis is characterized by irreversible inflammation created by the persistent accumulation of plaques. The blockage of vessels is associated with plaque-induced inflammation, ultimately leading to hypoxia and ischemia. Reprogrammed epigenetic profiles may play a role in the pathogenesis of atherosclerosis.

DNA Methylation

Studies have found that the DNA methylation pattern in atherosclerosis is different from that of cancer and autoimmune disease and is characterized by global DNA hypermethylation. Genome-wide studies have found that DNA methylation is positively correlated with the severity and disease grade of atherosclerosis, suggesting that the global DNA methylation pattern may be able to be used to monitor the progression of disease. Studies have also shown that the DNA methylation in cardiovascular diseases is modulated by inflammation-associated genes, such as TLR2.

Epithelial cells treated with low-density lipoprotein (LDL) showed overexpressed DNMT1 and hypermethylation at the promoter of KLF2, which is an anti-inflammatory gene. Suppression of KLF2 induces the development of inflammation (Tabaei and Tabaee 2019). DNMT inhibitors induce hypomethylation and have a positive effect on KLF2. The promoter of KLF4 can be methylated by overexpressed DNMT3A, leading to downregulation of KLF4 and vascular inflammation. In addition, the DNA methylation profile of coronary artery disease is characterized by global DNA hypermethylation in PBMCs, triggering an inflammatory process. A murine atherosclerosis model shows that 5-aza alleviates atherosclerosis (Cao et al. 2014).

Histone Modification

Using immunohistochemistry, it was demonstrated that the histone repressive marker H3K27me3 was downregulated in vascular smooth muscle cells (Wierda et al. 2015). The expression of JMJD3 was increased, and along with a decrease in the expression of H3K27me3 led to de-repressed target genes.

An atherosclerosis model showed that GSK-J4, an inhibitor of JMJD3, can downregulate inflammation-associated cytokines and chemokines, including TNF-α. HDAC5 and HDAC7 have a negative effect on the expression of KLF4 in epithelial cells, and the upregulation of HDACs can suppress KLF4 expression and contribute to the pathogenesis of atherosclerosis (Zheng et al. 2015). Another study found that HDAC3 knockout leads to upregulation of IL-4-associated genes, leading to the activation of anti-inflammatory effects, reversing atherosclerosis. A murine model demonstrated that lipopolysaccharides failed to induce inflammation and release cytokines after HDAC9 knockout (Azghandi et al. 2015). Another study revealed that upregulated HDAC can be stimulated chemically in small muscle cells and that epigenetic inhibition can disrupt abnormal cell proliferation and apoptosis, leading to inhibition of the development of atherosclerosis.

miRNA

Studies have showed that miRNAs play a crucial role in the pathogenesis of atherosclerosis. Recent studies have shown that miRNAs can regulate the accumulation and function of HDL and LDL, and the levels of lipoprotein. miR-128-1 and miR-301b have been found to have an impact on the regulation of lipoprotein-associated genes, including ABCA1 and LDLR (Laffont and Rayner 2017). Dysregulation of miR-148a plays a role in lipoprotein metabolism by targeting these genes. The use of an miR-148a inhibitor can potentially upregulate the expression of ABCA1 and LDLR, leading to upregulated HDL-C and downregulated LDL-C.

An abnormal level of cholesterol is a risk factor for atherosclerosis. miR-223 plays a role in cholesterol synthesis by activating ABCA1. A murine miR-223 knockout model showed upregulated HDL-C and cholesterol. miR-33 can also regulate the expression of ABCG1 and ABCA1 by targeting SREBP1, which plays a role in cholesterol metabolism. Altered miR-33 can affect cholesterol levels, contributing to the pathogenesis of atherosclerosis (Ouimet et al. 2017). In addition, let-7 g can deactivate inflammation-associated genes, such as TGF-β and LOX-1 by inhibiting SIRT1 and TGF-β. A murine model showed that downregulated let-7 g levels can increase expression of PAI-1, leading to endothelial inflammation (Wang et al. 2017).

5.6.2 Hypertension

Hypertension is characterized by persistent increased blood pressure. Hypertension is one of the leading public health issues worldwide and is a main risk factor for stroke, hypertensive nephropathy, and hypertensive ophthalmopathy. The mechanism of hypertension still remains unknown, but the pathogenesis is believed to be multifactorial. Epigenetics plays an important role in the pathogenesis of hypertension.

