Epigenetic Dysfunctional Diseases and Therapy for Infection and Inflammation

Abstract

Even though the discovery of the term ‘epigenetics’ was in the 1940s, it has recently become one of the most promising and expanding fields to unravel the gene expression pattern in several diseases. The most well studied example is cancer, but other diseases like metabolic disorders, autism, or inflammation-associated diseases such as lung injury, autoimmune disease, asthma, and type-2 diabetes display aberrant gene expression and epigenetic regulation during their occurrence. The change in the epigenetic pattern of a gene may also alter gene function because of a change in the DNA status. Constant environmental pressure, lifestyle, as well as food habits are the other important parameters responsible for transgenerational inheritance of epigenetic traits. Discovery of epigenetic modifiers targeting DNA methylation and histone deacetylation enzymes could be an alternative source to treat or manipulate the pathogenesis of diseases. Particularly, the combination of epigenetic drugs such as 5-Aza-2-deoxycytidine (Aza) and trichostatin A (TSA) are well studied to reduce inflammation in an acute lung injury model. It is important to understand the epigenetic machinery and the function of its components in specific diseases to develop targeted epigenetic therapy. Moreover, it is equally critical to know the specific inhibitors other than the widely used pan inhibitors in clinical trials and explore their roles in regulating specific genes in a more defined way during infection.

Graphical abstract

1. Introduction

The study of epigenetics involves genetic control by factors other than an individual’s DNA sequence. Genes can be switched on or off, and transcription can be regulated through epigenetic changes. These phenomena are heritable, alternative states of gene activity that are not explained by mutation or normal developmental regulation [1]. Many human diseases, including various cancers are caused as a result of epigenetic changes [2]. Epigenetic research is now becoming a rapidly developing and exciting area in biology. Studies are available in cataloguing systematically different epigenetic modifications in various tissues during developmental and pathological situations [3]. The present understanding of the mechanisms underlying epigenetic phenomena and their predominance as contributors to the development of human disease has led to a greatly enhanced interest in epigenetic research.

Epigenetic mechanisms rely on the modifications of cytosine bases in the DNA and changes in the nucleosome positioning of histones as well. They are DNA-protein interactions, suppression of transposable element mobility, cellular differentiation, embryogenesis, X-chromosome inactivation, and genomic imprinting. In multicellular organisms, the ability of epigenetic marks persist during development and can potentially be transmitted to offspring which may be necessary for generating the large range of different phenotypes that arose from the same genotype [4]. For example, cloned animals with the identical genotype may develop a different disease pattern [5]. Monozygotic twins are identical at the DNA sequence level, but have different DNA methylation and histone modification profiles [6] that might affect the occurrence of diseases, including cancer [7] or autoimmune disorders [8]. Epigenetic modification is also a key factor in cellular differentiation during development [9]. This review mainly focuses on epigenetic changes such as chromatin dynamics, the establishment of methylation patterns on DNA, and the role of RNA and proteins during gene regulation processes. Moreover, we will discuss the role of environment on epigenetic changes during infection and inflammation.

2. Epigenetic modification causes different types of diseases

Epigenetic modifications have been well established in cancer [7] and occur in several other diseases, including diabetes [10], lupus [11], asthma [12, 13], and a variety of neurological disorders. Rett syndrome is the neuro-developmental disorder most intensely studied with regard to epigenetic changes. In this disease one can see mutations in methyl CpG binding protein 2 (MeCP2) that binds to methylated DNA [14]. Other mental retardation disorders such as α-thalassemia, X-linked syndrome, Rubinstein-Taybi syndrome and Coffin-Lowry syndrome are also linked to the disruption of genes involved due to epigenetic changes [15]. Recently, research has been started on Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease to explore the possible epigenetic changes [16]. Studies on Huntington’s disease showed that histone modifications are responsible for the development of the disease [17]. Aberrant DNA methylation, altered histone modifications, and chromatin modifications coordinate a reprogramming of immune T-cell response, dendritic cell function, macrophage activation, and a breach of airway epithelial barrier in early-life that commands asthma risk and severity in later life [18]. Epigenetic modifications are also responsible for altered gene expression that could predispose individuals to the diabetic phenotype during intrauterine and early postnatal development, as well as throughout adult life [10, 19, 20].

Several inherited disorders among humans are due to defect in genomic imprinting, and parent-specific monoallelic expression of a gene, such as Angelman’s syndrome, Prader–Willi syndrome, and Beckwith–Wiedemann syndrome [21]. Epigenetic changes can also introduce genetic instability and have a major role in the development of human cancer. There is an overall change in the DNA methylation pattern in cancers [22, 23]. For example, patients with sporadic colorectal cancers show methylation and silencing of the gene encoding MLH1a [2]. Epigenetic alterations due to DNA methylation by modifications in histone, such as histone methylation and acetylation are responsible for genomic instability and developing cancer [24, 25]. Furthermore, there are autosomal dominant disorders with overgrowth syndrome, like Sotos syndrome, caused by the change in the histone methylation pattern [26]. Recently, it has been found that autoimmune diseases may also be generated by several alterations in the epigenetic mechanism. For example, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) have global hypomethylation in the cells they target at promoter regions of DNA [27]. The overall summary of different diseases caused by the dysfunction of the epigenetic changes is given in table 1. These studies clearly implicate that epigenetic changes occur not only in infection and inflammation, but also in other types of diseases. In the following sections, the epigenetic machineries and their components that are responsible for causing different types of diseases, including infection and inflammation will be discussed in this review.

Table 1.

