Inflammatory bowel disease (IBD) is comprised of 2 major disorders: Crohn's disease (CD) and ulcerative colitis (UC). IBD results from a continuum of complex interactions between host-derived and external elements that involve various aspects of the intestinal microbiota, the immune system, the genetic composition/susceptibility of the host, and specific environmental factors (e.g., diet, smoking, stress, and hygiene).1–3 Epigenetics is one of the most rapidly expanding fields in biology, interacting with genetic and environmental factors in affecting the immune system (Fig. 1). Epigenetics may be defined as changes in phenotype that persist through mitosis and even meiosis, but occur independently of changes to the underlying DNA sequence. Consequently, epigenetics is generally understood to be the study of mechanisms that control gene expression in a potentially heritable way.4 Complex epigenetic states are orchestrated by several converging and reinforcing signals, including transcription factors, noncoding RNAs, DNA methylation, and histone modifications.5 Great progress has been made in the description of epigenetic modifications in human health and disease. These advances have provided new insights into the role of epigenetic modifications in cancer, neurodevelopmental disorders, neurodegenerative and neurological diseases, and in autoimmune diseases.4 Epigenetic alterations are likely to be found in other human diseases. It was proposed, more than a decade ago, that inherited and/or acquired epigenetic marks may be of etiological and pathogenic importance in IBD.6 Many laboratories have worked on the problem, and some excellent reviews have appeared during the last 2 years that cover the emerging role of epigenetics in IBD.7–14 The fundamental principles of epigenetic modifications and their molecular machineries relevant to IBD have recently been described in detail.4,7,8 We therefore only summarize key features and new insights of relevant epigenetic modifications. We provide an overview of the epigenetic control of inflammation and, in particular, the epigenetic regulation of the differentiation and function of key effector cells of the innate and adaptive immune system. The article also focuses on recent advances in the understanding of the epigenetic control in IBD, which includes DNA methylation, histone modifications, and interplay with intestinal microbiota. Recent advances on the role of epigenetics in colitis-associated cancer (CAC) will also be discussed. Finally, we outline clinical and therapeutic implications and providing discourse on the challenges, perspectives, and outstanding questions for the emerging role of epigenetics in IBD.
CONCEPT AND PRINCIPLES OF EPIGENETICS
The term “epigenetics” was formally defined by Waddington15 as “the causal interactions between genes and their products, which bring the phenotype into being.” Waddington used the metaphor of an “epigenetic landscape” to articulate the concept of molecular mechanisms that can reinterpret the invariant genetic code into a multitude of different phenotypic outcomes.16 The modern concept of epigenetics refers to the heritable marking of DNA that leads to the alteration of gene expression independently of genetic information carried by the primary DNA sequence.
One of the earliest challenges was to elucidate how epigenetic mechanisms extract contrasting phenotypic identities from the same genetic code? It was evident that the DNA sequence must carry another layer of heritable information, although the corresponding molecular principles were not understood at the time. Epigenetic marking, or modification, of DNA is now universally accepted as the underlying mechanistic basis to this phenomenon. Epigenetic modifications possess the fundamental properties required to variably influence phenotype in a transmissible manner. They are heritable (i.e., mitotically stable) allowing their retention through successive cell divisions and have the capacity to directly, or indirectly, alter the transcriptional status of the underlying DNA sequence. Most importantly of all, perhaps, epigenetic modifications are fully reversible, allowing their erasure and subsequent reestablishment on passage through the parental germlines (although as we discuss below, incomplete “resetting” of the epigenome could explain the intergenerational inheritance of some adverse phenotypes). DNA methylation, histone posttranslational modifications, and nucleosome positioning are the best-characterized epigenetic modifications. However, during the past decade, roles of chromatin remodeling complexes, the much-vaunted polycomb group proteins17 and micro RNAs (miRNAs)18 among others, have risen to prominence as critical modifiers of epigenetic modifications. Collectively, epigenetic modifications and their respective modifiers co-ordinate a multitude of molecular functions including gene transcription,19 DNA-protein interactions,20 protein translation,21 and silencing of endogenous retrotransposons.22,23 Through these functions, epigenetic modifications act deterministically on key developmental processes including growth, cellular fate and differentiation, immunity, X-chromosome inactivation, and genomic imprinting.
EPIGENETIC SYSTEMS AND DISEASE MODELS
The overriding significance of epigenetics in the maintenance of normal biological functions is highlighted by the fact that human diseases often develop when epigenetic marks are incorrectly established, or are established at inappropriate times or locations. For example, monozygotic twins carry the same genetic code but, nonetheless, display discordant DNA methylation and histone modification profiles, which may alter the penetrance of, or susceptibility to, cancer and autoimmunity.24–26 The inherent plasticity and reversibility of epigenetic modifications, although highly desirable in the cellular context, also renders them vulnerable to alteration by environmental stimuli. Accordingly, epigenetic modifications are potential mediators of gene-environment interactions underlying complex multifactorial diseases, including IBD.27 Environmental factors known, or suspected, to have epigenetic effects include nutrition, stress, chemical exposure, pharmaceutical agents, and inflammation (reviewed in Ref. 28). Data from both humans and animal models suggest that environmental factors may elicit phenotypes associated with specific epigenetic modifications that can be inherited transgenerationally.29–34 Epigenetic modifications may therefore exert a profound influence on the pathogenesis of IBD by connecting host gene function to known environmental risk factors including intestinal microbiota.
Epigenetics may not only play a role in IBD pathogenesis, but may also determine the outcome of specific disease sequelae such as CAC35,36 discussed in detail in a subsequent section of this article. Nonetheless, the strong link between epigenetics, growth deregulation, and cancer is exemplified here by reference to the phenomenon of “genomic imprinting,” which, to date, remains one of the most informative epigenetic paradigms of human disease.37,38 The existence of imprinting came from seminal nuclear transplantation studies in mice, showing that reconstituted diploid embryos derived from 2 paternal or 2 maternal pronuclei are not viable.39,40 Therefore, despite carrying identical genetic information and residing within the same nucleus, the parental genomes are functionally nonequivalent because of differential marking by epigenetic modifications. An explanation for this form of noncomplementation was subsequently attributed to the existence of imprinted genes, which are differentially expressed or silenced in a parent-of-origin–dependent manner.41 This form of monoallelic expression is governed by the acquisition of differential epigenetic marks in the gametes, which are maintained and appropriately interpreted by the transcriptional machinery in somatic cells. Imprinted genes play pivotal roles in mammalian development with particular emphasis on the regulation of prenatal growth, postnatal behavior and metabolism.42–47 However, the unusual expression of imprinted genes from only 1 of the 2 parental alleles effectively renders them functionally haploid. Consequently, imprinted genes are especially vulnerable to a form of misregulation known as “loss of imprinting,” which manifests either as the inappropriate reactivation of the normally silent allele, causing “biallelic” overexpression, or silencing of the active allele, leading to loss of function. Because imprinted gene products are exquisitely sensitive to changes in dosage, their altered expression through loss of imprinting can lead to a number of growth disorders in humans, including cancer.48–53 Evidence for parent-of-origin effects on familial IBD transmission has emerged, suggesting a potential involvement of imprinting.54,55 Moreover, imprinting effects on the IBD susceptibility loci, NOD2, PRDM1, and IL12B were recently reported, although the influence of ethnicity was noted.56 Genomic imprinting is not further discussed here, but its inclusion serves as a powerful illustration of how epigenetics can elicit diametrically opposite functionalities on 2 identical DNA sequences, even within the context of a single nucleus.
The most widely studied epigenetic modification is DNA methylation that occurs in mammals by the covalent attachment of a methyl group to the 5′ carbon of the cytosine residue within symmetrical cytosine-guanine (CpG) dinucleotides.57 This symmetry, coupled with the preference of the principal DNA methyltransferase, DNMT1, for hemimethylated cytosines as a substrate,58 provides a mechanistic basis by which methylation patterns are stably inherited through successive cell generations. Specific attention is given below to the role of DNMTs. Methylation occurs at approximately 70% of CpG dinucleotides in the genome overall but shows regional differences in its distribution. CpG dinucleotides are significantly underrepresented in vertebrate genomes due to “CpG suppression,”59 a phenomenon in which the prevalence of CpGs is constrained because of their inherent propensity to undergo point mutation by means of the spontaneous deamination of 5-methylcytosine to thymine. Accordingly, in humans and mice, CpG dinucleotides are the least frequent dinucleotide, comprising less than 1% of all dinucleotide permutations. Exceptions to this rule are CpG-islands (CGI), CpG-dense regions typically, but not exclusively, associated with gene promoters. Unlike other regions of the genome where CpGs are mostly hypermethylated, CGIs are generally protected from methylation. Aberrant de novo methylation of CGI promoters, including those of tumor suppressor genes (TSGs), is a hallmark of many human diseases including cancer (reviewed in Refs. 60,61) and is one of the features discussed below in relation to CAC. However, there is growing appreciation that less CpG-dense, “non-CGI” promoter regions are more often affected by cell lineage-specific differences in methylation, which directly correlate with their transcriptional status, than canonical CGI promoters.62,63 Similarly, lineage- and cancer-specific methylation patterns are also much more prevalent at “CGI shores,” i.e., regions of lower CpG density that typically flank canonical CGI sequences.64,65
DNA methylation is strongly correlated with gene silencing and as such is widely believed to participate directly in transcriptional repression. Mechanisms by which this might occur are not fully understood, but are likely to involve the recruitment of methyl-CpG binding domain proteins and histone deacetylases (HDACs), which together orchestrate transcriptional repression through induction of heterochromatin.60,66 Methylation-dependent repression is also thought to play a critical role in preventing the inappropriate transcription of genomic repeat elements including retrotransposons, the failure of which can lead to genome instability.22,23 DNA methylation is therefore essential for normal cellular functioning through the correct regulation of gene expression and maintenance of genome structural integrity. Whether DNA methylation is definitively instrumental in transcriptional repression remains an area of intense debate, because many researchers hold the view that DNA methylation is a consequence rather than a cause of repression. Epigenomic data, showing that de novo methylation predominantly targets repressed CGI promoters, argue that methylation may, at least in some contexts, be a consequence and not the cause of repression.67–69
Much attention has been devoted to the identity of the trans-factors that initiate and maintain CpG methylation patterns both as biomarkers of disease activity and as potential therapeutic targets. An extensive body of literature assigns this role to the DNMTs. On the premise of protein sequence homology, the DNMT family was initially thought to consist of 5 members: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L;70 however, recognition post hoc that DNMT2 methylates RNA,71 sets it apart from the other DNMTs. Furthermore, DNMT3L lacks intrinsic 5-cytosine-methyl-transferase activity, instead serving as an accessory factor for DNMT3A function.71–74 Current literature broadly subdivides 5-cytosine-methyltransferase activity into “maintenance” and “de novo” modalities, the former predominantly regulated by DNMT1, reflecting its substrate preference for hemimethylated DNA,58 and the latter mostly attributed to DNMT3A/3B (and DNMT3L) which methylate either hemi- or un-methylated DNA.75–77 Neither of these subdivisions is absolute because DNMT1 may be additionally required for the de novo activities of DNMT3A/3B in some contexts.78,79 Furthermore, the ability of DNMT1 to maintain the correct fidelity of genome methylation may depend on the presence of DNMT3A/3B.80,81 The pivotal in vivo functions of the DNMTs have been formally demonstrated in mouse transgenic and knockout studies. Mice with genetic Dnmt1 deficiency, or transgenic overexpression, show embryonic lethality coupled with respective loss or gain of genome methylation.82,83 Similarly, Dnmt3a or Dnmt3b genetic deficiency led to postnatal and embryonic lethality, respectively, whereas Dnmt3a/3b compound deficiency additionally blocked normal de novo methylation of the genome during postimplantation development.84 Together, these studies highlight the essential requirement for correct genome methylation levels in normal growth, development, and physiological functioning. Altered DNA methylation and DNMT expression/regulation were among the first epigenetic changes to be reported in IBD,85–90 particularly in relation to CAC.91–93 Additional discussion of this topic is provided in later section of this article.