DNA Methylation

A genome-wide study was conducted on about 10,000 hypertensive individuals and normal controls identified dozens of CpGs sites that are involved in the pathogenesis of hypertension (Han et al. 2016). Loss of methylation at the promoter of TLR4 and Alu results in upregulation of encoded genes, contributing to the pathogenesis of hypertension. Abnormal DNA methylation of 11βHSD2 can dysregulate the expression of its encoded genes, leading to disruption in the regulation of mineralocorticoids (Bailey 2017). A murine hypertension model revealed that loss of methylation occurs in the promoters of AT1aR and NKCC1, which are regulators of the Na+ channel, leading to increased blood pressures. The DNA methylation pattern is globally downregulated and blood pressure is decreased by ACE inhibitors. Hypoxia can lead to a decrease in the DNA methylation levels of AGT and ACE1 in endothelial cells, resulting in increased blood pressure and decreased heart rate.

Histone Modification

Studies have found that upregulation of HDAC can decrease H3 and H4 activity and block the WNK4 pathway, leading to downregulation of WNK4 (Lee et al. 2018). Downregulation of WNK4 may play a role in the development of hypertension. A murine hypertension model revealed that exposure to high salt can decrease expression of LSD-1, resulting in loss of methylation of both H3K4 and H3K9, dysregulation of eNOS, and the initiation and progression of hypertension. Another murine hypertension model showed that Af17 knockout can upregulate the level of H3K79me2, leading to a decrease in sodium levels and resolution of hypertension. Yet another animal study revealed that upregulated β2-adrenergic receptors can deactivate WNK4, decrease the expression of HDAC8, and upregulate the level of histone acetylation, contributing to the pathogenesis of hypertension (Li et al. 2017).

miRNA

miR-126 is a key player in the development of endothelial cells. Dysregulated miR-126 can disrupt vascular integrity and affect vascular function. Deleted miR-126 is associated with damage to blood vessel structure and function, which contributes to the pathogenesis of hypertension (Yuan et al. 2019). Abnormal expression of miR-217 can target eNOS and FOXO1 of endothelial cells. Upregulated miR-143 has been observed in hypertensive patients, and inhibition of miR-143 can decrease blood pressure. A murine model found that decreased miR-181 is associated with hypertension (Han et al. 2018). Studies have shown that upregulated miR-637, miR-122, and let-7 correlate with hypertension. miR-27a and miR-150 are decreased and miR-92 and miR130 are increased in hypertensive patients compared with healthy controls (Nemecz et al. 2016).

5.6.3 Heart Failure

Heart failure (HF), or congestive heart failure (CHF), is one of the leading causes of death worldwide. Currently available drugs are limited to slowing the progression of the disease. The mechanism of HF remains unclear. Altered epigenetic profiles may play a role in the pathogenesis of HF.

DNA Methylation

A genome-wide study revealed that loss of methylation at the promoter region can upregulate encoded genes, such as GLUT1, in HF (Li et al. 2017). This study also identified several genes associated with angiogenesis, including PECAM-1 and ARHGAP24, which are modulated by DNA methylation status. Studies have also found that altered DNA methylation patterns have an impact on the expression of ADORA2A. Upregulated DNMT1 increases methylation levels of SERCA2a by targeting TNF-α signaling pathways. A study on murine HF models demonstrated that DNMT3 knockout mice have impaired cardiac function. A recent study found that DNMT3B is the main DNMT in cardiomyocytes, and the disease develops rapidly in DNMT3B knockout models, suggesting it plays a crucial role in the initiation and progression of HF (Nuhrenberg et al. 2015).

