Sage Journals: Discover world-class research

Abstract

The term ‘epigenetics’ loosely describes DNA-templated processes leading to heritable changes in gene activity and expression, which are independent of the underlying DNA sequence. Epigenetic mechanisms comprise of post-translational modifications of chromatin, methylation of DNA, nucleosome positioning as well as expression of noncoding RNAs. Major advances in understanding the role of DNA methylation in regulating chromatin functions have been made over the past decade, and point to a role of this epigenetic mechanism in human disease. Rheumatoid arthritis (RA) is an autoimmune disorder where altered DNA methylation patterns have been identified in a number of different disease-relevant cell types. However, the contribution of DNA methylation changes to RA disease pathogenesis is at present poorly understood and in need of further investigation. Here we review the current knowledge regarding the role of DNA methylation in rheumatoid arthritis and indicate its potential therapeutic implications.

Keywords

DNA methylation epigenetics rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory condition of the joints that affects approximately 1% of the world’s population. It is a systemic inflammatory disease with characteristic features of joint swelling, pain and stiffness [Feldmann et al. 1996]. Major advancements in understanding the development of RA originate from studies investigating the expression and regulation of pro-inflammatory cytokines within the affected synovial tissue. One of these pro-inflammatory cytokines, tumour necrosis factor (TNF)-α, is a pivotal factor in the inflammatory cascade and led to its identification as a target for therapeutic intervention [Brennan et al. 1992]. Subsequently, in 1992, the first open-label trial of a TNF-α blocking agent was initiated at the Charing Cross Hospital, United Kingdom [Elliott et al. 1993]. Anti-TNF therapy is one of the most successful treatments for RA and has paved the way for other biological therapies, targeting CTLA-4, CD20 and IL-6 (e.g. abatacept, rituximab and tocilizumab) [Malottki et al. 2011]. Despite their successes, these numerous new medicines for inflammatory arthritis all have broadly similar efficacy and benefit only a proportion of patients treated. Undoubtedly it is a difficult task to develop new treatment options, however it is hoped that an understanding of the complexity underpinning the regulation of gene expression in different cell types that are critically involved will yield an improved understanding of the causes of the disease. In this review we aim to collate the current knowledge on DNA methylation in autoimmunity with a particular focus on RA, its role in altering gene expression in different cells that contribute to the pathogenesis of RA, and discuss its therapeutic and diagnostic potential.

Genetic and nongenetic factors influencing rheumatoid arthritis onset

The etiological paradigm of RA is that an environmental trigger in a genetically predisposed individual can induce autoimmune responses. Most attention thus far has been given to determination of genetic factors governing the susceptibility of an individual to developing RA. A key observation made decades ago was that specific alleles of the class II major histocompatibility complex (MHC) confer susceptibility to RA [Winchester, 1994]. In particular, susceptibility to RA was found to be associated with certain HLA-DRB1 alleles, which encode for a similar amino acid sequence (QKRAA, QRRAA or RRRAA) providing the rationale for the aptly named ‘shared-epitope’ hypothesis (reviewed by Ollier and Thompson [1992] and Orozco et al. [2006]). Subsequently, with the advent of genome wide association studies (GWAS) a number of other non-HLA susceptibility alleles have also been implicated, such as PTPN22, PADI4, CD40 and CTLA-4, many of which are implicated in T cell signalling and function [Suzuki et al. 2011].

Although it is undeniable that genetic factors play a major role in the susceptibility to RA, the low concordance rate (12–20%) observed in RA between monozygotic twins suggests that environment factors play a significant role in the pathogenesis of RA [Aho et al. 1986; Silman et al. 1993; Svendsen et al. 2002]. Indeed, a number of potential environmental risk factors have been implicated, including smoking and periodontitis [De Pablo et al. 2008]. Porphyromonas gingivalis, the cause of periodontitis, expresses an enzyme with peptidylarginine deiminase (PAD) activity (termed PPAD in P. gingivalis), leading to citrullinated peptide residues. Autoantibodies against citrullinated peptides are present in the majority of RA patients and it has been suggested that PPAD activity, which enzymatically regulates the conversion of peptidyl-arginine to peptidyl-citrulline, can mediate the generation of autoantibodies that drive autoimmunity in RA [Lundberg et al. 2010]. Similarly, cigarette smoking, which is also associated with periodontitis, can influence not only disease onset but can contribute to the severity of RA in a dose-dependent fashion [Saag et al. 1997]. In addition to smoking, other known environmental factors include latent viral infections [Costenbader and Karlson, 2006], sex hormones [Cutolo et al. 2002] and vitamin D [Merlino et al. 2004]. It is thought that these environmental factors influence epigenetic modifications, which in concert with the individual genetic susceptibility status result in the development of RA symptoms. Since these environmental factors play a significant role in RA pathogenesis, understanding epigenetic modifications that constitute the underpinning link between environmental cues and disease will provide a better understanding of how to treat RA.