Genomic DNA in eukaryotic cells is packed together with special proteins, termed histones, to form chromatin. The fundamental unit of chromatin is the nucleosome. Individual nucleosomes each comprised a complex of 8 core histone proteins, 2 molecules each of the histones H2A, H2B, H3, and H4, encompassing 146 bases pairs of genomic DNA. The histone octamer forms the structural basis of the nucleosome on which the DNA strand is wrapped around. Core histone proteins are tightly folded, however residues in their protruding amino-terminal regions, or “tails,” can be altered covalently by numerous posttranslational modifications (reviewed in Ref. 94) of which acetylation and methylation are the most pertinent to this review article. These modifications are important for determining the accessibility of the DNA to the transcription machinery and for DNA replication, recombination, chromatin condensation, and mRNA splicing. Accordingly, histone modifications are major contributors to epigenetic patterns of inheritance and play key roles in determining the transcriptional state of the genome. Unlike DNA (cytosine) methylation, which is mainly associated with transcriptional repression, histone modifications are extremely diverse and can be associated either with active transcription or repression,95 providing an additional dimension to epigenetic regulation of gene expression. Histone acetylation and methylation are the best-characterized posttranslational epigenetic modifications of histone tails. While acetylation is generally considered an active mark, histone methylation can promote either transcriptional activation or repression, depending on the level of methylation (mono [me1]-, di [me2]- or tri [me3]-methylation) and the specific residues (lysines and/or arginine) involved. For example, highly transcribed, open “euchromatin” regions of the genome are enriched in active marks including histone (H)3 lysine (K)4, H3K36 and H3K79 trimethylation, and acetylation (ac). Conversely, transcriptionally repressed, closed “heterochromatin” regions are typically enriched in repressive marks, H3K9me3, H3K27me3, and H4K20me3 and show reciprocal depletion in acetylation levels. Although it is still not entirely clear how these histone marks impact mechanistically on transcription, their presence is highly predictive for gene expression or silencing (reviewed in Ref. 96). A multitude of different histone modifications, each with different activating or repressive effects, can be present simultaneously leading to the widely advocated histone code hypothesis.97,98 It was proposed that histone modifications, in addition to their individual functions, manifest a complex array of interdependencies allowing for dozens of different epigenetic landscapes to be established, thereby fine tuning higher order chromatin structural organization, gene expression, and repression.97,98 Therefore, a single mark, acting alone, is unlikely to dictate significant transcriptional effects. Rather, as recent data further attest, the combined effects of all modifications enriched within specific gene regions instruct the assembly of many different chromatin states, each with unique transcriptional and/or other functional outcomes.99
Current literature cites the existence of numerous enzymes that catalyze either the addition or the removal of histone posttranslational modifications. For example, histone acetylation, which is strongly correlated with regions of active transcription, is regulated by both histone acetyltransferases (HATs) and HDACs that respectively add or remove acetyl groups nonspecifically at a number of residues in histone tails. Accordingly, HAT and HDAC activities correlate with decreased and increased transcription, respectively. Mechanisms by which the acetylation/deacetylation of histones mediate transcription have been sought but are not well understood. However, one possibility is that acetylation neutralizes positively charged lysines, destabilizing the interaction with negatively charged DNA resulting in a more open chromatin structure accessible to transcription factors and other coactivator proteins.100 The process of histone methylation is much more specifically regulated than histone acetylation. Histone methyltransferases (HMTs) and the more recently discovered “demethylases” are the enzymes mediating this process, targeting specific residues within individual histone core proteins. Recent advances in knowledge in this area and their broad relevance to human disease have sparked renewed interest in epigenetic modifiers as potential therapeutic targets. Small molecule inhibitors have been designed against the key enzymes, thus far discussed here, including HMTs, HATs, HDACs, and DNMTs.101,102 The tremendous therapeutic potential of such inhibitors and their possible application in IBD treatment is briefly discussed here under “Diagnostic and Therapeutic Implications.”
NUCLEOSOME POSITIONING AND CHROMATIN REMODELING
Although the packaging of DNA by nucleosomes occurs in a highly regulated and ordered fashion, the overall structure of the nucleosomal array is not fixed, but rather shows a highly variable distribution along the DNA strand. One of the main influences on this irregular pattern is the binding of other “nonhistone” proteins, such as transcription factors and accessory proteins, to the DNA leading to the local displacement of nucleosomes. Accordingly, the precise positioning of nucleosomes can be altered in a highly regulated manner to elicit functional changes in gene expression (reviewed in Ref. 103). For example, nucleosomes act as a physical barrier to transcriptional initiation by preventing access of transcription factors to their consensus binding sites in the DNA sequence. Nucleosomes can also regulate transcript elongation by impeding the progression of bound RNA polymerases through the structural gene. Even relatively minor nucleosome displacements can bring about, or reflect, changes in RNA polymerase II activity. The 5′ and 3′ regions of genes are generally devoid of nucleosomes, thus providing space for the assembly and disassembly of multifactor transcription complexes.104 The eviction of a nucleosome from the region surrounding the transcription start site of genes correlates with active transcription. Conversely, occupation of the transcription start site by a nucleosome is the characteristic feature of repressed genes.105 Besides playing a direct role in transcription, nucleosome positioning has also been described to influence local DNA methylation patterns.105 In particular, nucleosome enrichment across gene exons and depletion within introns is remarkably similar to the distribution of DNA methylation. The findings support the view that nucleosome occupancy plays a key role in shaping the genomic methylation landscape. The functional significance of this relationship is unclear but it could act to define intron–exon boundaries or participate in mRNA splicing. Changes in nucleosome positioning are regulated by chromatin remodeling complexes, which are large multiprotein assemblies that alter the composition or organization of nucleosome core proteins. Current literature recognizes 5 families of chromatin remodeling complexes: SWI/SNF, ISWI, NuRD/Mi-2/CHD, INO80, and SWR1 based on conservation of their principal catalytic domains (reviewed in Ref. 106). These complexes regulate transcription by inducing adenosine triphosphate hydrolysis-dependent protein conformational changes, which, in turn, provoke reconfiguration of nucleosome positioning. Each of these families possesses both unique and partially overlapping functional specializations. SWI/SNF proteins are key regulators of gene expression,107 whereas ISWI family members participate in chromatin condensation and transcriptional repression.108 NuRD/Mi-2/CHD family proteins are functionally diverse. Some family members facilitate lateral sliding and eviction of nucleosomes, thus acting as transcriptional activators, while others promote repression by means of intrinsic HDAC and/or methyl-CpG binding domain activity.109,110 Finally, proteins of the INO80 group participate in a wide array of chromatin-related functions including transcription, DNA replication, and repair.111–113
Noncoding RNAs comprise endogenous small single-stranded miRNAs that are present at lower levels than mRNA. The miRNAs regulate gene expression at the posttranscriptional level and thereby numerous biological processes. The miRNAs bind to untranslated mRNAs and inhibit mRNA translation (partial sequence complementarity) or cause mRNA degradation (complete sequence complementarity). To date, 1872 precursor and 2578 mature miRNA sequences have been described in humans (http://mirbase.org, accessed February 3, 2014). Each miRNA may show complementarity with many different mRNAs, and each mRNA may be targeted by many different miRNAs. The miRNA-mediated gene regulation is critical for normal cellular processes such as cell cycle, differentiation, proliferation, apoptosis, and, most importantly in the context of this review article, innate, and adaptive immune functions.114 The expression of miRNAs themselves in the immune system can be regulated at different steps of their biogenesis by immunogenic stimuli.115 Altered miRNA expression has been associated with many diseases including IBD.14
EPIGENETIC REGULATION OF INNATE AND ADAPTIVE IMMUNITY
Phagocytic cells within the lamina propria (e.g., macrophages, dendritic cells [DCs], and neutrophils) and epithelial cell barrier represent the central components of the intestinal innate immune system.116 Antigens reaching the lamina propria activate innate immune cells followed by a response of T-lymphocytes, which are the prime effector cells of adaptive immunity. The innate and adaptive immunity arms are tightly cross regulated serving to uphold intestinal homeostasis, thereby controlling complex commensal–host crosstalk through the recruitment, maintenance, and regulation of effector functions of various intestinal immune cells (Fig. 1).117 Macrophages represent the most abundant mononuclear cell population of the intestine. Macrophages play an important role in intestinal antigen presentation to other immune cells in the lamina propria and in sustaining intestinal immune homeostasis.118 It has been suggested that blood monocytes are the exclusive source of macrophages in inflamed intestinal mucosa with both peripheral monocytes and their derivatives playing important roles in the pathophysiology of IBD.119 In addition, several DC subtypes form a further central part of the functional mucosal barrier of the intestine and play an important role in IBD pathogenesis.120 Recent advances have highlighted a fundamental role of DCs in intestinal innate immune homeostasis.121 DCs initiate immune responses during microbial invasion and inflammation through antigen presentation and also polarize subsequent adaptive immune responses.122
Emerging evidence suggests an important role for epigenetic mechanisms in modulating both the innate and adaptive immune systems. This includes the differentiation and function of monocytes/macrophages, DCs, neutrophils, and T-helper (Th) cell subsets. Importantly, inflammation may directly drive epigenetic reprogramming leading to aberrant immune responses. Chromatin-based events are potentially important in amplifying and perpetuating the inflammatory response as well as regulating inflammation-induced transcription, tolerance, and T-cell lineage commitment7 (and references cited therein).