Important enzymes and associated diseases involved in epigenetic modification

Epigenetic modifiers	Enzyme responsible	Function	Epigenetic Modification	Diseases	Reference
						DNA methyl transferase	DNMT1, DNMTA, DNMT3B, DNM3L	Maintenance and de novo methylation	Transcriptional repression and activation	Rett syndrome,	14
DNA hyper methylation	Diabetes	20
Global hypo methylation, hyper methylation of promoter.	Cancer	22,23
Defect in genomic imprinting	Prader–Willi syndrome (PWS)	21
Histone acetyl transferase	HATs and HDACs	Histone acetylation and deacetylation	Reduced histone acetylation	Rubinstein – Taybai Syndrome	15
			Altered histone acetylation / deacetylation	Diabetes	20
			Reduced HDAC expression and activity	Asthma	13
			Reduced histone acetylation	Cancer	26
			Decreased acetylation of histones	Huntington’s disease	19
Histone methylation	HMTs and HDMs	Histone methylation and demethylation	Low level of histone methylation	Cancer	24
			Diminished level of methylation of histone	Sotos syndrome	26
			Hyper methylation of histones	Huntington’s disease	16

3. Epigenetics machinery

Epigenetic changes are defined as modifications of DNA or chromatin that do not involve alterations or deletion of the DNA sequence. Epigenetic modifications can be grouped into four main categories: DNA methylation, histone modifications, nucleosome positioning, and microRNA mediated (Figure 1). All of these modifications are functionally linked. DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of its function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable.

Figure 1. Key mechanisms for epigenetic modifications.

DNA methylation and histone modifications are the key epigenetic modifications that are equally influenced by environmental factors, diet, and inflammation or infection. DNA methylation occurs in CpG islands near the promoter regions of the genes and results in gene repression (heterochromatin). DNA demethylation also occurs in the CpG island results in gene activation (euchromatin). Histone modifications alter chromatin structure through acetylation (associated with gene activation) or deacetylation (associated with gene repression). The miRNAs binds to a specific 3′ untranslated region of an mRNA and inhibits its translation to protein. They are encoded within the genome, and their expression is regulated by other epigenetic mechanisms.

3.1 DNA methylation

DNA methylation, which involves methylation and demethylation of the cytosine residue in DNA, is the most widely known epigenetic modification. DNA methylation is an essential process important for development like embryogenesis, proliferation and differentiation [28]. This also plays a major role for the development of different germ line and somatic mutations that finally lead to the generation of different diseases including cancer [29]. DNA methylation occurs in CpG dinucleotides that are mostly associated near the promoters of genes, in the first exon, and near the 3′ end [29]. The CpG dinucleotides tend to cluster in regions called CpG islands [30] where mostly DNA methylation takes place. CpG island shores, a new concept, are highly conserved and differentially methylated regions which lie in close proximity (~2 kb) of CpG islands where most of the tissue-specific DNA methylation seem to occur [31]. It has also been seen that CpG island shores are the most (~70%) methylated regions involved during reprogramming [32]. CpG-island methylation is most commonly associated with gene repression, except in gene bodies where DNA methylation is associated with activation [33]. CpG islands remain unmethylated in normal active cells and germ line cells. However, there are some, ~6% of genes, that become methylated during the early phase of development or in differentiated tissues in a tissue-specific manner [34].

There are different mechanisms in which DNA methylation can directly block transcription by inhibiting the binding of specific transcription factors to its target sequence. DNA methylation also indirectly blocks transcription by recruiting methyl-CpG-binding proteins (MBD) and other repressive chromatin remodeling complexes to the methylated sites [21]. DNA methyltransferases (DNMTs) are enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine to DNA [35]. DNMTs are also associated with histone modifying enzymes such as histone deacetylases (HDACs), histone methyltransferases (SUV(39)H1/2 and EZH2), and ATP dependent chromatin remodeling enzymes (hSNF2H and LSH) [35]. Five members of the DNMT family have been reported in mammals: DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. Among them, only three enzymes namely DNMT1, DNMT3a, and DNMT3b, possess methyltransferase activity. DNMT3a and DNMT3b are considered as de novo DNMTs as they are catalytically active members of the DNMTs. Whereas, DNMT1 is a maintenance enzyme that methylates hemi-methylated CpG dinucleotides in the nascent strand of DNA after DNA replication and is transcribed during the S phase of the cell cycle. The functions of DNMTs are essential for maintaining DNA-methylation patterns in proliferating cells [5]. Defect in DNMTs has been implicated in multiple developmental abnormalities and are embryonically lethal at E14.5–18.5, demonstrating that de novo methylation is an essential process for mammalian development [36]. De novo DNMTs are highly expressed in embryonic stem (ES) cells and down regulated in differentiated cells [37]. DNMT3L, the third member of the DNMT3 family, is expressed during gametogenesis. Although catalytically inactive, it is required for establishing maternal genomic imprinting [38]. It also interacts and activates DNMT3a and DNMT3b and colocalizes with them in the nucleus [39]. Recently, small siRNA-mediated DNA methylation has also been described mainly in plants that recruit DNMTs to catalyze de novo DNA methylation of specific regions including not only gene promoters but also repetitive sequences but the exact mechanism is still unclear [40]. Further studies are needed to explore the role of different DNMTs in DNA methylation and gene regulation.

3.2 Histone modifications

Chromatin is the dynamic component of a cell in which DNA is wrapped around an octamer formed by the four core histones H2A, H2B, H3, and H4 to build a nucleosome [41]. Nucleosome is the fundamental unit of chromatin. DNA that is wrapped around the histone octamer in 1.65 turns is 147-bp segment in length and is separated from neighboring nucleosomes by ~50 bp of free DNA. Histone H1 is called the linker histone that binds to the linker DNA (DNA separating two histone complexes) and does not form part of the nucleosome [41]. The histone cores are mostly globular except for their N-terminal tails, which have no definite structure [42]. At the time of histone modifications, N-terminal tails of histones that protrude from their own nucleosome make contact with adjacent nucleosomes and undergo modifications that would affect the overall chromatin structure [43]. All histones are subjected to post-transcriptional or translational modifications. There are several post-transcriptional histone modifications that occur, including histone acetylation/methylation, phosphorylation, ubiquitination, SUMOylation, and ADP-ribosylation [44]. Histone modifications play key roles in most cellular functions that are involved in the manipulation and expression of DNA [41, 45]. Histones are therefore one of the important modifiers in epigenetic regulation. The different types of histone modifications will be discussed in the following subsections.