Histone Modification

Studies have found that HDACs are key players in the development of HF (Evans and Ferguson 2018). HDAC5 and HDAC9 regulate cell proliferation in HF models. Deletion of these HDACs can accelerate the growth rate of cardiomyocytes by targeting and regulating the expression of Mef2c, resulting in the hypertrophy seen in HF. Furthermore, deleted HDAC4 murine models show cardiac muscle cell hyperproliferation, leading to the cardiac hypertrophy and the development of HF. In a HDAC2 knockout model, hypertrophy of cardiac muscle is ameliorated, while reintroduction of HDAC2 can accelerate cardiac muscle hypertrophy and contribute to the development of HF (Yoon et al. 2018). Emerging studies show that SIRT1 and SIRT3 have a positive effect on HF. P300, a HAT, and associated CBP genes function as a hypertrophic stimulator in the development of HF. Pharmaceutically inhibited p300 and CBP attenuate the progression of cardiac hypertrophy, and reintroduction of p300 and CBP stimulates the hypertrophy. Altered histone methylation of H3K4me3 and H3K9me3 has been observed in patient samples and murine models of HF. Upregulated JMJD2A can reduce the expression of H3K9me3 and H3K36me3 by targeting FHL1, leading to the proliferation of cardiomyocytes. JMJD2A deletion decreases the level of hypertrophy, suggesting that JMJD2A plays a key role in the pathogenesis of HF (Zhang et al. 2011).

miRNA

Studies have found that decreased miR-1 is associated with cardiac hypertrophy, and reintroduction of miR-1 can alleviate the development of cardiac muscle hypertrophy in a murine HF model (Al-Hayali et al. 2019). Studies have demonstrated that miR-1 functions by targeting several genes, including IGF-1, GATA4, and NCX1. Several muscle-associated miRNAs, including miR-133 and miR-378, are downregulated in HF patient samples. Furthermore, miR-23, miR195, and miR199 are upregulated in HF patients compared to normal controls (Vegter et al. 2016). Studies have shown that upregulated miR-195 and miR-499 can downregulate the expression of MO25 and HMGA. The latter two facilitate cell apoptosis, thus inhibition of these will result in cardiac hypertrophy in HF. Murine HF models have shown that overexpressed miR-499 activates the WNT pathway, resulting in cardiac hypertrophy. Murine studies have also shown that upregulated miR-199b targets Dyrk1a and contributes to the pathogenesis of HF (da Costa Martins et al. 2010).

5.7 The Role of Epigenetics in Gastrointestinal Tract Diseases

5.7.1 Gastric Cancer

Gastric cancer is one of the most aggressive cancers, with poor prognosis, and is one of the leading causes of death worldwide. Studies have found that both genetic background and environmental factors play an important role in the development of gastric cancer. Epigenetics is the bridge between genetics and the environment that can also contribute to the pathogenesis of gastric cancer. Emerging evidence demonstrates that altered epigenetic profiles can modulate gene silencing or activation that leads to the development and progression of gastric cancer.

DNA Methylation

Altered DNA methylation patterns have been identified in gastric cancer. Gastric cancer patients demonstrate hypermethylation in promoter regions and global hypomethylation of repetitive elements. Abnormal DNA methylation patterns occur at both advanced stages and early stages of gastric cancer.

H. pylori (HP) is an important factor in the pathogenesis of gastric cancer, and the interaction between HP infection and DNA methylation is a hotspot in gastric cancer research (Maeda et al. 2017). Studies have found that high DNA methylation levels correlate with HP infection in gastric cancer, and eradication of HP infections is associated with a decrease in DNA methylation in gastric cancer (Maekita et al. 2006; Miyazaki et al. 2007). Genome-wide studies have revealed that the oncogenes HAND1 and FLNC are activated in gastric cancer.

Tumor suppressor genes, including p16, COX- 2, and MGMT, have been found to be downregulated by DNA hypermethylation at their promoter regions in gastric cancer. These genes are associated with cell proliferation, DNA damage repair, and apoptosis. Studies have found that altered DNA methylation patterns at the promoter of these genes dysregulate the encoded genes and contribute to the pathogenesis of gastric cancer.

For example, hypermethylated homeobox D10, also known as HoxD10, which plays a role in cell apoptosis, has been observed in gastric cancer, leading to downregulation of the gene and aberrant apoptosis and gastric cellular hyperproliferation in gastric cancer. Studies have also found that 5mc and 5hmc are closely associated with gene expression, and that 5mc is downregulated in gastric cancer patients compared to normal controls. Decreased 5mc is correlated with dismal prognosis in gastric cancer (Necula et al. 2015).