Epigenetic modification of chromatin plays an important role in macrophage polarization and function. However, current literature on the epigenetic regulation of macrophage polarization and functional consequences for macrophage gene expression and phenotype is limited. Epigenetic analyses of macrophage polarization at various tissue sites during the initial inflammatory activation, immune homeostasis and the resolution of inflammation have primarily focused on histone acetylation and methylation, with limited analysis of nucleosome remodeling123 (and references cited therein). Nevertheless, evidence supports the hypothesis that epigenetic changes fundamentally reprogram macrophages to exhibit altered gene expression in response to environmental stimuli. Briefly, defined combinations of active and repressive histone marks regulate the chromatin states of inflammatory cytokine gene loci relevant for polarized M1/M2 macrophage phenotypes and thus transcription rates in response to acute stimulation and polarizing stimuli. This also includes the epigenetic regulation of key inflammatory cytokine genes, which allows a fine-tuned rapid and effective immune response and cytokine production, respectively. Classically activated M1 macrophages produce proinflammatory cytokines, which mediate resistance to pathogens and contribute to tissue destruction, whereas alternatively activated M2 macrophages produce anti-inflammatory cytokines, which promote tissue repair and remodeling.124 Furthermore, epigenetic mechanisms might also explain the gene-specific signature of tolerant macrophages following the state of acute immune activation. However, the identity of epigenetic mechanisms regulating macrophage tolerance in response to environmental cues requires further clarification.123 A better understanding of the epigenetic mechanisms that mediate repression of inflammatory cytokine gene expression in (human) macrophages represents an important area for future research and for potential new therapeutic approaches. Given that intestinal macrophages derive from peripheral blood monocytes, it will also be important to analyze epigenetic marking of monocytes. Recent studies have begun to explore the epigenetic control of monocyte differentiation and function. Importantly, this includes persistent enhanced effector immune functions of monocytes after a primary infection or vaccination and subsequent protection and resistance of the host against a secondary (re-)infection independent of adaptive immunity. These adaptive features of innate immunity have recently been described as trained immunity.125 For example, a NOD2-mediated epigenetic change at the level of an active histone mark (H3K4me3) is the mechanism through which mycobacterial components (bacille Calmette-Guérin) enhance innate immune responses.126 The modified methylation status of cytokine promoters after bacille Calmette-Guérin vaccination in human monocytes and the blockade of the in vitro training effects with methyltransferase inhibitors suggests that the innate immune response in humans can be reprogrammed epigenetically. Thus, epigenetic reprogramming has potential preventive and therapeutic purposes in inflammation and autoimmunity. One example is systemic lupus erythematosus, where monocytes play a central pathophysiological role and have been described as having aberrant behavior in a number of assays. Furthermore, cytokines can induce changes in the epigenome of systemic lupus erythematosus monocytes and persistence of these changes might lead to heritable changes in gene expression, which drive many of these aberrant functions.127 Altered monocyte-derived macrophage functions (spontaneous and lipopolysaccharide [LPS]-induced tumor necrosis factor alpha release) and modified DNA methylation in white blood cells have also been described in women treated with oral contraceptives.128
Furthermore, during the transition of human immature monocyte-derived DCs into activated (LPS-conditioned) and tolerized (transforming growth factor beta–conditioned) DCs, changes in the modification of histones (H3K4me3 and H2K27me3) and alteration in these epigenetic marks may have a role in the resulting gene regulation of these cells (e.g., chemokines, cytokines, cell surface molecules, and transcription factors).129 In addition, the chemotactic activity of monocyte-derived immature DCs and M1 macrophages can be altered by chromatin modulation in these cells. For example, simvastatin, a widely used statin that blocks cholesterol synthesis but also has pleiotropic immunomodulatory and anti-inflammatory properties, can induce repressive chromatin at the chemokine (C-C motif) ligand 2 (CCL2) promoter. CCL2, also known as monocyte chemotactic protein 1 (MCP1), recruits monocytes, memory T cells, and DCs to sites of injury and inflammation. The reduced gene expression and secretion of CCL2 in monocyte-derived cells was accompanied by enrichment of repressive marks, H3K27me3/H3K9me3 and depletion of active marks, H3ac and H3K4me3 at the CCL2 promoter.130 Downregulation of CCL2 in these cells may affect their chemotactic activity leading to reduced recruitment of monocyte-derived DCs and proinflammatory M1 macrophages to sites of tissue injury and/or inflammation. It has been argued that immunoparalysis/immunosuppression observed after sepsis is mediated by DC dysfunction arising from the perpetuation of an aberrant gene expression program.131 In this regard, it has been reported that chronic repression of Il12, which encodes a key host defense cytokine, in DCs from postseptic mice correlates with promoter enrichment of bivalent (i.e., active and repressive) marks H3K4me3 and H3K27me2.132 Although epigenomic studies on DCs are limited, some of these suggest a role for histone modification and DNA methylation in DC differentiation and function. Furthermore, epigenetic perturbations mediated by HDAC inhibitors are likely to modify the function of DCs from immunostimulatory to immunomodulatory. Regulation of H3K9me2/me3 marks by cell type-specific HMTs or histone demethylases is essential for the development and differentiation of DCs.133 Furthermore, several reports indicate the involvement of DNA methylation in regulating DC-specific gene expression programs. The majority of DNA methylation changes seem to arise early during hematopoietic linage commitment, and only a few are acquired during terminal differentiation133 (and references cited therein).
Beside monocytes/macrophages and DCs, neutrophils also play a critical role in the maintenance of intestinal immune homeostasis. They are critical for bacterial clearance, release cytokines and antimicrobial proteins, and phagocytize invading microbes that translocate across the intestinal epithelial cell (IEC) layer. During the inflammatory response, neutrophils also contribute to the recruitment of other immune cells and facilitate mucosal healing by releasing mediators necessary for the resolution of inflammation.134 Neutrophil migration into the infectious site is markedly impaired in severe sepsis and associated with depletion of active H3ac marks at the C-X-C chemokine receptor type 2 (CXCR2) promoter in neutrophils.135 CXCR2 encodes a receptor for interleukin 8 (IL8) and mediates neutrophil migration to sites of inflammation. Another example of epigenetic control of neutrophil differentiation and function arises from the observation that neutrophils from mice that lack the transcription factor Jun dimerization protein 2 (Jdp2), which regulates histone modification, displayed impaired bactericidal function, apoptosis, and surface expression of lymphocyte antigen 6 complex, locus G (Ly6G).136
Accumulated evidence shows that epigenetic mechanisms are key determinants of CD4+ Th cell differentiation and function. Th cells exist in a variety of epigenetic states that determine their function, phenotype, and capacity for persistence. These polarization states include Th1, Th2, Th17, and T-regulatory (Treg) cells. Briefly, Th1, Th2, and Th17 cells are important for eradicating intracellular pathogens, helminthes, and extracellular bacteria/fungi, respectively. Th1 and Th17 cells are also involved in many types of autoimmune diseases, whereas Th2 cells contribute to allergic responses. Treg cells are critical in maintaining self-tolerance and in modulating immune responses to infections.137 The state of chromatin and DNA methylation at lineage-restricted cytokine and transcription factor genes, as well as their regulatory elements in Th cells, both reflects and affects their function in transcription138 (and references cited therein). Different profiles of DNA methylation, active and repressive histone marks, RNA interference, and methyl-CpG binding domain proteins are associated with active and accessible, inactive but poised, as well as silenced gene loci in Th cells. Consequently, these distinguishing epigenetic marks ensure that transcription of Th1-type cytokines (e.g., IFNγ) and Th2-type cytokines (e.g., IL4, IL5, IL13) is restricted to the appropriate lineage.137–140
Th1/Th2 development is also epigenetically regulated, involving chromatin modifications of the IFNγ gene locus and the epigenetic control of the Th2 cytokine locus.141 Less is known about the epigenetic processes controlling Th17-cell differentiation and transcription of lineage defining Th-17 cytokines (e.g., IL17, IL21, IL22, and IL26). However, recent developments describe epigenetic mechanisms that can explain the constrained plasticity of Th17 cells by the epigenetic status of genes encoding for the master transcriptional regulators of polarization and canonical cytokines.137,142,143 The discovery that forkhead box P3 (FoxP3) is the transcription factor that specifies the Treg cell lineage has facilitated recent progress in understanding Treg cell biology.144 Treg cells do not express a lineage-defining cytokine; on the contrary, production of most inflammatory cytokines is repressed.143 Constitutive FoxP3 expression is required to maintain the immunosuppressive activity of Treg cells.137 FoxP3 both activates and represses target genes by epigenetically regulating histone modifications through recruitment of several histone-modifying proteins.145 For example, genes activated by FoxP3 show enrichment of active marks, H3K4me3, H3K9/14ac, and H4K16ac. Conversely, genes repressed by FoxP3 show enrichment of the repressive mark, H3K27me3.146,147 Treg cells, and also conventional CD4+ T cells, show lineage-specific methylation of regions overlapping methylation-sensitive enhancers within the vicinity of immunologically relevant genes. These findings argue that DNA methylation plays key role in establishing and maintaining cell type-specific gene expression by restricting the lineage activity of cell type-specific enhancers.148
In summary, epigenetic regulation of key cytokines and transcription factors specific to cells of the innate and adaptive immune systems plays an important role in the development, differentiation, phenotype and function of monocyte/macrophages, DCs, neutrophils, and Th cells (Fig. 1). The dynamic interactions between the genome and epigenome have important implications for a better understanding of human inflammatory diseases (including IBD) and their pathophysiology, thereby enabling the development of gene-specific therapeutic approaches.
EPIGENETIC CONTROL AND INTESTINAL MICROBIOTA
Intestinal microbiota profoundly affect host immune composition under physiologic conditions and are likely the most important environmental factor in IBD as targets of the inflammatory response.149 New evidence indicates that intestinal bacteria can regulate epithelial gene expression and the intestinal immune response through epigenetic mechanisms (Fig. 1). Similarly, bacterial and host (self-DNA) epigenetics can also directly affect host genetics to trigger inflammatory processes.12 Several mechanisms have been proposed to link epigenetic modifications with inflammation, involving the innate immune response against microbiota.
Butyrate is an endogenous metabolite formed during fermentation of dietary fibers by the intestinal microbiota and is a potent inhibitor of histone deacetylase (HDAC) activity. Butyrate-dependent HDAC inhibition has been shown to upregulate expression of the pattern recognition receptor protein NOD2 by increasing histone acetylation.150 Likewise, sodium butyrate increases the production of intestinal alkaline phosphatase, an endogenous protein responsible for detoxification of bacterial LPS.151 A number of other studies have similarly inferred that bacteria induce histone modifications, thereby modulating the inflammatory response and key cellular processes of the epithelium in the gastrointestinal tract (Fig. 1).152–160 Additionally, commensal probiotic bacteria demonstrably modulate the IL23/Th17 axis, which has parallel significance to the IL12/Th1 axis in regulating IBD pathogenesis.3 Conversely, Bifidobacterium breve and Lactobacillus rhamnosus (LGG) may exert their anti-inflammatory effects in the gut, at least in part, by modulating IL23 and IL17 endogenous synthesis through inhibition of histone acetylation and enhancement of DNA methylation.161 However, Th1 mucosal immunity is characterized by overproduction of IFNγ, and it has been further shown that levels of IFNγ promoter methylation in peripheral T cells correlate with immune response to microbial components and expression/secretion of IFNγ in patients with UC.162
Mucosal DNA methylation can also react to changes in commensal microbiota.11 Oral treatment with a genetically modified strain of Lactobacillus acidophilus deficient in lipoteichoic acid effectively ameliorated inflammation-induced colitis and restored intestinal homeostasis in experimental models.163 Lipoteichoic acid is a major immunostimulatory cell wall component of Gram-positive bacteria, which can specifically bind to toll-like receptors (TLRs) on host cells. L. acidophilus bacteria also protect mice from CAC presumably by reversal of cancer-related DNA methylation within promoters of genes that normally block or restrain intestinal cancer progression.164 Bacterial gene products may dampen detrimental gut inflammation and protect against inflammatory conditions, including IBD and CAC, acting not only through immune cell modulation, but also through direct interactions with the gut epithelium (Fig. 1).165 For example, the pattern recognition receptor, TLR4 senses the presence of LPS from Gram-negative bacteria, and it has been shown that TLR4 transcription is epigenetically repressed in IECs to prevent excessive inflammatory responses to commensal bacteria.166 Furthermore, Tlr4 methylation levels are significantly lower in IECs of the large (but not small) intestine of germ-free (GF) mice than in those of conventional mice. These findings argue that commensal bacteria contribute to the maintenance of intestinal symbiosis by controlling the epigenetic modification of the host gene.167 Contrariwise, the mucosal microbiome composition is significantly altered in Tlr2-deficient mice compared with wild-type mice but similarly associated with epigenomic and transcriptomic modifications.168 Immune-related gene expression is significantly altered by Tlr2-deficiency and correlates with DNA methylation changes. Bacterial presence also results in epigenetic modifications in gingival epithelia and bacteria-induced expression of epithelial antimicrobial molecules human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20).169 Challenge of neonatal GF mice with conventional microbiota normalized Cxcl16 promoter hypermethylation to levels typically observed in specific pathogen-free mice.170 CXCL16 is expressed at high levels by human epithelial cells and plays an important role in invariant natural killer T (iNKT) cell recruitment during inflammation. The iNKT cells probably play an important role in the pathogenesis of UC.1 Cxcl16 protein levels in serum and mRNA expression in the colon are significantly higher in GF mice compared with specific pathogen-free mice. Hence, numbers of iNKT cells have been shown to be persistently increased in the colonic lamina propria of GF mice compared with specific pathogen-free mice, resulting in increased morbidity in oxazolone-induced colitis, a murine model of UC.170 The findings argue that microbial exposure drives epigenetic mechanisms that determine both Cxcl16 gene expression and consequent iNKT cell recruitment in the colon. Interestingly, colonization of adult GF mice with conventional microbiota did not protect against mucosal iNKT accumulation and related pathology.170 These results indicate that age-sensitive contact with commensal microbes is critical for colitis susceptibility. Consequently, colonic mucosal epigenetic maturation continues through early postnatal development in mice and may contribute to the age-associated increase in colitis susceptibility.171 In this regard, a marked increase in sensitivity to dextran sulfate sodium (DSS)–induced colitis was observed in mice after prenatal maternal exposure to epigenetically active (methyl-donor [MD]) diet.172 This phenotype was associated with changes in mucosal DNA methylation, gene expression, and the intestinal microbiome. Interestingly, the same MD diet regimen did not alter colitis susceptibility in young adult mice when administered postnatally. These results provide evidence that prenatal epigenetic reprogramming of mucosal immunology through maternal dietary factors, but not postnatal diet, creates a persistent effect on the enteric microbiome by inducing longstanding modification during early development relevant to mammalian colitis. Developmental dietary intervention induced alteration of normal colonic mucosa-associated microbiota shifts may have contributed to the observed increase in colitis susceptibility in the MD supplemented offspring.173 Thus, prenatal nutritional programming can modulate the mammalian host to harbor a colitogenic microbiome.174
These studies provide important insights into microbe-specific immunity through epigenetic regulation. They highlight the intimate interrelationship between expression of immune-related genes and immunity pathways in the host with compositional and functional differences of the microbiome. Based on this promising data, future research should aim to further elucidate potential epigenetic mechanisms underlying the interplay between the microbiome and host immunity.