3.2.1. Histone acetylation/methylation

The chromatin present either into more loosen, and actively transcribed euchromatin or more tighten, transcriptionally inactive heterochromatin form. In general, euchromatin is characterized by high levels of acetylation and trimethylated H3K4, H3K36, and H3K79, and heterochromatin is characterized by low levels of acetylation and high levels of H3K9, H3K27, and H4K20 methylation [34]. Recent advance studies identified that histone modification levels are predictive for actively transcribed genes and are associated with high levels of H3K4me3, H3K27ac, H2BK5ac and H4K20me1in the promoter. Whereas, H3K79me1 and H4K20me1 are along the gene body [5]. In general, acetylation of histones is one of the important post-translational modifications where the lysine residue of histones is reversibly modified by two sets of enzymes–histones acetyl transferases (HATs) and histone deacetylases (HDACs).

The HATs use the cofactor acetyl CoA to catalyze the transfer of an acetyl group to the ε-amino group of lysine side chains of histones. This neutralizes the lysine’s positive charge and therefore loosens the interactions between histones and DNA. Thus, chromosomal domains are more accessible for the transcription factors to the gene promoters. Moreover, this unfolding of chromosomal domains also enhances the process of transcriptional elongation [46].

Two major types of HATs are responsible for histone acetylation: type-A and type-B. The type-B HATs mainly acetylate newly synthesized histone H4 at K5 and K12, but also certain sites within H3. However, they only acetylate free histones and not those already deposited into chromatin. Type-B pattern of acetylation is important for deposition of histones, is highly conserved, and generally found in the cytoplasm. Type-A HATs are more diverse and are classified into 3 groups according to amino acid sequence homology – GNAT, MYST, and CBP/p300 families [47]. Another important group of histone modifying enzymes, HDACs, are the predominant transcriptional repressor enzymes that act to reverse the effects of HATs and deacetylates lysine (K) to restores the positive charge of the lysine. There are four different classes of HDACs. Class I contain the enzymes HDACs 1, 2, 3, and 8, and class II contain the enzymes HDACs 4, 5, 6, 7, 9, and 10. Class IV has only a single member, HDAC11, while class III (referred to as sirtuins) are homologous to yeast scSir2. This latter class, in contrast to the other three classes, require a specific cofactor NAD+ for its activity [45]. Thus, the dynamic state of chromatin results from the crosstalk between HDACs and HATs. HATs and HDACs are therefore vital as they play a key-controlling role in developmental processes, and their deregulation has been linked to the progression of cancers (leukemia, colorectal and breast cancer), inflammatory diseases and different human disorders, like the Rubinstein–Taybi and Fragile X syndromes [48].

The next important histone modification is histone methylation. Histone methylation occurs on the side chains of lysine and arginine residues at histone tails of H3 and H4 [49]. Histone methyl transferases (HMTs) and histone demethylases (HDMs) are the two important factors that control or regulate DNA methylation through chromatin dependent transcription repression or activation. Histone methylation and demethylation modifications are essential and associated with either transcriptional repression or activation. This modification does not alter the charge of the histone protein, unlike acetylation and phosphorylation. In this modification, lysine may be mono-, di-, or tri-methylated, whereas arginine may be mono-, symmetrically or asymmetrically dimethylated [50]. Studies have shown that monomethylations of H3K27, H3K9, H4K20, H3K79 and H2BK5, and trimethylation of H3K4 (H3K4me3), H3K79me3 are responsible for gene activation, while H3K27me3, H3K9me3 are responsible for gene repression [51, 52]. It has been observed that histone methylation and other epigenetic regulation mechanisms are associated with aging and neurodegenerative diseases [53]. These studies further underscore that histone methylation is an important modification that takes place at all time.

3.2.2. Histone Phosphorylation

Primary sites for histone phosphorylation are the N-terminal tails, even though other regions within the core are also present. Histone tails are phosphorylated by different kinases and the phosphate groups are removed by phosphatases. This is a highly dynamic process that commonly takes place on serine, threonine, and tyrosine residues of histone tails [54]. As in the case of phosphorylation of H3Y41, which is phosphorylated by the non-receptor tyrosine kinase JAK2 [45]. Various cellular processes has been associated with phosphorylation of histones, including transcriptional regulation, apoptosis, cell cycle progression, DNA repair, chromosome condensation, and developmental gene regulation [55–57]. Phosphorylation of serine and threonine residues of the histone tails in general is associated with chromatin condensation during mitosis and meiosis. It has been found that C-terminal phosphorylation of Thr119 in histone H2A is linked to regulation of chromatin structure and function during mitosis [55]. Phosphorylation of H3 Ser10 (H3S10) has been observed – in chromatin compaction during mitosis. In addition, phosphorylation of H3S10 also plays a key role in transcriptional regulation of the NF-κB mediated pathway, including early genes like c-jun and c-fos [52]. Phosphorylation of H2A at Ser139 (γ-H2AX) is an important histone modification that plays a major role in DNA double-strand break by helping recruit DNA damage repair proteins to the site. Moreover, phosphorylation of γ-H2AX has been identified as one of the early events after DNA damage [58]. Thus, in parallel with other histone modifications, histone phosphorylation plays an important role in cells.