Histone Modification

Early studies showed that an increase in H3K9me3 was associated with an aggressive course and dismal prognosis in gastric cancer. It has also been found that gastric cancer cells are sensitive to EZH2 inhibitors in vitro. Further analysis revealed there is abnormal accumulation of H3K27me3 and that PRC2 is a target of the epidrug Tazemetostat, suggesting that it may be a potential therapeutic target for the treatment of gastric cancer (Choi et al. 2014). Recent studies found that both DNA methylation and histone modification aberrations contribute to the pathogenesis of gastric cancer.

It has been shown that EZH2 interacts with DNMTs to increase DNA methylation levels in a series of genes, such as MT1F, BHMT, and ACSL1. Furthermore, H3K27me3 regions are frequently hypermethylated in AGS127, a gastric cancer cell line. The co-existence of hypermethylation and histone modification, including downregulated H3K9ac and upregulated H3K9me3, has been observed at the promoter of MLH1, leading to downregulation of the encoded gene. After treatment with a DNMT inhibitor, the expression of MLH1 is restored. This was not true with the HDAC inhibitor, but combination treatment has a synergistic effect on gene expression, suggesting that DNA methylation is the foremost player in deactivating this gene, and histone modification may also play an important role in this pathway (Guo and Yan 2015).

miRNA

miRNAs can be categorized into tumor suppressive and oncogenic miRNAs, both of which are involved in the regulation of gene expression. Studies have found that overexpressed miR-106b can target and decrease the expression of CDKN1B and BCL2L11, which are involved in cell apoptosis, leading to cancer cell hyperproliferation in gastric cancer (Zhu et al. 2019). A murine gastric cancer model showed that miR-16, miR-126, and miR-106a are associated with drug resistance. Genome-wide studies identified several miRNAs, including miR-99a, miR-202, and miR-133a, that are upregulated in gastric cancer. Decreased miR-let7 may be a clinical biomarker for the prognosis of gastric cancer. Another study revealed that dysregulated miR-let7, miR-21, and miR-99a can target PKM2 and PTEN and play an important role in the pathogenesis of gastric cancer (Wang et al. 2015). Furthermore, miRNAs can interact with DNA methylation, contributing to disease initiation and progression. Dysregulated miR-331-3p can target HER2, leading to hyperproliferation of cancer cells in gastric cancer, suggesting it plays a key role in the pathogenesis of gastric cancer.

5.7.2 Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a systemic and chronic autoimmune disease. The major inflammatory bowel diseases include Crohn’s disease (CD) and ulcerative colitis (UC). IBD may affect the entire digestive tract, but most commonly involves the small intestine and the colon. IBD adversely impacts quality of life and is often life-threatening. Genetic variation and environmental factors play significant roles in IBD. Altered epigenetic profiles bridge the gap between genomic and environment factors.

DNA Methylation

Studies have found that hypermethylation occurs in the promoter of TRAF6, causing a decrease in gene expression in PBMCs in patients with CD and UC. Genome-wide studies showed that the promoters of AATK, BGN, and SERPINA5 are hypermethylated in inflamed mucosa samples of CD compared to normal controls, and the expression of encoded genes is deactivated (McDermott et al. 2016). Altered DNA methylation patterns occurring in the promoter of DOK2 have been observed, which can regulate cell growth. Thus, dysregulated expression of this gene results in abnormal cell proliferation. The promoter of CDH1 is hypermethylated and the gene is downregulated in inflamed tissue samples of CD and UC. On the other hand, normal areas demonstrate no such changes, suggesting that this dysregulation contributes to the pathogenesis of CD and UC. A recent study revealed that FANCC, THRAP2, and GBGT1 are methylated only in inflamed mucosa of CD, but not in UC, and normal areas show no significant change in methylation of these genes, indicating that methylation may play a key role in the initiation and progression of CD (Cooke et al. 2012).