EPIGENETIC MODIFICATIONS IN IBD
To date, relatively little is known about the role of epigenetics in IBD. Initial DNA methylation studies have been directed predominantly towards IBD-related cancer (see Epigenetics in Colitis-associated Cancer section below). More recent genome-wide DNA methylation studies used peripheral blood or intestinal biopsy specimens of patients with IBD and healthy controls to identify methylation differences in genes regulating several pathways associated with IBD.85–90 A significant number of differentially methylated loci contained genes linked directly to the immune response, host response to bacteria, and IL23/Th17 and IL12/Th1 pathways. Additionally, genes identified as being differentially methylated in epigenome-wide methylation-association studies have also been identified as susceptibility genes in genome-wide association studies including TNF, NOD2, IL19, IL27, CARD9, ICAM3, and IL8RB.88,89 Furthermore, mucosal genome-wide methylation profiling showed evidence of differential methylation between (1) active CD and controls, (2) active UC and controls, (3) inactive UC and controls, and (4) inactive CD and inactive UC. Interestingly, differences were not found in methylation profiles between (1) quiescent CD and controls and (2) active CD and active UC.89 Future epigenome-wide methylation association studies are widely anticipated to confirm the diagnostic and prognostic potential of DNA methylome profiling in IBD.175 Importantly, the function of epigenetic marks and their role in IBD pathogenesis in individual cell types remains to be comprehensively defined.
Histone modifications are less extensively studied in IBD than DNA methylation. However, patterns of histone acetylation in the colon of rats with colitis and humans with CD have been described.176 Relative enrichment of the active marks, H4K8/K12ac and H4K5/K16ac, was observed in the inflamed mucosa and in Peyer's patches, respectively in comparison with nondiseased tissue. A gene-specific and temporal-specific pattern of histone modifications on the activated gene for collagen type I, the most abundant component of the fibrotic extracellular matrix, can be induced by fibrosis relevant cytokines (IL1β, transforming growth factor beta, and tumor necrosis factor alpha) suggesting that epigenetic factors regulate fibrotic gene transcription relevant to IBD with a fibrostenosing phenotype.177 Nevertheless, current understanding of the role played by histone modifications in IBD derives mostly from experimental or clinical trials of HDAC inhibitors, which are discussed below.
Studies in animal models have shown that intestinal miRNAs regulate gut homeostasis.178 Other studies have sought miRNA expression profiles in the peripheral blood and gut biopsy specimens of healthy controls versus adult/pediatric patients with active and inactive IBD. The miRNA expression profiles were found to be dysregulated at both the tissue level and in peripheral blood of UC and CD patients. In addition, studies have shown that IBD-related glucocorticoid therapy can modify the expression profile of different miRNAs; however, the data are conflicting.179 A number of comprehensive reviews have been published on the potential value of miRNAs in IBD during recent years to which we refer the reader.8,13,180 Specific miRNA profiles and identification of associated targets might provide additional insight into IBD pathogenesis and help to predict IBD susceptibility, progression, relapse, and response to therapy. However, miRNA profiling in IBD is in its infancy and additional studies are required to identify reliable and consistent IBD-associated miRNA profiles and to attribute-associated functions.
EPIGENETICS IN COLITIS-ASSOCIATED CANCER
A direct link between chronic inflammation and cancer susceptibility is well established; however, causal molecular mechanisms remain poorly understood. Advances in knowledge have nonetheless been forged through the study of several gastrointestinal cancers and have uncovered evidence to support inflammation-dependent transformation of epithelial cells as a mechanism of neoplasia. For example, studies of the Helicobacter pylori-associated gastric cancer paradigm181 have illuminated key genetic and epigenetic pathways underlying inflammation-induced cancer.182–185 Focusing here on cancer susceptibility in IBD, we highlight recurrent themes of classical cancer genetics and cytokine-dependent perturbation of gastrointestinal epigenetic inheritance, which resonate strongly through emerging research in this field.
Individuals with IBD show a significantly increased risk of acquiring CAC for which the most prominent risk factors are the localization, duration, and severity of gastrointestinal inflammation. CAC remains a significant complication in the long-term management of IBD; thus, the elucidation of molecular mechanisms underlying CAC is of high priority. As discussed above, accumulated evidence points to a role for inflammation in the pathogenesis of numerous cancers. Therefore, CAC provides an ideal human disease system through which fundamental concepts of inflammation-related carcinogenesis can be explored and widely disseminated. Similar to the pathogenesis of IBD per se, the development of CAC is highly heterogeneous and is likely to be driven by the converging effects of host genetic and epigenetic factors, as well as the influence of environmental factors, such as the intestinal microbiome (reviewed in Refs. 186,187).
As discussed above, DNA methylation and histone modification together establish stable and heritable patterns of gene expression in normal cells. However, alterations in these key epigenetic modifications, leading to global deregulation of gene expression, are frequently observed in both inflammation and cancer, as well as in inflammation-related cancer.92,188 Aberrant DNA methylation is one of the earliest molecular changes in cancer and may correlate with the early stages of both IBD and CAC.187 In cancer cells, normally hypermethylated sequences become globally hypomethylated, which can lead to genomic instability, a characteristic feature of colorectal cancers (CRC).189 Conversely, normally unmethylated CGI promoters, including those at some TSG loci, become aberrantly hypermethylated leading to their repression. Inactivation of TSGs through promoter hypermethylation provides a persuasive epigenetic mechanism of cancer pathogenesis.60 Hypermethylated TSGs have been suggested as potential epigenetic biomarkers of cancer risk on which new approaches to clinical screening might be based.190 For example, specific gene methylation patterns are highly predictive of precancerous pathology in the gastrointestinal tract and can signify the presence of inflammation and dysplasia, as well as cancer. These data strongly implicate a role for epigenetic alterations in inflammation-dependent carcinogenesis.
The available literature supporting roles for epigenetic modification in CAC remains significantly underdeveloped. Moreover, the DNA methylation studies that have recently emerged have often made contradictory findings, although these have largely been in respect of the CpG-island hypermethylator phenotype (CIMP) phenomenon. CIMP is a well-described epigenetic phenomenon in a subset of sporadic CRC involving the coordinate methylation-dependent silencing of TSG loci. However, “CIMP-positive” CRC cases frequently display molecular correlates of microsatellite instability and secondary genetic mutations, which complicates interpretation of CIMP as a driver of CRC. Nonetheless, CIMP has been widely advocated as an epigenetic marker of CRC risk, progression, and survival,191 prompting investigators to search for “CIMP” signatures as both a mechanism and marker of CAC. Age-related increases in CGI methylation levels are also known to occur in the colonic epithelium and may act as confounding factors in this regard. One study reported that age-related CGI methylation may show accelerated progression in IBD because of increased cell turnover in the context of inflammation.91 The findings were argued in evidence of an epigenetic mechanism for cancer susceptibility in UC. Nevertheless, in a subsequent study, the same investigators analyzed methylation levels of 11 genes in UC-associated cancers, UC-associated dysplasias, and sporadic CRC cases to determine if accelerated age-related methylation in dysplastic UC translates into subsequent cancer. They reported that CIMP was infrequent in UC-associated cancers compared with sporadic CRC, thus arguing against a major role for DNA methylation (CIMP) as a driver of CAC.192
Conversely, other studies have unequivocally shown hypermethylation of loci previously linked to cancer pathogenesis, CDH1, GDNF, HPP1, and MYOD1, in active UC compared with quiescent UC.92 Other studies have replicated the latter finding of CDH1 hypermethylation in both dysplastic and tumor tissue taken from subjects with UC, compared with nondysplastic tissues.93,193 Furthermore, aberrant CGI promoter hypermethylation of the TSGs, P53, P14AR, P16INK4a, P21CIP1, and MLH1 in UC lacking neoplasia has been collectively reported by several studies.194,195 The concept was further developed by Dhir et al who investigated DNA methylation levels within canonical WNT/beta-catenin signaling pathway components downstream of the intestinal TSG, adenomatous polyposis coli (APC). Deregulation of WNT/beta-catenin signaling in association with APC genetic inactivation is a key mechanism of sporadic CRC pathogenesis; however, somatic mutations within this pathway are not commonly reported in CAC. They instead found that methylation-dependent inactivation of several WNT pathway genes is a frequent, early event in both IBD and IBD-related cancer,196 providing additional evidence to support a potential epigenetic route to CAC. Another potentially important mechanism relates to the CGI hypermethylation and inactivation of the suppressor of cytokine signaling 3 (SOCS3) locus leading to hyperactivation of the oncogenic IL6/STAT3 pathway in UC and CAC.197
Although progress has undoubtedly been made, the candidate gene approach favored by many of the above-described studies provides a limited view of potentially widespread epigenetic deregulation in CAC. Consequently, the full extent of epigenetic alterations that may precede the emergence of CAC remains poorly understood. Genome-wide approaches to comprehensively determine DNA methylation (and histone modification) patterns will be critical to an improved understanding of the epigenetic principles underlying CAC and their active deployment in IBD disease management. Studies of this genre have yet to emerge en masse; however, a recent microarray-based genome-wide analysis revealed that extensive CGI hypermethylation is much less prevalent in CAC compared with sporadic CRC cases. Consistent with the findings of earlier candidate gene approaches,192 Olaru et al198 reported the existence of a CIMP-positive CAC subset, showing key similarities to sporadic CRC in age of cancer onset and extensive cancer-specific CGI hypermethylation. However, the authors attributed the acquisition of CIMP in this subset of CAC to the effects of aging rather than inflammation per se, further concluding that CGI methylation plays a limited role in CAC pathogenesis.