3.2.3. Ubiquitination and epigenetic modification

Histone ubiquitination was identified 28 years ago [59], but remains one of the least understood histone modifications. Until recently, it has been known that ubiquitination is a process that regulates a variety of cellular events, including protein degradation, stress response, cell-cycle, protein trafficking, endocytosis signaling, and transcriptional regulation [60]. Now, research on the role of ubiquitination on gene regulation is being considered as an important epigenetic modification and a new field of study. Ubiquitin is a 76 amino acid protein that can either direct the conjugated protein to proteasomal degradation or act as a modifier for protein function. Typically, ubiquitin receptors recognize a protein that is tagged with ubiquitin and remove it from the cell through the proteasomal degradation pathway. Otherwise, it modifies protein function by three-step enzymatic reaction, involving ubiquitin-activating, -conjugating and -ligating enzymes E1, E2 and E3, respectively. These enzymatic reactions conjugate either one (monoubiquitination) or multiple (polyubiquitination) ubiquitin moieties on the target polypeptides or the lysine residues in which ubiquitin attaches, depending on the substrate-specific combination of used E2 and E3 [61, 62]. More recent studies have revealed that reversible ubiquitylation of histone H2B by E2 and E3 has been associated in transcriptional activation and gene silencing. Ubiquitylation and subsequent deubiquitylation of H2B are both required for transcriptional elongation of certain stress-inducible genes, and removal of the ubiquitin tag from H2B enables the establishment of telomeric silencing through the association of Sir factors along with chromatin [63]. During transcription, ubiquitination of H2A is generally associated with gene silencing whereas ubiquitination of H2B has been related to both gene activation and silencing [61]. Ubiquitination of H3 and H4 are less abundant and as yet, no functional consequence has been assigned to this modification. It has been reported that murine HDAC6 is associated with two proteins, p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3) is involved in ubiquitination. A zinc-finger domain at the C-terminal of HDAC6, called “ubiquitin carboxyl-terminal hydrolase-like zinc finger” (ZnF-UBP) domain, signifies a possible link between histone acetylation and histone ubiquitination [64]. Moreover, studies have shown that H2B ubiquitination is upstream of H3-K4 methylation [65]. Aberration in the ubiquitin system plays a significant role in neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s disease, although the detailed role of epigenetic changes mediated by ubiquitination is poorly understood.

3.2.4. SUMOylation

SUMOylation is the attachment of a small ubiquitin-related modifier (SUMO) moiety of around 11-kDa protein, covalently present at specific lysine residues in a variety of target proteins. SUMOylation cause modulation of protein functions by changing its stability, localization, or interaction with other proteins and has important roles in cell proliferation, differentiation, and apoptosis [66]. SUMOylation is an enzyme-catalyzed, multistep, reversible process that specifically targets proteins harboring a SUMO interaction motif [67, 68]. SUMOylation takes place mostly in transcription factors or transcription regulators and leads to transcriptional repression [69, 70]. A number of transcriptional repressors, such as the histone deacetylases HDAC1, HDAC4, HDAC6, and HDAC9 and the repressor C-terminal binding protein (CtBP), have shown to be subjected to sumoylation [71]. However, the precise molecular mechanisms of these repressions are not well known. In rheumatoid arthritis (RA), there is evidence that SUMO is overexpressed in synovial tissue and synovial fibroblasts (SF) [72]. Recent studies have shown that de-SUMOylation in rheumatoid arthritis synovial fibroblasts (RASFs) decreased the levels of histone acetylation, which later cause a reduction in the expression of certain matrix metaloproteinases and interleukins, thereby reducing the destructive potential of the fibroblasts [73]. Another study also showed that telomeric silencing in S. cerevisiae has been associated with decreased levels of acetylation where sumoylation of histones may enhance its silencing effect [74]. Sir2, one of the members of the conserved sirtuin family of NAD+-dependent histone deacetylase undergo sumoylation at several lysine residues by SUMO ligase Siz2, which therefore affects its function in telomere silencing [75]. Taken together, although there are some studies in this area, still it needs a better understanding of the underlying epigenetic impact of these modifications.

3.2.5. ADP-ribosylation

ADP-ribosylation of proteins is a cellular process in which the reversible post-translational modification of proteins are catalyzed by poly-ADP-ribose polymerases (PARPs), a family of nuclear enzymes that transfer and polymerize ADP-ribose units from NAD+ on to a variety of nuclear proteins to form a branched polymer known as poly-ADP-ribose (PAR). ADP-ribosylation therefore, plays an important role in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling. In recent years, it has been found that ADP-ribosylation also has nuclear functions, including histone modification, transcriptional regulation, DNA repair pathways, maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis [76]. Until recently, there is less information on the direct regulation of epigenetic modifications by ADP-ribosylation. A growing body of evidence supports a role for PARP in heterochromatin organization through the establishment and maintenance of DNA methylation patterns. Few studies have shown that binding of PARP1, a major PARP, to the broken DNA ends induces significant PARylation, which results in recruitment and modification of the chromatin-associated proteins and therefore, changes the chromatin microenvironment [77]. Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein that is greatly abundant within the brain and mutation of which causes the neurodevelopmental disorder Rett syndrome. This MeCP2 protein undergoes different post-translational modifications and recent studies have shown that it also undergoes polyADP-ribosylation mediated modifications. MeCP2 can bind to both the methylated and non-methylated regions of chromatin [78], although the exact molecular mechanism of this interaction is still unclear. Another study has revealed that endogenous MeCP2 from mouse brain shows polyADP-ribosylation, and deletion of the domains responsible for polyADP-ribosylation results in increased heterochromatin clustering. Furthermore, it was found that PARP1 deficiency increases the ability of MeCP2 to aggregate and to bind to pericentric heterochromatin [79]. A recent study has shown the role of ADP-ribosylation as an element of the histone code. Mass spectrometry and electron-transfer dissociation studies showed that K13 of histone H2A, K30 of H2B, K27 and K37 of H3 as well as K16 of H4 as ADP-ribose acceptor sites catalyzed by PARP1 [80]. Studies also showed that PARP1-dependent ribosylation of histones influences their subsequent methylation pattern, with the inhibition of H1 histone [81]. These data illustrate the possible role of ADP-ribosylation mediated chromatin modifications.