Histone Modification

Studies have shown that the interaction between histone modification and gut microbiome is critical for the pathogenesis of IBD. The gut microbiome can produce a small molecule, n-butyrate, which functions as a HDAC inhibitor (Chang et al. 2014). A recent study demonstrated that bacteria in the gut can metabolize propionate, and the resulting metabolite plays a role in regulating gene expression of PPARγ and Gpr43. The levels of HDAC are also downregulated, suggesting that HDAC inhibitors can be a marker of inflammation in CD and UC (Felice et al. 2015). Helminths have been found to negatively regulate cell differentiation by targeting the HDAC level.

miRNA

Several miRNAs, including miR-21, miR-16, and miR-594, were found to be overexpressed in inflamed mucosal samples from CD patients compared to samples from normal areas in CD patients and in healthy controls, suggesting that these miRNAs participate in the pathogenesis of CD (Soroosh et al. 2018). Genome-wide studies have shown that miR-106a, miR23b, and miR-191 are upregulated, while miR-629 and miR-19b are downregulated in both CD and UC (Ni et al. 2018). Another study compared active CD, inactive CD, and normal controls and found that miR-126, miR-9, and miR-130a, are overexpressed and were only found in active patients, but not in inactive patients and normal controls, suggesting that these miRNAs may be a good marker for the treatment and that they may contribute to the initiation and progression of CD. In both human and murine models, it has been shown that expression of miR-141 is repressed in patients with CD. The study found that miR-141 can target CXCL12β to modulate migration of leukocytes in the inflamed area, and that dysregulated miR-141 plays an important role in the pathogenesis of CD. Various miRNAs, including let-7b, miR-30e, miR-16, and miR-192, are upregulated in PBMCs from CD patients, indicating that they may contribute to the pathogenesis of IBD (Moein et al. 2019).

5.7.3 Hepatic Cirrhosis

Hepatic cirrhosis is a degenerative disorder characterized by normal liver tissue being replaced by fibrosis tissue. Hepatitis B and C and alcohol abuse are the most common causes of cirrhosis. In cirrhosis, hepatic stellate cell (HSC) is converted into myofibroblasts and accelerates fibrosis, but the mechanism of cirrhosis is still incompletely elucidated. Epigenetics plays a role in the pathogenesis of cirrhosis (Fig. 1.9).

Fig. 1.9
figure 9

Epigenetic modifications take part in the gene expression in hepatic stellate cells

Full size image

DNA Methylation

Genome-wide studies in patients with cirrhosis have shown that the DNA methylation of 10% of all genes is altered, which results in either gene activation or deactivation (Urabe et al. 2013). MeCP2, a DNA methylation modifier, binds to the promoter of PPARγ, and then induces gene repression of H3K9me3, resulting in silencing of the expression of PPARγ, activating HSC and triggering fibrosis (Urabe et al. 2013). A murine study showed that the expression of αSMA, a marker of fibrosis, is decreased in HSCs of MeCP2 knockout mice. Furthermore, MeCP2 activates the expression of fibrosis-associated genes by targeting the histone modifier, ASH1. This leads to an increase in expression of αSMA, TIMP1, and collagen I, contributing to the pathogenesis of cirrhosis. Another study showed that the DNA methylation pattern at the promoter of PPP1R18 and HoxA2 is associated with the severity of disease. Human cirrhosis samples and murine models demonstrated upregulation of DNMT3A and DNMT3B and downregulation of TET, leading to alteration of the DNA methylome profile in HSCs. Knockdown of these DNA modifiers leads to inhibition of fibrosis, suggesting that these DNA modifiers play a key role in the initiation and progression of cirrhosis (Oh et al. 2007).

Histone Modification

Upregulation of HDAC has been observed in cirrhosis samples and animal models, and pharmaceutical inhibition of HDAC can decrease expression of HDAC and attenuate hepatic stellate cell proliferation (Khan et al. 2016). Studies have also found that HDAC inhibitors can repress the expression of fibrotic genes and inhibit cell growth, leading to attenuation of fibrosis. Murine studies have revealed that alcohol exposure can increase levels of H3K9ac and MLL1, resulting in extensive activation of fibrosis-associated genes. Furthermore, murine studies have shown that JMJD1A knockdown mice overexpress H3K9me2 at the promoter of PPARγ, increasing the expression of profibrogenic genes and accelerating fibrosis. Recent studies have demonstrated that HMT inhibitors can also attenuate fibrosis (Jiang et al. 2015).