The conclusions of Olaru et al are supported by epigenomic evidence that CGI hypermethylation in human malignancies (including colon cancer) may, in general, be less extensive than initially hypothesized.62 Furthermore, revisions to the much-vaunted model of CGI methylation-dependent cancer pathogenesis have been proposed. For example, although CGI hypermethylation generally correlates with repression of TSGs, the assumption that methylation per se is instrumental in their repression may not always be correct. On the contrary, recent studies show that cancer-related de novo methylation is attracted predominantly to CGI promoters that are already repressed in normal tissues. In these instances, methylation does not directly participate in repression and therefore is not necessarily a driver of cancer.67–69 With the above caveats noted, future research should address whether extensive CGI hypermethylation might still inactivate rare but nonetheless pivotal protective genes in CAC. Alternatively, there is growing recognition of an important role for less CpG-dense, non-CGI promoters, and “CGI shores” as targets of lineage-specific differential methylation in cancer.65 Mapping the methylation profiles of these regions on a genome-wide scale has the potential to unravel many of the unresolved epigenetic questions surrounding CAC pathogenesis and should be an immediate priority of IBD research.
Definitive mechanisms to explain the inflammation-associated perturbation of gastrointestinal epigenetic inheritance in CAC and in other gut inflammatory cancers have not been firmly established in vivo. However, fundamental roles for specific proinflammatory cytokines and the reactive oxygen/nitrogen species induced as a consequence of their bioactivity have begun to crystallize. Among the best available evidence, the proinflammatory cytokine, IL1β, a key determinant of inflammation-related cancer risk in humans, was shown to stimulate DNMT1 activity in a nitric oxide (NO)-dependent manner through upregulation of inducible nitric oxide synthase (iNOS).199 Similarly, IL6, acting through DNMT1, was shown to promote hypermethylation of genes associated with tumor suppression, adhesion, and apoptosis, thereby enhancing the oncogenic phenotype of colon cancer cells. These results argue that cytokine-dependent, DNMT-mediated gene silencing is a key mechanism of inflammation-related colon tumorigenesis.200 This concept is further supported by evidence that exogenous IFNγ exposure can elicit genome hypermethylation in concert with increased expression of the de novo methyltransferase, DNMT3B, in human colonic epithelial cells.201
Oxidative stress, which is a key component of inflammatory reactions, was elegantly shown to provoke recruitment of a repressive complex containing DNMT1, DNMT3B, HMT proteins of the polycomb group and HDAC to CGI promoters in human colon cancer cells. The investigators further showed that DNMT1, polycomb, and HDAC proteins were similarly enriched on CGI promoters in a mouse model of colitis.202 Therefore, oxidative stress can promote recruitment of a repressive complex that may explain the de novo hypermethylation and loss of gene function that accompanies the progression of inflammation-related neoplasias, including CAC. A recent study showing that IL6 promotes increased DNMT1 expression leading to epigenetic inactivation of the STAT3-negative regulator, SOCS3, in human CAC provides direct clinical relevance for many of the above-discussed mechanisms (summarized in Fig. 2).203 A number of small molecule inhibitors of DNMTs are currently being evaluated in clinical trials, the outcomes of which could engender novel therapeutic approaches to the challenging complication of CAC in patients with IBD.
DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS
Epigenetic research could provide IBD biomarkers to (1) confirm diagnosis and disease phenotype; (2) predict the course of the disease and relapses; (3) evaluate the response to therapy; and (4) prediction and detection of CAC. This includes DNA methylation analyses92,204–209 and miRNA profiling210–216 in fecal samples, mucosal biopsy specimens, and/or peripheral blood/serum. It has been proposed that routine DNA methylation profiling in colonic tissue from patients with IBD might serve as a potential biomarker of disease, intestinal inflammation, disease phenotype, and/or the early detection of CAC. Several marker genes have been advocated for this purpose including methylated-in tumor-1 (MINT1),208 cyclooxygenase-2 (COX2),208 runt-related transcription factor 3 (RUNX3),208 signal transducer and activator of transcription 4 (STAT4),209 protease-activated receptor 2 (PAR2),206 slit protein homolog 2 (SLIT2),204 transmembrane protein with EGF-like and 2 follistatin-like domains 2 (TMEFF2),204 forkhead box protein E1 (FOXE1),205 synaptic nuclear envelope protein 1 (SYNE1),205 and multidrug resistance protein 1 (MDR1).207
Besides these potential diagnostic applications, further studies of IBD-associated epigenetics might lead to new therapeutic approaches. Chromatin regulators, as therapeutic compounds that suppress proinflammatory cytokine gene induction and inflammation, offer potential for gene- and patient-specific immunomodulatory therapy that can even induce remission in patients with chronic IBD. Anti-inflammatory effects of HDAC inhibitors have been firmly established in several models of intestinal inflammation and colitis.217,218 However, these compounds may confer additional protective benefits by promoting the survival and barrier function of IECs.219 A range of small molecule inhibitors of chromatin-modifying enzymes are being efficacy-tested in clinical trials for use in non-IBD conditions.7 However, the therapeutic effects of these compounds in IBD have not yet been confirmed. Targeting individual HAT/HMT/HDAC enzymes with high specificity has shown immense therapeutic promise in other human diseases and could form part of a new strategy in IBD therapy. Specificity remains a key priority here because some global inhibitors of HDACs and DNMTs may have off-target effects that promote both pro- and anti-inflammatory sequelae and increase the risk of side effects and/or malignancy.7
In addition, several compounds targeting DNA methylation status have been demonstrated to have potential therapeutic effects in experimental colitis models and/or patients with IBD. This includes compounds that act as methyl donors, which increase global methylation levels or small compounds that affect methylation-dependent gene expression. Black raspberries, for example, are a natural food rich in protective antioxidants and anti-inflammatory compounds including folic acid. Dietary intake of black raspberries may suppress colonic ulceration by correcting promoter hypermethylation of homeostatic genes that systematically regulate inflammation in a model of DSS-induced colitis.220 Likewise, inhibition of DNA methylation in DSS-induced colitis results in disease exacerbation, whereas folate supplementation to promote methylation partially ameliorates the severity of colitis.201 Dietary folate did not significantly affect the intestinal microbiome and inflammation in DSS-induced colitis in another study.221 However, prenatal and lactational exposure to a methyl-deficient maternal diet (folate, vitamin B12, and choline) was found to aggravate DSS-induced colitis in rats.222 Dietary (pro-)oxidants can cause epigenetic changes in antioxidant defense and an upregulation of inflammatory processes in IECs as evidenced by altered promoter methylation of superoxide dismutase (SOD) 2 and glutathione peroxidase (GPx). Treatment of IECs with a demethylating agent or antioxidant normalized the activities of SOD2 and GPx and prevented inflammation.223 Furthermore, IEC-specific deficiency of DNMT or HDAC activity leads to an altered mucosal inflammatory response and barrier function.224,225
Other therapeutic approaches aim to target gene expression mediated by DNA methylation more specifically. For example, the tylophorine analog W-8 was found to induce FoxP3 promoter demethylation, thus upregulating FoxP3 expression. This effect was sufficient to drive naive CD4+ T-cell differentiation to correctly functional and immunosuppressive Treg cells with protective properties in murine experimental colitis.226 Increased differentiation of Treg cells and inhibition of Th17 cells were also observed in DSS-induced colitis after treatment with a potent ligand of the aryl hydrocarbon receptor (AhR), which has been shown to modulate Treg- and Th17-cell differentiation.227 Interestingly, analysis of mesenteric lymph nodes and lamina propria cells in allied colitis experiments revealed reciprocal hyper- and hypo-methylation effects on FoxP3 and Il17 promoters respectively, which ameliorated with AhR ligand treatment.
CONCLUSIONS AND FUTURE PERSPECTIVES
Significant advances in knowledge of IBD pathogenesis have been achieved during the past decade. The literature reviewed here serves to illustrate that epigenetic mechanisms, underlying the immunological and oncogenic aspects of IBD, have already contributed extensively to this progress in understanding. The advent of epigenome-wide methylation association studies has been exploited to great effect both in cancer biology and in nonmalignant complex diseases such as autoimmunity or diabetes (reviewed in Ref. 175) and its undoubted utility is becoming palpable in IBD research.85,88,90 Further large scale studies of this type are imminently anticipated and will impact profoundly on our understanding of IBD by adding a second major dimension of disease heritability to complement, and perhaps reintegrate, the extensive genetic data garnered from genome-wide association studies. Better access to and reduced cost of massively parallel/next generation sequencing technologies will facilitate exploration beyond DNA methylation, prospectively allowing clinical translation of the much-vaunted histone code paradigm into novel approaches to the management of IBD and its associated comorbidities, including CAC.
Many of the earliest epigenetic studies in IBD research focused exclusively on CAC; however, it has become increasingly apparent that epigenetic mechanisms exert a pivotal influence on all facets of IBD pathogenesis. Heritable programs of gene transcription established by DNA methylation, histone modification, and RNA interference regulate the development and function of innate and adaptive immunity, pathogen recognition, and host microbiome interactions as well as mucosal homeostasis and integrity. Deregulated epigenetic inheritance within each of these key modalities has now been firmly established in both clinical IBD and experimental models. Mouse genetic models have proven indispensable in evaluating gene-specific effects and their functional consequences, yet the picture is far from complete. Convergent themes of transgenerational epigenetic inheritance and gene-environment interaction have emerged through rodent models of experimental colitis, suggesting that maternal diet may, through modification of the intestinal epigenome, influence the penetrance and/or severity of IBD-related inflammatory pathology. Further investigation of this exciting phenomenon is nonetheless required to determine the extent of its potential therapeutic value.
Finally studies of cytokine- and oxidative stress-dependent carcinogenesis have thrown light on epigenetic mechanisms linking deregulated immunity to IBD-related cancer. These findings will not only inform IBD management but may also have broad application in other human cancers for which inflammatory provenance has been firmly established. The inherent stability and plasticity of epigenetic modifications will be the key factors to their prospective translation as predictive biomarkers or as targets for pharmacologic intervention in IBD therapy.
1. Kaser A, Zeissig S, Blumberg RS. Inflammatory bowel disease. Annu Rev Immunol. 2010;28:573–621.
2. Khor B, Gardet A, Xavier RJ. Genetics and pathogenesis of inflammatory bowel disease. Nature. 2011;474:307–317.
3. Di Sabatino A, Biancheri P, Rovedatti L, et al.. New pathogenic paradigms in inflammatory bowel disease. Inflamm Bowel Dis. 2012;18:368–371.
4. Portela A, Esteller M. Epigenetic modifications and human disease. Nat Biotechnol. 2010;28:1057–1068.
5. Bonasio R, Tu S, Reinberg D. Molecular signals of epigenetic states. Science. 2010;330:612–616.
6. Petronis A, Petroniene R. Epigenetics of inflammatory bowel disease. Gut. 2000;47:302–306.
7. Scarpa M, Stylianou E. Epigenetics: concepts and relevance to IBD pathogenesis. Inflamm Bowel Dis. 2012;18:1982–1996.
8. Ventham NT, Kennedy NA, Nimmo ER, et al.. Beyond gene discovery in inflammatory bowel disease: the emerging role of epigenetics. Gastroenterology. 2013;145:293–308.