Epigenetics and chromatin modification

The British developmental biologist Conrad Waddington originally coined the term ‘epigenetics’ in 1942 when he attempted to link genotypic and phenotypic changes during embryogenesis [Waddington, 1942, 2012]. Epigenetics was initially broadly defined as the ‘causal mechanisms’ that link the genetic information of an organism and how it interacts with the environment in order to produce observable traits. More recently the Epigenetic Roadmap Project has defined epigenetics as referring to both DNA-independent heritable changes in gene activity and expression, and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable, thereby including much broader chromatin-templated processes. DNA is wrapped around an octamer of histone proteins H3, H4, H2A and H2B, thus forming the complex structure of chromatin [Li, 2002]. Posttranscriptional modification of the histone tails and methylation of cytosine bases in the DNA can modify the accessibility of chromatin and alter gene transcription by allowing transcription factors to bind at promoter sites and initiate transcription [Ernst et al. 2011; ENCODE Project Consortium et al. 2012]. The dynamic nature of these modifications means that these changes are not always stable, and they can be altered in response to stimuli [Bird, 2007; Lund et al. 2012]. Moreover, the sequence and modification-specific context of chromatin modifications, and the particular composition and recognition potential of regulatory factors led to the hypothesis of a ‘histone code’ that controls specific DNA-templated systems such as gene transcription, genome stability, imprinting and X-chromosome inactivation [Jenuwein and Allis, 2001]. The complexity, dynamics and specific roles of these post-translational modifications on histones and other nuclear proteins, and the associated recognition systems in genome and chromatin biology is subject of current studies, revealing a multitude of covalent modifications whose significance is only partially understood (reviewed by Rothbart and Strahl [2014]). Since epigenetic modifications are less stable than genome sequences, these processes are subject to variable responses and hence can lead to altered development and disease. Epigenetics and the study of chromatin modifications has gained increased interest because it has been realized that the study of epigenetic modifications specific to disease may give a mechanistic insight into age-related autoimmunity [Hirst and Marra, 2009]. Therefore, when investigating RA in the context of epigenetics it may provide an indication as to why genetically predisposed individuals develop disease while others do not, and may also explain the heterogeneity of symptoms and therapeutic responses observed [Oppermann, 2013].

DNA methylation

Methylation of the carbon 5 position of cytosine (5mC) is one of the best-studied and mechanistically understood epigenetic mechanisms, however it is clear that knowledge of DNA methylation is still incomplete [Smith and Meissner, 2013]. In mammals, cytosine methylation occurs largely in the context of CpG dinucleotides, which are often clustered in 5’ regulatory regions termed CpG islands [Deaton and Bird, 2011], typically encompassing the first exons of genes [Yoder et al. 1997]. In mammals, most CpG dinucleotides are unmethylated and are often clustered in 5’ regulatory regions termed CpG islands [Deaton and Bird, 2011], typically encompassing the first exons of genes [Yoder et al. 1997]. Typically, an area is termed a CpG island when, on average, a region of 1000 base pairs long shows an elevated C+G base composition and an absence of DNA methylation [Deaton and Bird, 2011]. The precise mechanism of how CpG islands are protected against methylation is not completely understood, but transgenic mice experiments suggest that protection may be directed by recognition of common cis-acting sequences located in CpG islands [Siegfried et al. 1999]. However, when methylated, CpG islands can constitute a powerful gene silencing mechanism (described below).

Other regions such as the gene body show a high degree of methylation, which is often associated with a higher level of gene expression in a dividing cell [Hellman and Chess, 2007]. In contrast to gene bodies, promoter regions are typically lacking DNA methylation [He et al. 2011]. IN addition, DNA methylation also plays a major role in suppressing transposable elements, by providing long-term heritable gene silencing [Wu and Zhang, 2010]. Around 45% of the mammalian genome consists of transposable and viral elements [Schulz et al. 2006]. These viral insertions require long-term silencing because if expressed they are potentially harmful because their replication and insertion can lead to aberrant gene expression.