miRNA

Numerous miRNAs have been studied in cirrhosis (Starlinger et al. 2019). The most studied miRNA in cirrhosis is the miR-29 family. Studies have shown that the expression of miR-29 is downregulated by NFκB and TGFβ in activated HSC. Human cirrhosis samples and murine models have demonstrated that the expression of miR-29 is decreased, and that it is regulated by TGF-β. An increase in the expression of miR-29 decreases production of collagen I. Collagen I contributes to the pathogenesis of cirrhosis. Upregulated miR-29a reduces cell growth and fibrosis by targeting fibrosis-associated genes in HSC. Furthermore, studies revealed that miR-29a can inhibit the expression of DNMTs, including DNMT3B and DNMT1, resulting in demethylation of targeted genes and downregulation of fibrosis-associated genes in HSC. A recent study demonstrated that miR-29 can regulate IGF-1 and PDGF-C, leading to inhibition of mitosis and migration capacity (Deng et al. 2017).

6 Discussion

Epigenetics is a rapidly developing field, although the clinical application of epigenetics is still in its early stage. However, there are several epidrugs and biomarkers that have been approved by FDA and are already widely used in clinical practice. Oncology and autoimmune disorders are the main fields targeted by epigenetic studies, although epigenetic aberrations can affect any disease or organ system.

Novel epigenetic-based technologies, such as the infinium humanmethylation450K (HM450K), whole genome bisulfite sequencing (WGBS), methylated DNA immunoprecipitation (MeDIP), and chromatin immunoprecipitation sequencing (ChIP-Seq), have helped in the development of this field. Along with the explosion of epigenetic studies, large amounts of complicated and cumbersome data have been generated. Luckily, the ability to analyze such “big data” is now available as a result of rapid technological advances in computing power. Entire bioinformatics departments have been established in academic institutions to tackle these issues. Furthermore, a number of databases, including the Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), are continually adding population-based data to help better understand disease pathogenesis. All of these resources have contributed to the development of epigenetics.

Numerous epigenetic challenges remain unmet. Multiple studies have demonstrated that an altered epigenetic profile can disrupt biological pathways and play a key role in the pathogenesis of human diseases. But translating this knowledge to clinical applications remains a significant problem. Biomarkers are being identified, but they are often of low sensitivity and specificity, rendering them poor surrogates to track disease status. Furthermore, variable and non-standardized patient samples pose problems due to the sample heterogeneity of epigenetic modifications (Morente et al. 2008).

Clearly, further studies are required to answer these questions regarding the role of the epigenetic landscape on human health. The role of epigenetics in cell destiny, including differentiation, proliferation, apoptosis, and metabolism is still incompletely understood. Detailed analysis of DNA methylation, histone modification, and miRNAs in healthy and diseased human subjects is necessary for better understanding of the mechanism of diseases. Clarifying the interplay among genetic variation, environmental factors, and epigenetic aberration is critical to understanding the initiation and progression of human diseases.

7 Conclusion

Genetic variations and environmental factors only contribute a portion of the risk for developing disease. Epigenetic modification can be influenced by environmental factors and provides hereditable but reversible changes in gene expression in the absence of DNA sequence changes. Great efforts have been focused on the epigenetic study of human diseases in the past decades, and this has resulted in the development of new innovative strategies for the diagnosis, monitoring, prognostication, and management of diseases. Extensive studies on DNA methylation, histone modification, and miRNAs have contributed to a better understanding of the pathogenesis of human diseases.

It has been found that an altered epigenetic profile that is associated with the development of a disease state can be taken advantage of in a clinical setting, as these changes can be used as biomarkers in some cases. Epigenetic changes may also be reversed pharmaceutically, and hence the development of agents that affect DNA methylation or histone modifications, as well as miRNAs that can change gene expression. Discoveries that address epigenetic disruption put us on the cusp of fulfilling the promise of personalized medicine and tailored therapy that has been a holy grail for clinical practitioners for many years now. From the first epidrug for the treatment of hematological malignancies approved by the FDA, new emerging epigenetic medications and biomarkers are continually being tested in clinical trials. Other technological advances such as high throughput and highly sensitive means of analyzing epigenetic changes, optimized databases, and the computing ability to analyze large datasets are invaluable tools to achieving these goals.