9. Jenke AC, Zilbauer M. Epigenetics in inflammatory bowel disease. Curr Opin Gastroenterol. 2012;28:577–584.
10. Stylianou E. Epigenetics: the fine-tuner in inflammatory bowel disease? Curr Opin Gastroenterol. 2013;29:370–377.
11. Kellermayer R. Epigenetics and the developmental origins of inflammatory bowel diseases. Can J Gastroenterol. 2012;26:909–915.
12. Low D, Mizoguchi A, Mizoguchi E. DNA methylation in inflammatory bowel disease and beyond. World J Gastroenterol. 2013;19:5238–5249.
13. Pekow JR, Kwon JH. MicroRNAs in inflammatory bowel disease. Inflamm Bowel Dis. 2012;18:187–193.
14. Iborra M, Bernuzzi F, Invernizzi P, et al.. MicroRNAs in autoimmunity and inflammatory bowel disease: crucial regulators in immune response. Autoimmun Rev. 2012;11:305–314.
15. Waddington CH. An introduction to modern genetics. New York: Macmillan; 1939.
16. Waddington CH. The epigenotype. 1942. Int J Epidemiol. 2012;41:10–13.
17. Sauvageau M, Sauvageau G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell. 2010;7:299–313.
18. Chuang JC, Jones PA. Epigenetics and microRNAs. Pediatr Res. 2007;61:24R–29R.
19. Eccleston A, Cesari F, Skipper M. Transcription and epigenetics. Nature. 2013;502:461.
20. Bell AC, Felsenfeld G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature. 2000;405:482–485.
21. Tuorto F, Liebers R, Musch T, et al.. RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis. Nat Struct Mol Biol. 2012;19:900–905.
22. Greally JM. Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome. Proc Natl Acad Sci U S A. 2002;99:327–332.
23. Bourc'his D, Bestor TH. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature. 2004;431:96–99.
24. Fraga MF, Ballestar E, Paz MF, et al.. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci U S A. 2005;102:10604–10609.
25. Javierre BM, Fernandez AF, Richter J, et al.. Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res. 2010;20:170–179.
26. Kaminsky ZA, Tang T, Wang SC, et al.. DNA methylation profiles in monozygotic and dizygotic twins. Nat Genet. 2009;41:240–245.
27. Renz H, von Mutius E, Brandtzaeg P, et al.. Gene-environment interactions in chronic inflammatory disease. Nat Immunol. 2011;12:273–277.
28. Feil R, Fraga MF. Epigenetics and the environment: emerging patterns and implications. Nat Rev Genet. 2011;13:97–109.
29. Painter RC, Osmond C, Gluckman P, et al.. Transgenerational effects of prenatal exposure to the Dutch famine on neonatal adiposity and health in later life. BJOG. 2008;115:1243–1249.
30. Pembrey ME, Bygren LO, Kaati G, et al.. Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet. 2006;14:159–166.
31. Tobi EW, Lumey LH, Talens RP, et al.. DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific. Hum Mol Genet. 2009;18:4046–4053.
32. Morgan HD, Sutherland HG, Martin DI, et al.. Epigenetic inheritance at the agouti locus in the mouse. Nat Genet. 1999;23:314–318.
33. Waterland RA, Jirtle RL. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol. 2003;23:5293–5300.
34. Chamorro-Garcia R, Sahu M, Abbey RJ, et al.. Transgenerational inheritance of increased fat depot size, stem cell reprogramming, and hepatic steatosis elicited by prenatal exposure to the obesogen tributyltin in mice. Environ Health Perspect. 2013;121:359–366.
35. Bernstein CN, Blanchard JF, Kliewer E, et al.. Cancer risk in patients with inflammatory bowel disease: a population-based study. Cancer. 2001;91:854–862.
36. Itzkowitz SH, Yio X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol. 2004;287:G7–G17.
37. Cattanach BM, Kirk M. Differential activity of maternally and paternally derived chromosome regions in mice. Nature. 1985;315:496–498.
38. Ferguson-Smith AC. Genomic imprinting: the emergence of an epigenetic paradigm. Nat Rev Genet. 2011;12:565–575.
39. McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell. 1984;37:179–183.
40. Surani MA, Barton SC, Norris ML. Nuclear transplantation in the mouse: heritable differences between parental genomes after activation of the embryonic genome. Cell. 1986;45:127–136.
41. Wood AJ, Oakey RJ. Genomic imprinting in mammals: emerging themes and established theories. PLoS Genet. 2006;2:e147.
42. Plagge A, Gordon E, Dean W, et al.. The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet. 2004;36:818–826.
43. Smith ER, Lee MG, Winter B, et al.. Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase II. Mol Cell Biol. 2008;28:1041–1046.
44. Ferguson-Smith MA, Yang F, Rens W, et al.. The impact of chromosome sorting and painting on the comparative analysis of primate genomes. Cytogenet Genome Res. 2005;108:112–121.
45. Garfield AS, Cowley M, Smith FM, et al.. Distinct physiological and behavioural functions for parental alleles of imprinted Grb10. Nature. 2011;469:534–538.
46. Ferron SR, Charalambous M, Radford E, et al.. Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis. Nature. 2011;475:381–385.
47. Cowley M, Garfield AS, Madon-Simon M, et al.. Developmental programming mediated by complementary roles of imprinted grb10 in mother and pup. PLoS Biol. 2014;12:e1001799.
48. Yamazawa K, Kagami M, Nagai T, et al.. Molecular and clinical findings and their correlations in Silver-Russell syndrome: implications for a positive role of IGF2 in growth determination and differential imprinting regulation of the IGF2-H19 domain in bodies and placentas. J Mol Med (Berl). 2008;86:1171–1181.
49. Weksberg R, Shuman C, Beckwith JB. Beckwith-Wiedemann syndrome. Eur J Hum Genet. 2010;18:8–14.
50. Cassidy SB, Dykens E, Williams CA. Prader-Willi and Angelman syndromes: sister imprinted disorders. Am J Med Genet. 2000;97:136–146.
51. Soejima H, Nakagawachi T, Zhao W, et al.. Silencing of imprinted CDKN1C gene expression is associated with loss of CpG and histone H3 lysine 9 methylation at DMR-LIT1 in esophageal cancer. Oncogene. 2004;23:4380–4388.
52. Lim DH, Maher ER. Genomic imprinting syndromes and cancer. Adv Genet. 2010;70:145–175.
53. Cui H, Cruz-Correa M, Giardiello FM, et al.. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk. Science. 2003;299:1753–1755.
54. Akolkar PN, Gulwani-Akolkar B, Heresbach D, et al.. Differences in risk of Crohn's disease in offspring of mothers and fathers with inflammatory bowel disease. Am J Gastroenterol. 1997;92:2241–2244.
55. Zelinkova Z, Stokkers PC, van der Linde K, et al.. Maternal imprinting and female predominance in familial Crohn's disease. J Crohns Colitis. 2012;6:771–776.
56. Fransen K, Mitrovic M, van Diemen CC, et al.. Limited evidence for parent-of-origin effects in inflammatory bowel disease associated loci. PLoS One. 2012;7:e45287.
57. Chen T, Li E. Establishment and maintenance of DNA methylation patterns in mammals. Curr Top Microbiol Immunol. 2006;301:179–201.
58. Hermann A, Goyal R, Jeltsch A. The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites. J Biol Chem. 2004;279:48350–48359.
59. Cooper DN, Gerber-Huber S. DNA methylation and CpG suppression. Cell Differ. 1985;17:199–205.
60. Clark SJ, Melki J. DNA methylation and gene silencing in cancer: which is the guilty party? Oncogene. 2002;21:5380–5387.
61. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 2012;13:484–492.
62. Weber M, Hellmann I, Stadler MB, et al.. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet. 2007;39:457–466.
63. Han H, Cortez CC, Yang X, et al.. DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter. Hum Mol Genet. 2011;20:4299–4310.
64. Irizarry RA, Ladd-Acosta C, Wen B, et al.. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet. 2009;41:178–186.
65. Doi A, Park IH, Wen B, et al.. Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet. 2009;41:1350–1353.
66. Bird AP, Wolffe AP. Methylation-induced repression–belts, braces, and chromatin. Cell. 1999;99:451–454.
67. Keshet I, Schlesinger Y, Farkash S, et al.. Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet. 2006;38:149–153.
68. Sproul D, Nestor C, Culley J, et al.. Transcriptionally repressed genes become aberrantly methylated and distinguish tumors of different lineages in breast cancer. Proc Natl Acad Sci U S A. 2011;108:4364–4369.
69. Sproul D, Kitchen RR, Nestor CE, et al.. Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns. Genome Biol. 2012;13:R84.
70. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet. 2000;9:2395–2402.
71. Goll MG, Kirpekar F, Maggert KA, et al.. Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science. 2006;311:395–398.
72. Bourc'his D, Xu GL, Lin CS, et al.. Dnmt3L and the establishment of maternal genomic imprints. Science. 2001;294:2536–2539.
73. Chedin F, Lieber MR, Hsieh CL. The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc Natl Acad Sci U S A. 2002;99:16916–16921.
74. Van Emburgh BO, Robertson KD. Modulation of Dnmt3b function in vitro by interactions with Dnmt3L, Dnmt3a and Dnmt3b splice variants. Nucleic Acids Res. 2011;39:4984–5002.
75. Gowher H, Jeltsch A. Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites. J Mol Biol. 2001;309:1201–1208.
76. Suetake I, Miyazaki J, Murakami C, et al.. Distinct enzymatic properties of recombinant mouse DNA methyltransferases Dnmt3a and Dnmt3b. J Biochem. 2003;133:737–744.
77. Yokochi T, Robertson KD. Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a. J Biol Chem. 2002;277:11735–11745.
78. Fatemi M, Hermann A, Gowher H, et al.. Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA. Eur J Biochem. 2002;269:4981–4984.
79. Lorincz MC, Schubeler D, Hutchinson SR, et al.. DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol Cell Biol. 2002;22:7572–7580.
80. Chen T, Ueda Y, Dodge JE, et al.. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol. 2003;23:5594–5605.
81. Liang G, Chan MF, Tomigahara Y, et al.. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol. 2002;22:480–491.
82. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992;69:915–926.
83. Biniszkiewicz D, Gribnau J, Ramsahoye B, et al.. Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Mol Cell Biol. 2002;22:2124–2135.
84. Okano M, Bell DW, Haber DA, et al.. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999;99:247–257.
85. Harris RA, Nagy-Szakal D, Pedersen N, et al.. Genome-wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases. Inflamm Bowel Dis. 2012;18:2334–2341.
86. Lin Z, Hegarty JP, Cappel JA, et al.. Identification of disease-associated DNA methylation in intestinal tissues from patients with inflammatory bowel disease. Clin Genet. 2011;80:59–67.
87. Lin Z, Hegarty JP, Yu W, et al.. Identification of disease-associated DNA methylation in B cells from Crohn's disease and ulcerative colitis patients. Dig Dis Sci. 2012;57:3145–3153.
88. Nimmo ER, Prendergast JG, Aldhous MC, et al.. Genome-wide methylation profiling in Crohn's disease identifies altered epigenetic regulation of key host defense mechanisms including the Th17 pathway. Inflamm Bowel Dis. 2012;18:889–899.
89. Cooke J, Zhang H, Greger L, et al.. Mucosal genome-wide methylation changes in inflammatory bowel disease. Inflamm Bowel Dis. 2012;18:2128–2137.
90. Hasler R, Feng Z, Backdahl L, et al.. A functional methylome map of ulcerative colitis. Genome Res. 2012;22:2130–2137.