The process of cytosine methylation is catalysed by DNA methyltransferases (DNMTs), which use S-adenosylmethionine (SAM) as the methyl donor [Chiang et al. 1996]. DNMTs are divided into two classes based upon their physiological function. One category comprises the de novo methyltransferases DNMT3A and DNMT3B, which establish methylation patterns in both unmethylated and hemimethylated DNA with equal efficiencies [Okano et al. 1998, 1999; Yanagisawa et al. 2002; Chen and Li, 2004]. The other category comprises the ‘maintenance’ methyltransferase DNMT1, which recognizes and copies methylated CpGs to newly synthesized DNA [Bestor and Ingram, 1983; Bestor et al. 1988]. DNMT1 is upregulated during S phase and is recruited to DNA replication forks and is preferentially active on hemi-methylated CpG substrates [Pradhan and Esteve, 2003]. DNMT1 then methylates CpG sites on new daughter strands, thereby maintaining the methylation patterns that are complementary to the parent strand [Wilson et al. 2005]. The importance of these enzymes is demonstrated by the loss of DNMT1 or DNMT3b in mice resulting in embryonic lethality around day (E) 9 and loss of DNMT3a leads to severe runting and death around 1 month after birth [Li et al. 1992; Okano et al. 1999]. Moreover, in humans mutations in DNMT3B are responsible for the immunodeficiency, centromere instability and facial abnormalities (ICF) disorder [Hansen et al. 1999; Xu et al. 1999; Jin et al. 2008], while mutations in DNMT3A have been reported in acute myeloid leukaemia [Ley et al. 2010; Yan et al. 2011].

DNA demethylation

Although DNA cytosine methylation has been regarded in the past as a stable epigenetic mark in adult vertebrates, more recent studies have revealed that this mark is not as static as previously thought. In particular, evidence that efficient DNA demethylation systems exist is highlighted during development, where DNA methylation following fertilization is nearly completely erased, and specifically re-installed during embryonic development. DNA demethylation can be reversed through passive and active processes. Passive DNA demethylation refers to the relative lack of DNMT activity across cell divisions, leading to a gradual reduction in global DNA methylation levels [Monk et al. 1987], whereas active DNA demethylation refers to the enzymatic process through which the methyl group on the cytosine is removed. Several mechanisms have been proposed to explain the process of active DNA demethylation. The simplest of these mechanisms is the direct enzymatic removal of the methyl group from 5mC. Although this reaction was initially reported for methyl-CpG-binding domain protein 2 (MBD2) [Bhattacharya et al. 1999], its proposal as a DNA demethylase enzyme is controversial and other laboratories have not been able to reproduce the reported enzymatic activity [Wu and Zhang, 2010]. Regardless of this, it is still conceivable that a DNA demethylase enzyme exists, however at present no other enzyme has been described to fit this role.

Another proposed mechanism by which DNA demethylation occurs is through oxidative modification followed by removal of the base. Two seminal papers published in 2009 described the presence of 5-hydroxymethylcytosine (5hmC) and gave the first insights into the process of active DNA demethylation in both mouse and human [Kriaucionis and Heintz, 2009; Tahiliani et al. 2009]. The ten–eleven translocation (TET) enzymes, belonging to a large family of Fe2+ and alpha-ketoglutarate dependent dioxygenase enzymes have now been demonstrated as the DNA hydroxylases responsible for the conversion of 5mC to 5hmC [Johansson et al. 2014]. Furthermore, TET enzymes have been described as being responsible for the further oxidation of 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (reviewed by Shen et al. [2014]). Following this, an unmodified C can be generated through either a replication dependent dilution of the 5hmC or through base excision repair (BER) processes, mediated by the excision of 5fC or 5caC by thymine DNA glycosylase (TDG) (Figure 1) [Kohli and Zhang, 2013].

Figure 1.

The DNA demethylation cycle. Cytosine methylation is catalyzed by DNA methyltransferases (DNMTs). 5mC is hydroxylated by the ten–eleven translocation enzymes (TETs) to yield 5-hydroxymethylcytosine (5hmC), which in turn is oxidized to 5fC and 5caC. During passive demethylation, 5hmC, 5fC and 5caC are diluted in a replication dependent manner to regenerate an unmodified C. In a process of active restoration, 5fC and 5caC can be excised by TDG generating an abasic site as part of the base excision repair (BER) process that regenerates unmodified C. Red arrows denote passive DNA demethylation mechanisms and black arrows denote active DNA demethylation.

Genome-wide mapping has revealed that 5hmC plays a role in both transcriptional activation and silencing [Wu and Zhang, 2011]. Recent progress in identification of different types of protein modules that recognize unmethylated CpG dinucleotides (by CxxC domain containing proteins), 5mC and/or its oxidation products 5hmC, 5fC, 5caC (such as MeCp binding domains, Zn-finger and SET and RING-associated (SRA)) sheds new light on the complexity of DNA methylation (reviewed by Rothbart and Strahl [2014]) as well as its interaction with posttranslational histone modifications in chromatin regulation [Rose and Klose, 2014].