3.3 Nucleosome positioning

The nucleosome is the smallest unit of chromatin that takes part in DNA packaging as well as regulation of gene expression. Nucleosomes are a barrier to transcription, which impairs the binding of transcription factors to the DNA, as well as inhibit the transcriptional elongation by polymerases. In particular, the precise position of nucleosomes around the transcription start sites (TSSs) is important and influences the initiation of transcription. Promoter regions, just upstream of TSS at the 5′ end of a gene are mostly devoid of nucleosomes by poly-dA/dT sequences and are called “nucleosome free regions” (NFRs). NFRs are associated with active regulatory elements like enhancers, insulators, and silencers are profoundly important for transcription initiation. In the 5′ NFR, there are two highly positioned nucleosomes, termed the −1 and +1 nucleosomes, which are precisely localized. The −1 nucleosome is located upstream of the NFR, whereas +1 nucleosome is located downstream of the NFR in the yeast genome. Downstream of the +1 nucleosome, nucleosomes are less precisely localized and beyond ~1 kb from the TSS, there is an increased tendency for random nucleosome positions [82]. Nucleosomes strongly localized at the downstream of TSS at the 5′ end of a gene causes gene suppression [83]. If nucleosomes are displaced by even 30 bp at TSS, there are changes implicated in the activity of RNA polymerase II. Like 5′ ends, 3′ ends are also marked by distinct nucleosome positioning as well as an NFR region at the 3′ end. The 3′ NFR specifies the position where RNA polymerase II terminates transcription ([82]). Importantly, nucleosome positioning can also influence DNA methylation because DNMTs preferentially target nucleosome-bound DNA [27]. Methylated DNA is associated with condensed heterochromatin domains, where nucleosomes are strictly positioned, and thereby obstructing transcription. Recent studies have shown the connections between nucleosome positioning, DNA methylation, and other epigenetic factors during gene regulation [84]. Given above studies suggest that nucleosome positioning play an important role during gene regulation along with other epigenetic factors.

3.4. MicroRNA (miRNA)-mediated epigenetic modification

MiRNAs are non-coding RNAs, endogenously encoded in the genome and the size ranges from 17–25 nucleotides. The detailed biosynthesis of miRNA processing and gene regulation was previously discussed [85]. Sequential miRNAs processing have an important role in gene regulation and has done either by mRNA degradation or by translational repression [86]. MiRNAs are mostly located in the CpG island and therefore, are likely to be controlled by DNA methylation machinery. For example, miRNA like miR-127 was found to be expressed more in cancer cell lines after the treatment with Aza and 4 phenyl buteric acid (PBA) which lower DNA methylation levels and therefore open the chromatin structures to induce the expression of genes that had been silenced epigenetically [87]. It has been found that in colorectal cancer, there is an increased expression of miR-143 and reduced DNMT3A levels. Meanwhile, in lung cancer, the expression of miR-29 is increased and DNMT3B levels are decreased. In both cases, there is decreased cell growth and colony formation [88]. Another recent study has shown that miRNA-34b targets DNMT1, HDAC1, HDAC2 and HDAC4 in prostate cancer cell lines. More interestingly, miR-34b itself is epigenetically silenced by DNA methylation. The over expression of miR-34b in prostate cancer cells resulted in partial demethylation of miR-34b gene at an 5′ upstream sequence, as well as showed enrichment of trimethylated histone H3 lysine 4 (H3K4me3) mark for an active chromatin [89].

Regarding inflammation, a recent study has shown that miRNA 199 was found to be hyper methylated in chronic obstructive pulmonary disorder [90]. Furthermore, in obesity and metabolic diseases associated inflammation, the genes are epigenetically regulated by miRNAs. Similar to other diseases, miRNA primarily down regulates genes associated with proinflammatory pathways. For example, miRNA-146b-5 is decreased in monocytes from obese and type 2 diabetic mellitus patients. It has also been found that toll-like receptor 4 (TLR4)-induced inhibition of miRNA-107 is impaired in obesity, resulting in an increased inflammatory response in macrophage [91]. Moreover, miRNA-146a confines TLR-mediated signaling by blocking the signaling molecule TRAF6, whereas miRNA-155 down regulates the lipid phosphatase SHIP1, responsible for macrophage activation. MiRNA-132 binds to acetylcholine (ACH) mRNA and exerts its anti-inflammatory effects by reducing peripheral inflammation. Macrophages in culture when exposed to LPS leads to up-regulation of miRNA-155, which targets the mRNA for CCAAT/enhancer binding protein Beta (C/EBP Beta), is implicated in the regulation of pro-inflammatory cytokines during acute macrophage activation [92]. All of these studies therefore confirm that miRNA mediated epigenetic regulation of genes play an important role not only in cancer but also in inflammatory diseases.

3. Epigenetic mechanisms of infection and inflammation

The epigenome, the overall epigenetic state of an organism, is just as important as the genome to normal development. Importantly, environmental factors such as nutrients, pollution, toxins, infections, and hypoxia can have profound effects on the epigenetic signature and trigger susceptibility to diseases [92, 93] (Figure 2). For example, recently it has been found that the in-utero environment can also change the epigenome, with long-term adaptable phenotypes for gene regulation and age-associated diseases [94]. The studies by Bobetsis et al. showed that periodontal infection can lead to placental-fetal exposure and when coupled with fetal inflammatory response, leads to preterm delivery [95].

Figure 2. Environmental factors and epigenetic modulation in humans.

The figure demonstrates a sum of the various sources presents in the environment that regulates epigenetic parameters on humans. Among them, some might be advantageous for body health and performance such as, exercise, healthy dietary practice, others, like toxic chemicals, stress and misuse of drugs might be detrimental and impede body’s balance that manifest as a disease or psychological disorder. Long-term exposure to inflammation and immune system of the body could also trigger epigenetic alterations.