91. Issa JP, Ahuja N, Toyota M, et al.. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res. 2001;61:3573–3577.
92. Saito S, Kato J, Hiraoka S, et al.. DNA methylation of colon mucosa in ulcerative colitis patients: correlation with inflammatory status. Inflamm Bowel Dis. 2011;17:1955–1965.
93. Wheeler JM, Kim HC, Efstathiou JA, et al.. Hypermethylation of the promoter region of the E-cadherin gene (CDH1) in sporadic and ulcerative colitis associated colorectal cancer. Gut. 2001;48:367–371.
94. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705.
95. Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet. 2007;8:286–298.
96. Zentner GE, Henikoff S. Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol. 2013;20:259–266.
97. Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000;403:41–45.
98. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:1074–1080.
99. Ernst J, Kellis M. Discovery and characterization of chromatin states for systematic annotation of the human genome. Nat Biotechnol. 2010;28:817–825.
100. Henikoff S, Shilatifard A. Histone modification: cause or cog? Trends Genet. 2011;27:389–396.
101. Selvi BR, Mohankrishna DV, Ostwal YB, et al.. Small molecule modulators of histone acetylation and methylation: a disease perspective. Biochim Biophys Acta. 2010;1799:810–828.
102. Rius M, Lyko F. Epigenetic cancer therapy: rationales, targets and drugs. Oncogene. 2012;31:4257–4265.
103. Szerlong HJ, Hansen JC. Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure. Biochem Cell Biol. 2011;89:24–34.
104. Parmar JJ, Marko JF, Padinhateeri R. Nucleosome positioning and kinetics near transcription-start-site barriers are controlled by interplay between active remodeling and DNA sequence. Nucleic Acids Res. 2014;42:128–136.
105. Chodavarapu RK, Feng S, Bernatavichute YV, et al.. Relationship between nucleosome positioning and DNA methylation. Nature. 2010;466:388–392.
106. Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010;463:474–484.
107. Reisman D, Glaros S, Thompson EA. The SWI/SNF complex and cancer. Oncogene. 2009;28:1653–1668.
108. Erdel F, Rippe K. Chromatin remodelling in mammalian cells by ISWI-type complexes—where, when and why? FEBS J. 2011;278:3608–3618.
109. Bowen NJ, Fujita N, Kajita M, et al.. Mi-2/NuRD: multiple complexes for many purposes. Biochim Biophys Acta. 2004;1677:52–57.
110. Denslow SA, Wade PA. The human Mi-2/NuRD complex and gene regulation. Oncogene. 2007;26:5433–5438.
111. Vincent JA, Kwong TJ, Tsukiyama T. ATP-dependent chromatin remodeling shapes the DNA replication landscape. Nat Struct Mol Biol. 2008;15:477–484.
112. Shimada K, Oma Y, Schleker T, et al.. Ino80 chromatin remodeling complex promotes recovery of stalled replication forks. Curr Biol. 2008;18:566–575.
113. Conaway RC, Conaway JW. The INO80 chromatin remodeling complex in transcription, replication and repair. Trends Biochem Sci. 2009;34:71–77.
114. Lu LF, Liston A. MicroRNA in the immune system, microRNA as an immune system. Immunology. 2009;127:291–298.
115. Bronevetsky Y, Ansel KM. Regulation of miRNA biogenesis and turnover in the immune system. Immunol Rev. 2013;253:304–316.
116. Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell. 2010;140:859–870.
117. Kelsall BL. Innate and adaptive mechanisms to control [corrected] pathological intestinal inflammation. J Pathol. 2008;214:242–259.
118. Mowat AM, Bain CC. Mucosal macrophages in intestinal homeostasis and inflammation. J Innate Immun. 2011;3:550–564.
119. Zhou L, Braat H, Faber KN, et al.. Monocytes and their pathophysiological role in Crohn's disease. Cell Mol Life Sci. 2009;66:192–202.
120. Cader MZ, Kaser A. Recent advances in inflammatory bowel disease: mucosal immune cells in intestinal inflammation. Gut. 2013;62:1653–1664.
121. Tezuka H, Ohteki T. Regulation of intestinal homeostasis by dendritic cells. Immunol Rev. 2010;234:247–258.
122. Kayama H, Takeda K. Regulation of intestinal homeostasis by innate and adaptive immunity. Int Immunol. 2012;24:673–680.
123. Ivashkiv LB. Epigenetic regulation of macrophage polarization and function. Trends Immunol. 2013;34:216–223.
124. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–795.
125. Netea MG, Quintin J, van der Meer JW. Trained immunity: a memory for innate host defense. Cell Host Microbe. 2011;9:355–361.
126. Kleinnijenhuis J, Quintin J, Preijers F, et al.. Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc Natl Acad Sci U S A. 2012;109:17537–17542.
127. Zhang Z, Maurer K, Perin JC, et al.. Cytokine-induced monocyte characteristics in SLE. J Biomed Biotechnol. 2010;2010:507475.
128. Campesi I, Sanna M, Zinellu A, et al.. Oral contraceptives modify DNA methylation and monocyte-derived macrophage function. Biol Sex Differ. 2012;3:4.
129. Huang Y, Min S, Lui Y, et al.. Global mapping of H3K4me3 and H3K27me3 reveals chromatin state-based regulation of human monocyte-derived dendritic cells in different environments. Genes Immun. 2012;13:311–320.
130. Zanette DL, van Eggermond MC, Haasnoot G, et al.. Simvastatin reduces CCL2 expression in monocyte-derived cells by induction of a repressive CCL2 chromatin state. Hum Immunol. 2014;75:10–14.
131. Wen H, Schaller MA, Dou Y, et al.. Dendritic cells at the interface of innate and acquired immunity: the role for epigenetic changes. J Leukoc Biol. 2008;83:439–446.
132. Wen H, Dou Y, Hogaboam CM, et al.. Epigenetic regulation of dendritic cell-derived interleukin-12 facilitates immunosuppression after a severe innate immune response. Blood. 2008;111:1797–1804.
133. Kim HP, Lee YS, Park JH, et al.. Transcriptional and epigenetic networks in the development and maturation of dendritic cells. Epigenomics. 2013;5:195–204.
134. Fournier BM, Parkos CA. The role of neutrophils during intestinal inflammation. Mucosal Immunol. 2012;5:354–366.
135. Ishii M, Asano K, Namkoong H, et al.. CRTH2 is a critical regulator of neutrophil migration and resistance to polymicrobial sepsis. J Immunol. 2012;188:5655–5664.
136. Maruyama K, Fukasaka M, Vandenbon A, et al.. The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation. Immunity. 2012;37:1024–1036.
137. Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol. 2010;28:445–489.
138. Wilson CB, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol. 2009;9:91–105.
139. Allan RS, Zueva E, Cammas F, et al.. An epigenetic silencing pathway controlling T helper 2 cell lineage commitment. Nature. 2012;487:249–253.
140. Zhu J. Transcriptional regulation of Th2 cell differentiation. Immunol Cell Biol. 2010;88:244–249.
141. Janson PC, Winerdal ME, Winqvist O. At the crossroads of T helper lineage commitment-epigenetics points the way. Biochim Biophys Acta. 2009;1790:906–919.
142. Muranski P, Restifo NP. Essentials of Th17 cell commitment and plasticity. Blood. 2013;121:2402–2414.
143. Kanno Y, Vahedi G, Hirahara K, et al.. Transcriptional and epigenetic control of T helper cell specification: molecular mechanisms underlying commitment and plasticity. Annu Rev Immunol. 2012;30:707–731.
144. Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol. 2012;30:531–564.
145. Katoh H, Zheng P, Liu Y. FOXP3: genetic and epigenetic implications for autoimmunity. J Autoimmun. 2013;41:72–78.
146. Katoh H, Qin ZS, Liu R, et al.. FOXP3 orchestrates H4K16 acetylation and H3K4 trimethylation for activation of multiple genes by recruiting MOF and causing displacement of PLU-1. Mol Cell. 2011;44:770–784.
147. Zheng Y, Josefowicz SZ, Kas A, et al.. Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature. 2007;445:936–940.
148. Schmidl C, Klug M, Boeld TJ, et al.. Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity. Genome Res. 2009;19:1165–1174.
149. Manichanh C, Borruel N, Casellas F, et al.. The gut microbiota in IBD. Nat Rev Gastroenterol Hepatol. 2012;9:599–608.
150. Leung CH, Lam W, Ma DL, et al.. Butyrate mediates nucleotide-binding and oligomerisation domain (NOD) 2-dependent mucosal immune responses against peptidoglycan. Eur J Immunol. 2009;39:3529–3537.
151. Malo MS, Biswas S, Abedrapo MA, et al.. The pro-inflammatory cytokines, IL-1beta and TNF-alpha, inhibit intestinal alkaline phosphatase gene expression. DNA Cell Biol. 2006;25:684–695.
152. Angrisano T, Pero R, Peluso S, et al.. LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes. BMC Microbiol. 2010;10:172.
153. Schmeck B, Beermann W, van Laak V, et al.. Intracellular bacteria differentially regulated endothelial cytokine release by MAPK-dependent histone modification. J Immunol. 2005;175:2843–2850.
154. Schmeck B, Lorenz J, N'Guessan PD, et al.. Histone acetylation and flagellin are essential for Legionella pneumophila-induced cytokine expression. J Immunol. 2008;181:940–947.
155. Slevogt H, Schmeck B, Jonatat C, et al.. Moraxella catarrhalis induces inflammatory response of bronchial epithelial cells via MAPK and NF-kappaB activation and histone deacetylase activity reduction. Am J Physiol Lung Cell Mol Physiol. 2006;290:L818–L826.
156. Haller D, Holt L, Kim SC, et al.. Transforming growth factor-beta 1 inhibits non-pathogenic Gram negative bacteria-induced NF-kappa B recruitment to the interleukin-6 gene promoter in intestinal epithelial cells through modulation of histone acetylation. J Biol Chem. 2003;278:23851–23860.
157. Hamon MA, Cossart P. Histone modifications and chromatin remodeling during bacterial infections. Cell Host Microbe. 2008;4:100–109.
158. Fehri LF, Rechner C, Janssen S, et al.. Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric epithelial cells reflects its impact on cell cycle regulation. Epigenetics. 2009;4:577–586.
159. Ding SZ, Fischer W, Kaparakis-Liaskos M, et al.. Helicobacter pylori-induced histone modification, associated gene expression in gastric epithelial cells, and its implication in pathogenesis. PLoS One. 2010;5:e9875.
160. Angrisano T, Lembo F, Peluso S, et al.. Helicobacter pylori regulates iNOS promoter by histone modifications in human gastric epithelial cells. Med Microbiol Immunol. 2012;201:249–257.
161. Ghadimi D, Helwig U, Schrezenmeir J, et al.. Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system. J Leukoc Biol. 2012;92:895–911.
162. Gonsky R, Deem RL, Landers CJ, et al.. Distinct IFNG methylation in a subset of ulcerative colitis patients based on reactivity to microbial antigens. Inflamm Bowel Dis. 2011;17:171–178.
163. Mohamadzadeh M, Pfeiler EA, Brown JB, et al.. Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid. Proc Natl Acad Sci U S A. 2011;108(suppl 1):4623–4630.
164. Lightfoot YL, Yang T, Sahay B, et al.. Targeting aberrant colon cancer-specific DNA methylation with lipoteichoic acid-deficient Lactobacillus acidophilus. Gut Microbes. 2013;4:84–88.