Although there have been some major advancements made into investigating the role of DNA methylation in disease, little is known about the role of 5hmC, 5fC or 5caC. However, the processes that regulate DNA demethylation have been implicated in cancer. For example, fusion of MLL to TET1 is important for acute myeloid leukaemia [Ono et al. 2002], and inactivating TET2 mutations are frequently found in myeloid lineage malignancies [Delhommeau et al. 2009; Langemeijer et al. 2009]. Interestingly, in nonmyeloid cancers, downregulation of TET expression has been observed in human breast, liver, pancreatic and prostate cancer [Yang et al. 2013]. These disruptions in TET are associated with a decrease in 5hmC, which is in keeping with findings from combined TET1 and TET2 knockout mice that show decreases in 5hmC and increased 5mC expression [Dawlaty et al. 2013].

DNA methylation and gene regulation

It is well established that hypermethylation of promoter elements results in transcriptional silencing [Nakano et al. 2013]. This inverse correlation between DNA methylation and transcriptional activation has been verified in numerous experiments in which plasmid DNA is in vitro methylated and transfected into target cells [Yisraeli et al. 1988; Kass et al. 1997]. Similarly, increased gene expression is also demonstrated following culture with the demethylating agent 5-azacytidine [Christman, 2002]. There are two proposed hypotheses for explaining the mechanism for transcriptional silencing mediated by DNA methylation. One hypothesis that has been proposed is that methylation can directly inhibit binding of transcriptional regulators. The use of reporter constructs in which in vitro methylation has been shown to reduce the binding of a number of different transcription factors [Eden and Cedar, 1994]. This interference has been demonstrated for a number of transcription factors such as c-Myc [Prendergast and Ziff, 1991], E2F [Di Fiore et al. 1999; Campanero et al. 2000], nuclear factor of activated T cells (NFAT) [Cribbs et al. 2014] and NF-κB [Bednarik et al. 1991], all of which have a CpG dinucleotide within their core binding motif. A second hypothesis proposes that methylation-specific binding proteins (MBP) interact with methylated DNA and bind to prevent transcriptional initiation complexes [Boyes and Bird, 1991]. The family of MBP proteins is composed of five members, methyl-CpG-binding protein 2 (MeCp2), MBP1, MBP2, MBP3 and MBP4. With the exception of MBP4, which is involved in the DNA repair process, these proteins associate with histone deacetylases (HDACs) and result in transcriptional silencing through altered chromatin structure [Wade et al. 1999; Momparler, 2003]. DNA methylation also directs dimethylation of histone 3 lysine 9 (H3K9me2), a repressive chromatin mark, through the interaction of DNMT1 with the replication complex [Hashimshony et al. 2003; Esteve et al. 2006]. In addition, there is also evidence to suggest that DNA methylation inhibits histone 3 lysine 4 (H3K4) methylation, a mark indicative of active transcription, however the precise mechanism underpinning this is currently unknown [Hashimshony et al. 2003; Okitsu and Hsieh, 2007]. Therefore, it seems likely that a DNA methylation profile acts as a scaffold to maintain transcriptional repression patterns through modification of active marks on histone tails.

DNA methylation in RA

Whole genome analysis of aged individuals has highlighted a number of hypomethylated regions that may contribute to age-related diseases, including some autoimmune diseases such as RA [Heyn et al. 2012]. The first evidence to suggest that DNA methylation may play a role in ageing and autoimmunity came from studies investigating the effect of the DNA methyltransferase inhibitor 5-azacytidine that can induce symptoms associated with autoimmunity [Richardson, 1986; Richardson et al. 1986]. Moreover, there is now a large body of evidence to suggest that patients with certain autoimmunity syndromes such as systemic lupus erythematosus (SLE) display a globally hypomethylated genome [Liu et al. 2011a, 2011b]. Specifically for RA, differential DNA methylation has been demonstrated in the IL-6 promoter [Nile et al. 2008]. In this paper, Nile and colleagues isolated DNA from peripheral blood mononuclear cells (PBMCs) and determined the methylation status of the IL-6 promoter. They found that IL-6 promoter methylation reduced transcriptional activity and identified a single CpG within the IL-6 promoter that was key to regulating IL-6 gene expression. Similarly, another study identified an association between smoking and hypomethylation of a CpG motif in the IL-6 promoter of chronic periodontitis patients [Ishida et al. 2012]. However, a note of caution needs to be made when interpreting studies where the data has been generated using DNA isolated from whole PBMC populations. A large proportion of RA patients can present with leukopenia, with altered PBMC cellular frequencies and compositions. Since methylation patterns are cell-type specific, and the balance of T cells in the RA patients can be significantly altered, results from unsorted cell populations may skew results and interpretations from whole blood methylome analyses [Martens et al. 1997; Lawson et al. 2006].