3.1. Epigenetics of environment associated infection and inflammation

Genome-wide epigenetic marks of DNA as well as chromatin modifications do not persist throughout life, but undergo defined, synchronized changes during different stages of development, predominantly in mammals. These alterations lead to the lineage- and tissue-specific expression of genes [96, 97]. Mostly in mammals and occasionally in plants, there is a change in epigenetic pattern after birth. Besides the developmental ontogeny of epigenetic modifications, the variation in epigenetic pattern is thought to be largely because of extrinsic or environmental factors and intrinsic factors, but their relative contributions remain largely unknown [98]. Recently, the possible impact of the environment on epigenetic regulation has drawn significant interest. To date, most studies done in ‘environmental epigenetics’ has been on DNA methylation, which is essential in development and is involved in processes such as genomic imprinting and silencing transposable elements [96, 99]. However, the role of environment in the change of the epigenome is not very straightforward. Developmental and pathological phenotypes could change the measured levels of epigenetic modifications at specific loci. Studies have been done to explore the role of environmental factors and diet in the regulation of genes by DNA methylation and covalent histone modifications. All of these studies are more nascent and work is still going on to understand the mechanism. Few experiments have been done to understand the role of stress on epigenetic changes and found that when laboratory rodents experience prenatal stress, including maternal separation, maternal care, abusive caregiving in infancy, juvenile social housing, and adult social stress, there are variations in DNA methylation and histone modification [100].

Epigenetic states are also modified by the emotional health of an individual as studies were done with psychosis, psychotic disorders, and severe depression [101]. Anti-androgenic compound ‘vinclozolin’, an anti-fungal agent used in vineyards or chemical methoxychlor, an oestrogenic compound used as a pesticide, can cause reproductive abnormalities in laboratory animals. These groups of chemicals also have the potential to cause cancer in humans as well as reproductive abnormalities [102]. Several other human population studies report that the nutritional status of grandparents can have phenotypic consequences in their grandchildren. These trans-generational effects are not because of mutations but could be related to epigenetic inheritance [103, 104]. An effect of diet on the methylation status of DNA and thereby on the phenotype in humans comes from a recent study on patients with hyper-homocysteinaemia where there is an unbalanced DNA methylation status. Normal methylation is restored at the imprinted IGF2-H19 locus when folate is included in the diet because folate is needed for the methionine cycle, and therefore, for the synthesis of S-adenosylmethionine, the methyl donor for DNA methylation. Decrease of folate in the cells lead to an increase in S-adenosylhomocysteine, which is a powerful inhibitor of S-adenosylmethionine-dependent methyltransferases like DNMT1- and makes the cell have an increased level of methylation. [104, 105].

Recently, it has been reported that exercise has a positive effect, and a six-month exercise program can influence the genome wide methylation pattern in adipose tissue affecting adipocyte metabolism [106, 107]. Exposures to early life risk factors like gestational diabetes, maternal smoking, and maternal obesity are putative early-life predictors of adult cardiovascular disorders associated with epigenetic changes [108]. Recent studies depict that environment plays a crucial role in the global or local epigenetic changes in immune cells that are frequently observed in obesity and type-2 diabetes mellitus, which further perpetuate with changes in the phenotype and function of these immune cells. Diseases like obesity and metabolic disorders are linked to environmental factors in addition to the genetic factors. Furthermore, studies have shown that the environmental factors play a significant role in the expression of genes related to metabolic, endocrine and neural functions and therefore, reprogram immune cells to the diseases [91]. Taken together, studies discussed above suggest that environmental factors serve a potential role in epigenetic regulation of genes during different disease conditions.

3.2. Epigenetics of microbial mediated inflammation

Inflammation is a complex set of physiological responses to different noxious stimuli like infection, trauma, injury, toxins, and other imbalances [109]. An acute inflammatory response is associated with the recruitment of leukocytes, including the mast cells followed by macrophages from the blood to the damaged sites [109]. Whereas, in chronic inflammation, the condition is persistent and underlies many diseases, including periodontal disease and diabetes mellitus [110].

Macrophages are therefore one of the critical regulators in the inflammatory responses. Immediately after the microbial invasion, microbial products signal through extracellular and intracellular pattern-recognition receptors (PRRs) or sensors (e.g., Toll-like receptors, or TLRs, and nitric oxide dismutase or NOD proteins), which recognize small molecular sequences referred to as “pathogen-associated molecular patterns (PAMPs)”. Thus, signaling pathways are triggered which initiate inflammatory activation to produce histamine and pro-inflammatory cytokines like IL-1, TNFα, and IL-6. Inflammatory genes that encode for cytokines, chemokines, and other molecules responsible for inflammation that are found to be regulated by pro-inflammatory transcription factors, including nuclear factor kappa B (NF-κB) and activator protein (AP)-1, FOXP3, IRF, and STAT families. Transcription factors along with the inflammatory genes are regulated by epigenetic mechanisms including DNA methylation, histone modifications, and RNA-associated silencing by small non-coding RNAs [92, 111]. Thus, the body undergoes profound alterations in systemic homeostasis to support host defense.

3.3. DNA Methylation and Inflammation

DNA methylation is also an important mechanism of regulation of inflammatory genes. Recent studies have revealed that LPS cause down regulation of Thy-1 gene expression by activating HDACs via TLR4- signaling [112]. In disease like cystic fibrosis, TLR2 gene undergoes promoter hypo methylation, which is associated with enhanced pro-inflammatory response to bacterial peptidoglycan by the bronchial epithelial cells [113]. Intestinal homeostasis is maintained by methylation of DNA and acetylation of histone regulates TLR4 in intestinal epithelial cells [114]. Epigenetic modifications including DNA methylation and histone modifications play an important role in the regulation of TNFα and therefore inflammation associated with it [115]. Bacterial infections can alter the epigenetic machinery and therefore can alter the status of the genome which contribute host defense or pathogen load [116]. Microarray studies in mice explained that maternal health including infection by both bacteria ad virus contributes to changes in expression of key developmental genes in the placenta and therefore, linked to the development of the offspring [117]. Infection by Helicobacter pylori (HP) causes inflammation and induces alterations in the DNA methylation pattern in gastric epithelial cells showed that HP-induced DNA methylation in the Runx3 locus causes loss of expression in gastric epithelial cells responsible for the development of the disease [118].