165. Lightfoot YL, Mohamadzadeh M. Tailoring gut immune responses with lipoteichoic acid-deficient Lactobacillus acidophilus. Front Immunol. 2013;4:25.
166. Takahashi K, Sugi Y, Hosono A, et al.. Epigenetic regulation of TLR4 gene expression in intestinal epithelial cells for the maintenance of intestinal homeostasis. J Immunol. 2009;183:6522–6529.
167. Takahashi K, Sugi Y, Nakano K, et al.. Epigenetic control of the host gene by commensal bacteria in large intestinal epithelial cells. J Biol Chem. 2011;286:35755–35762.
168. Kellermayer R, Dowd SE, Harris RA, et al.. Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice. FASEB J. 2011;25:1449–1460.
169. Yin L, Chung WO. Epigenetic regulation of human beta-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria. Mucosal Immunol. 2011;4:409–419.
170. Olszak T, An D, Zeissig S, et al.. Microbial exposure during early life has persistent effects on natural killer T cell function. Science. 2012;336:489–493.
171. Kellermayer R, Balasa A, Zhang W, et al.. Epigenetic maturation in colonic mucosa continues beyond infancy in mice. Hum Mol Genet. 2010;19:2168–2176.
172. Schaible TD, Harris RA, Dowd SE, et al.. Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes. Hum Mol Genet. 2011;20:1687–1696.
173. Nagy-Szakal D, Ross MC, Dowd SE, et al.. Maternal micronutrients can modify colonic mucosal microbiota maturation in murine offspring. Gut Microbes. 2012;3:426–433.
174. Mir SA, Nagy-Szakal D, Dowd SE, et al.. Prenatal methyl-donor supplementation augments colitis in young adult mice. PLoS One. 2013;8:e73162.
175. Rakyan VK, Down TA, Balding DJ, et al.. Epigenome-wide association studies for common human diseases. Nat Rev Genet. 2011;12:529–541.
176. Tsaprouni LG, Ito K, Powell JJ, et al.. Differential patterns of histone acetylation in inflammatory bowel diseases. J Inflamm (Lond). 2011;8:1.
177. Sadler T, Scarpa M, Rieder F, et al.. Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition. Inflamm Bowel Dis. 2013;19:1354–1364.
178. McKenna LB, Schug J, Vourekas A, et al.. MicroRNAs control intestinal epithelial differentiation, architecture, and barrier function. Gastroenterology. 2010;139:1654–1664, 1664 e1651.
179. De Iudicibus S, Lucafo M, Martelossi S, et al.. MicroRNAs as tools to predict glucocorticoid response in inflammatory bowel diseases. World J Gastroenterol. 2013;19:7947–7954.
180. Coskun M, Bjerrum JT, Seidelin JB, et al.. MicroRNAs in inflammatory bowel disease—pathogenesis, diagnostics and therapeutics. World J Gastroenterol. 2012;18:4629–4634.
181. Correa P, Haenszel W, Cuello C, et al.. A model for gastric cancer epidemiology. Lancet. 1975;2:58–60.
182. Peterson AJ, Menheniott TR, O'Connor L, et al.. Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans. Gastroenterology. 2010;139:2005–2017.
183. Tomita H, Takaishi S, Menheniott TR, et al.. Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing. Gastroenterology. 2011;140:879–891.
184. Cheng AS, Li MS, Kang W, et al.. Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis. Gastroenterology. 2013;144:122–133.e129.
185. Schmid CA, Muller A. FoxD3 is a novel, epigenetically regulated tumor suppressor in gastric carcinogenesis. Gastroenterology. 2013;144:22–25.
186. Cho JH. The genetics and immunopathogenesis of inflammatory bowel disease. Nat Rev Immunol. 2008;8:458–466.
187. Hartnett L, Egan LJ. Inflammation, DNA methylation and colitis-associated cancer. Carcinogenesis. 2012;33:723–731.
188. Hahn MA, Hahn T, Lee DH, et al.. Methylation of polycomb target genes in intestinal cancer is mediated by inflammation. Cancer Res. 2008;68:10280–10289.
189. Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009;1:239–259.
190. Duffy MJ, Napieralski R, Martens JW, et al.. Methylated genes as new cancer biomarkers. Eur J Cancer. 2009;45:335–346.
191. Ogino S, Nosho K, Kirkner GJ, et al.. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58:90–96.
192. Konishi K, Shen L, Wang S, et al.. Rare CpG island methylator phenotype in ulcerative colitis-associated neoplasias. Gastroenterology. 2007;132:1254–1260.
193. Azarschab P, Porschen R, Gregor M, et al.. Epigenetic control of the E-cadherin gene (CDH1) by CpG methylation in colectomy samples of patients with ulcerative colitis. Genes Chromosomes Cancer. 2002;35:121–126.
194. Moriyama T, Matsumoto T, Nakamura S, et al.. Hypermethylation of p14 (ARF) may be predictive of colitic cancer in patients with ulcerative colitis. Dis Colon Rectum. 2007;50:1384–1392.
195. Wang FY, Arisawa T, Tahara T, et al.. Aberrant DNA methylation in ulcerative colitis without neoplasia. Hepatogastroenterology. 2008;55:62–65.
196. Dhir M, Montgomery EA, Glockner SC, et al.. Epigenetic regulation of WNT signaling pathway genes in inflammatory bowel disease (IBD) associated neoplasia. J Gastrointest Surg. 2008;12:1745–1753.
197. Li Y, de Haar C, Chen M, et al.. Disease-related expression of the IL6/STAT3/SOCS3 signalling pathway in ulcerative colitis and ulcerative colitis-related carcinogenesis. Gut. 2010;59:227–235.
198. Olaru AV, Cheng Y, Agarwal R, et al.. Unique patterns of CpG island methylation in inflammatory bowel disease-associated colorectal cancers. Inflamm Bowel Dis. 2012;18:641–648.
199. Hmadcha A, Bedoya FJ, Sobrino F, et al.. Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production. J Exp Med. 1999;190:1595–1604.
200. Foran E, Garrity-Park MM, Mureau C, et al.. Upregulation of DNA methyltransferase-mediated gene silencing, anchorage-independent growth, and migration of colon cancer cells by interleukin-6. Mol Cancer Res. 2010;8:471–481.
201. Kominsky DJ, Keely S, MacManus CF, et al.. An endogenously anti-inflammatory role for methylation in mucosal inflammation identified through metabolite profiling. J Immunol. 2011;186:6505–6514.
202. O'Hagan HM, Wang W, Sen S, et al.. Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Cancer Cell. 2011;20:606–619.
203. Li Y, Deuring J, Peppelenbosch MP, et al.. IL-6-induced DNMT1 activity mediates SOCS3 promoter hypermethylation in ulcerative colitis-related colorectal cancer. Carcinogenesis. 2012;33:1889–1896.
204. Azuara D, Rodriguez-Moranta F, de Oca J, et al.. Novel methylation panel for the early detection of neoplasia in high-risk ulcerative colitis and Crohn's colitis patients. Inflamm Bowel Dis. 2013;19:165–173.
205. Papadia C, Louwagie J, Del Rio P, et al.. FOXE1 and SYNE1 genes hypermethylation panel as promising biomarker in colitis-associated colorectal neoplasia. Inflamm Bowel Dis. 2014;20:271–277.
206. Tahara T, Shibata T, Nakamura M, et al.. Promoter methylation of protease-activated receptor (PAR2) is associated with severe clinical phenotypes of ulcerative colitis (UC). Clin Exp Med. 2009;9:125–130.
207. Tahara T, Shibata T, Nakamura M, et al.. Effect of MDR1 gene promoter methylation in patients with ulcerative colitis. Int J Mol Med. 2009;23:521–527.
208. Garrity-Park MM, Loftus EV Jr, Sandborn WJ, et al.. Methylation status of genes in non-neoplastic mucosa from patients with ulcerative colitis-associated colorectal cancer. Am J Gastroenterol. 2010;105:1610–1619.
209. Kim SW, Kim ES, Moon CM, et al.. Abnormal genetic and epigenetic changes in signal transducer and activator of transcription 4 in the pathogenesis of inflammatory bowel diseases. Dig Dis Sci. 2012;57:2600–2607.
210. Zahm AM, Thayu M, Hand NJ, et al.. Circulating microRNA is a biomarker of pediatric Crohn disease. J Pediatr Gastroenterol Nutr. 2011;53:26–33.
211. Fasseu M, Treton X, Guichard C, et al.. Identification of restricted subsets of mature microRNA abnormally expressed in inactive colonic mucosa of patients with inflammatory bowel disease. PLoS One. 2010;5.
212. Wu F, Zikusoka M, Trindade A, et al.. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. Gastroenterology. 2008;135:1624–1635.e1624.
213. Duttagupta R, DiRienzo S, Jiang R, et al.. Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis. PLoS One. 2012;7:e31241.
214. Wu F, Guo NJ, Tian H, et al.. Peripheral blood microRNAs distinguish active ulcerative colitis and Crohn's disease. Inflamm Bowel Dis. 2011;17:241–250.
215. Coskun M, Bjerrum JT, Seidelin JB, et al.. miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis. World J Gastroenterol. 2013;19:4289–4299.
216. Iborra M, Bernuzzi F, Correale C, et al.. Identification of serum and tissue micro-RNA expression profiles in different stages of inflammatory bowel disease. Clin Exp Immunol. 2013;173:250–258.
217. Glauben R, Siegmund B. Inhibition of histone deacetylases in inflammatory bowel diseases. Mol Med. 2011;17:426–433.
218. Edwards AJ, Pender SL. Histone deacetylase inhibitors and their potential role in inflammatory bowel diseases. Biochem Soc Trans. 2011;39:1092–1095.
219. Rosen MJ, Frey MR, Washington MK, et al.. STAT6 activation in ulcerative colitis: a new target for prevention of IL-13-induced colon epithelial cell dysfunction. Inflamm Bowel Dis. 2011;17:2224–2234.
220. Wang LS, Kuo CT, Stoner K, et al.. Dietary black raspberries modulate DNA methylation in dextran sodium sulfate (DSS)-induced ulcerative colitis. Carcinogenesis. 2013;34:2842–2850.
221. Macfarlane AJ, Behan NA, Matias FM, et al.. Dietary folate does not significantly affect the intestinal microbiome, inflammation or tumorigenesis in azoxymethane-dextran sodium sulphate-treated mice. Br J Nutr. 2013;109:630–638.
222. Chen M, Peyrin-Biroulet L, George A, et al.. Methyl deficient diet aggravates experimental colitis in rats. J Cell Mol Med. 2011;15:2486–2497.
223. Yara S, Lavoie JC, Beaulieu JF, et al.. Iron-ascorbate-mediated lipid peroxidation causes epigenetic changes in the antioxidant defense in intestinal epithelial cells: impact on inflammation. PLoS One. 2013;8:e63456.
224. O'Gorman A, Colleran A, Ryan A, et al.. Regulation of NF-kappaB responses by epigenetic suppression of IkappaBalpha expression in HCT116 intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2010;299:G96–G105.
225. Turgeon N, Moore Gagne J, Blais M, et al.. The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis. Am J Physiol Gastrointest Liver Physiol. 2014;306:G594–G605.
226. Meng X, Zhang Y, Jia Z, et al.. A novel tylophorine analog W-8 up-regulates forkhead boxP3 expression and ameliorates murine colitis. J Leukoc Biol. 2013;93:83–93.
227. Singh NP, Singh UP, Singh B, et al.. Activation of aryl hydrocarbon receptor (AhR) leads to reciprocal epigenetic regulation of FoxP3 and IL-17 expression and amelioration of experimental colitis. PLoS One. 2011;6:e23522.