3.4. Histone Methylation in Inflammation

Regulation of gene expression of CD4+ T cells and CD8+ T cells are primarily done by histone methylation, including H3K4me3, H3K9me2, H3K9me3, and H3K27me3 [119, 120]. Studies revealed regulatory regions of gene loci in T cells undergo dynamic and complex changes in histone methylation during their differentiation to distinct subsets of effector T cells [121]. Studies have shown that Jmjd3, a member of the Jumonji family, is expressed in macrophages when exposed to pathogens and pro-inflammatory cytokines, where it activates the polycomb-group (PcG) target genes and regulates their H3K27me3 levels and transcriptional activity. Some of the PcG proteins responsible for inflammation are subjected to aberrant DNA methylation. PcG proteins bind to the regulatory regions of target genes and recruit DNMTs for more efficient repression. Inflammation during sepsis also causes NF-κB/RelB- pathway mediated repression of pro-inflammatory genes inducing H3K9me3 [122]. Jmjd3 erases histone marks and has been found to regulate differentiation and cell identity in macrophages, thereby controls inflammation by reprogramming of the epigenome. Continuous exposure to IL-4 activates Jmjd3 and the release of H3K27me3 repressive marks from the STAT6 promoter. Activated STAT6 then positively regulates Jmjd3 by binding to its promoter. Removal of H3K27 methylation marks by Jmjd3 trigger expression of specific inflammatory genes [123]. These studies clearly indicate the dynamic role of histone methylation during inflammatory response.

3.5. Histone Acetylation and Inflammation

Genes are activated by the acetylation of histone by HATs and repressed by the activity of HDACs during inflammation caused by different agents like LPS, IL-1b, TNFα, etc. Many of these stimuli act through induction of pro-inflammatory transcription factors such as AP-1 and NF-κB, but other intracellular kinase pathways such as MAPKs and PI3K are also important [124]. Recent reports have shown the role of HDACs in the regulation of pro-inflammatory genes. For example, corticosteroids switch off inflammatory genes in asthma by the recruitment of HDAC2 to the promoter region NF-κB stimulated inflammatory gene complex and inhibition of HAT activity. There is a reduction in HDAC activity and HDAC2 expression in chronic obstructive pulmonary disease (COPD), a corticosteroid insensitive disease, where amplified inflammation and resistance to the actions of corticosteroids were observed [18]. CBP/p300 (cAMP-response element binding (CREB)- protein) has the intrinsic HAT activity and also recruit other HATs in the acetylation of histones at the promoter region of inflammatory genes (IL-1, IL-2, IL-8, and IL-12), thus regulating gene expression via transcriptional activation as well as displayed decreased HDAC activity. Transcription of both pro- and anti-inflammatory cytokines are regulated by the recruitment of HDACs to gene promoters via co-repressor complexes and transcription factors such as FOXP3, STATs, GATAs, ZEB1, and NF-κB [125]. In response to cytokine treatment, IKK-α, a member of IκB kinase complex binds to the NF-κB promoters with the aid of polymerase II and CBP, where it acetylates histone H3 at Lys9 and phosphorylates histone H3 at Ser10. [126, 127]. This is further responsible for the subsequent CBP-mediated acetylation of histone H3 on Lys14 ([128]. The p38 protein plays an important role in the induction of inflammatory and immune responses by enhancing the H3 phosphorylation marked promoters and thereby, enhance the recruitment to selected chromatin targets [129].

4. Clinical relevance of Epigenetic Modifiers

With the advancement of new diagnostic tools, it reveals that there are many diseases that are caused by epigenetic modifications. Development of ‘epigenetic therapies’ now has the immense potential towards the treatment of many diseases including cancer, cardiovascular disorders, and also inflammation. Recently, many agents have been found to modify epigenetic patterns by changing DNA methylation patterns or cause modification of histones. Many of these agents are currently being tested in clinical trials [2].

Two general types of HDAC inhibitors are commonly used for treatment, pan inhibitors that are broad acting and inhibitors that target a specific class of HDAC enzyme. DNA methyltransferase (DNMT) inhibitors, 5-azacytidine (clinical name Vidaza) and 5-Aza-2′deoxy-5-azacytidine (clinical name decitabine) are pan inhibitors and are now used to increase fetal hemoglobin production by causing hypo methylation of γ-globin genes in patients with sickle cell disease [130]. HDAC inhibitors have been found to be – a novel category of anti-cancer drugs. To date, the United States Food and Drug Administration have approved four HDAC inhibitors, Vorinostat, Romidepsin, Panobinostat, and Belinostat. More specifically, these HDAC inhibitors have the least severe side effects and are mostly used for hematologic cancers. More clinical trials are continuously expanding to address other types of cancer and also nonmalignant diseases. HDAC inhibitors are also used in various types of neurodegenerative diseases, inflammation disorders, and cardiovascular diseases [131]. Similar to Aza, Zebularine is another cytidine analog that inhibits DNMT1 activity. Although not yet FDA approved, this isoform has found good results in mouse models [132].

Three naturally occurring small molecules such as curcumin, anacardic acid, and garcinol have been found to have HAT inhibitory activity. Curcumin, the active ingredient in turmeric, is in phase II trials in advanced pancreatic, breast, and colorectal cancer (NCT00094445, NCT01740323, NCT01490996). HMT and HDM inhibitors, such as BIX-01294, chaetocin, and UNCO224, DZNep, are primarily in a preclinical stage of development. However, the HDM inhibitor phenelzine sulfate, a monoamine oxidase A inhibitor, is in phase II trials in combination with docetaxel in prostate cancer (NCT01253642) (Figure 3, 4). Recent studies have suggested that epigenetic modulation with chromatin-modifying agents can induce stemness and dedifferentiation and increase developmental plasticity. For instance, valproic acid (VPA), an HDAC inhibitor, has been shown to promote self-renewal/expansion of hematopoietic stem cells and facilitate the generation of induced pluripotent stem cells (iPSC) [133]. More recent studies show that the epigenetic reprogramming of adult somatic cells into pluripotent stem cells could be a safe method to generate iPSCs and may provide an attractive source of stem cells for regenerative medicine [134]. Recently, studies have shown that combinations of epigenetic modifiers are also found to play an important role in the reduction of inflammation. A single dose of combinatorial administration of Aza+TSA after the onset of acute lung injury (ALI) has been found to significantly attenuate lung vascular hyper permeability and inflammatory lung injury. Moreover, the combinatorial treatment with Aza+TSA reduces inflammation and promotes anti-inflammatory M2 macrophages during ALI [135, 136]. These two studies clearly demonstrated that epigenetic modifiers have a therapeutic potential for patients with sepsis-induced vascular injury and inflammation.

Figure 3. Clinical trials in aberrant DNA methylations associated with disease outcome.

This diagram represents the drugs mostly used in different phases of clinical trial that are used to alter DNA methylation associates with diseases in an individual. Aberrant DNA methylation is associated with gene repression in the promoter and activation in the gene body results in the manifestation of various diseases, including a variety of cancers and neurodegenerative diseases. Several molecules have been screened for altering the DNA methylation status associated with the disease outcome and are currently in different phases of clinical trial.

Figure 4. Clinical trials in aberrant histone modifications associated with disease outcome.

Histone modifications by HATs lead to active chromatin state associated with the activation of different genes, including inflammatory genes or genes responsible for neuronal functions. HDACs are responsible for repressed chromatin and therefore, transcriptionally inactive state of genes. Aberrant expression of HDACs might also shut down different essential genes responsible to maintain normal function of the body and thereby results with diseased state. Thus, various small molecules and drugs are now under screening and trials to use them clinically to modify the altered epigenetic state and reduce the diseased condition.

5. Summary and Future perspective

Previously for several decades, genetics was key to understand human diseases, but in recent years, epigenetics has taken the most important place to describe human diseases where environment, lifestyle, and diet play important roles in epigenetic modulation. Changes in the epigenetic landscape give rise to several significant human diseases, and the focus is to find therapies that can reverse gene expression. But despite the promise of epigenetic therapy, the clinical use of these agents may cause the nonspecific activation of genes and transposable elements in normal cells, as well as potential mutagenicity and carcinogenicity are the major concerns regarding their clinical use. Still, it is good that trials are mainly on life-threatening diseases like cancer where the target is to hit abnormally methylated CpG island with a single drug. Most commonly, drugs used in the clinical trial are inhibitors of DNMTs and/or HDACs, which has a broad spectrum of inhibition rather than specific activity. This could result in wide range change in the epigenetic pattern that could also upregulate several other genes including diseased genes or proto-oncogenes. Therefore, it is important to focus on individual inhibitors that will have more specific inhibition of a particular gene or a set of genes that will result in a more specific inhibition. At the end, methylation of CpG islands increases with age and could be important for the development of chronic diseases like inflammation in addition to cancer. Drugs and lifestyle changes could bring reversionor slow gradual epigenetic silencing.

6. Concluding Remarks

The study of epigenetics, therefore, has profound clinical significance. Understanding the role of epigenetic aberrations in disease progressions still is primitive. Future studies are needed to understand the relationship between epigenetic mechanisms, gene expression and the environment, and more emphasis is to be given on clinical trials of novel epigenetic therapies directed at a wide variety of diseases. Moreover, it is important to decrease the risk of epigenetic instability and epigenetic abnormalities that may be caused by broad-spectrum inhibitors later in life. Therefore, recent studies are focusing on identifying disease specific inhibitors rather than pan inhibitors. The promise for epigenetic therapies specific for diseases is as an essential treatment goal needed to be fulfilled. In this review, we have summarized the investigations that are critical to understand the complex epigenetic mechanisms to provide new ways for treating diseases associated with infection and inflammation.

Highlights.

• Epigenetic state plays an important role in disease manifestation.

• DNA methylation, Histone modification, nucleosomal positioning and miRNA, mediate major epigenetic regulation.

• Environmental factors are also responsible for associate alteration of the epigenetic state.

• Targeting the epigenetic machinery is a newer approach to change the altered epigenetic state.

Abbreviations

Aza

5-Aza 2 deoxycytidine

TSA

Trichostatin A

MeCP2

Methyl CpG binding protein 2

SLE

Systemic lupus erythematosus

RA

Rheumatoid arthritis

DNMTs

DNA methyltransferases

HDACs

Histone deacetylases

HAT

Histones acetyl transferases

HMT

Histone methyl transferases

HDM

Histone demethylases

MLH1

MutL homolog 1

CBP

CREB-binding protein

RASF

Rheumatoid arthritis synovial fibroblasts

PBA

Phenyl butyric acid

FOXO3

Forkhead box O3

IRF1

Interferon regulatory factor 1

STAT

Signal transducer and activator of transcription

LPS

Lipopolysaccharide

PcG

Polycomb-group proteins

MAPK

Mitogen-activated protein kinases

PI3K

Phosphatidylinositol-4,5-bisphosphate 3-kinase

ZEB1

Zinc finger E-box-binding homeobox 1

SUMO

Small ubiquitin-related modifier

TSS

Transcription start sites

NFR

Nucleosome free region

PRRs

Pattern-recognition receptors

PAMP

Pathogen-associated molecular patterns

NOD

Nitric oxide dismutase

APR

acute phase response

NF-κB

Nuclear factor kappaB

AP

Activator protein

Jmjd

Jumonji family

COPD

Chronic obstructive pulmonary disease

PARP

Poly-ADP-ribose polymerases

ACH

Acetylcholine

TLR4

Toll-like receptor 4

EBP

Enhancer binding protein

TNFα

Tumor necrosis factor alpha

HP

Helicobacter pylori

CREB

cAMP-response element binding

VPA

Valproic acid

iPSCs

Induced pluripotent stem cells

ALI

Acute lung injury