More recently, studies have begun to utilize new technologies and take into account that some of these differences could be attributed to the altered frequency of cell populations. Liu and colleagues performed a genome-wide epigenome analysis on PBMC populations using the Illumina 450K methylation bead array and used a series of statistical algorithms to reduce the confounding factors that have hampered previous analysis [Liu et al. 2013]. Using this approach they were able to filter out methylation changes that are likely to cause disease, rather than those changes that are a consequence of disease. The consequence of this analysis was the identification of two clusters within the MHC region whose differential methylation pattern potentially mediates genetic risk for RA. In the general context of disease pathogenesis, this study is important because it suggests that DNA methylation is a critical mechanism for conferring disease in already identified associated genes such as the MHC region. However, one limitation of this study is that the contribution of other disease correlated cell types, such as synovial fibroblasts are missing from their analysis. Moreover, since the study relies on the use of computational approaches to correct for differences in cell type, the results from this study should be validated in isolated populations. Therefore, although this study gives us the first glimpse of the relationship between DNA methylation and genetic susceptibility, further investigations exploring the functional consequences of these changes need to be conducted.

DNA methylation in RA fibroblasts

Epigenetic investigations into synovial fibroblasts isolated from RA patients have so far provided the best evidence that epigenetic modifications contribute to the disease pathology of RA. RA synovial fibroblasts (RASF) are intensively studied cells primarily because they are abundant in the joint and are key contributors to the high levels of cytokine expression seen in RA. RASF display a DNA methylome pattern distinctive from osteoarthritis synovial fibroblasts (OASF) that contributes to their pathogenic function [Nakano et al. 2013]. Early reports have suggested that RASF display a hypomethylated genome [Karouzakis et al. 2009]. This was specifically shown in a number of key genes regulating cell migration, adhesion and extracellular matrix interactions. Intriguingly, DNMT1 expression was reduced in RASF compared to OASF and may provide a mechanism for the reduction in methylation [Karouzakis et al. 2009]. However, despite the apparent global hypomethylation seen in RASF from early studies, later studies have not confirmed this finding [Takami et al. 2006; De La Rica et al. 2013; Nakano et al. 2013; Whitaker et al. 2013]. Specifically, a number of differentially methylated regions, including both hypomethylated and hypermethylated regions were observed. In addition, the reduction of DNMTs shown in the earlier studies has also not been confirmed in later studies [Nakano et al. 2013]. Notwithstanding these results, a note of caution should be made regarding the findings from RASFs because many studies remove the RASF cells from their environmental niche and passage them in vitro. However, although Nakano and colleagues have shown that the methylation pattern in RASF are stable following multiple passages [Nakano et al. 2013], more work is required to determine whether some of these observed differences are also present in freshly isolated RASFs.

DNA methylation in RA T cells

The role of T cell DNA hypomethylation in the autoimmune disease SLE is well established (reviewed by Zhang et al. [2013]), however little is currently known about the role of DNA methylation in T cells from RA affected individuals. The first evidence to suggest that RA patients exhibit dysregulated T-cell methylation patterns was provided by Richardson and colleagues who showed global hypomethylation in CD4+ RA T cells when compared to healthy controls [Richardson et al. 1990]. Looking at gene-specific methylation patterns at critical factors, it was more recently discovered that CD4⁺CD28^- T cells, a population of pro-inflammatory T cells found frequently in RA, have a significantly hypomethylated IFNG promoter than conventional CD4⁺CD8⁺ T cells and produced higher levels of IFN-γ following TCR stimulation [Pieper et al. 2014]. DNA demethylation also plays a role in the regulation of the X linked gene CD40L in CD4⁺ T cells and may in part explain the female prevalence of RA [Liao et al. 2012]. Similarly, DNA methylation dynamics plays a key role in the differentiation of different Th cell subsets [Cohen et al. 2011]. Janson and colleagues demonstrated that CD4⁺ T cells in the synovial joint are committed towards a Th1 and a regulatory T-cell phenotype. They demonstrated that IL17A gene expression was regulated by promoter methylation however Th17 commitment was not a common feature found in RA synovial joints [Janson et al. 2011]. Work from our own laboratory has shown that DNA methylation contributes to dysregulated regulatory T-cell (Treg) function [Cribbs et al. 2014]. In untreated RA patients, we found that reduced CTLA-4 protein expression was a consequence of increased methylation of a single CpG dinucleotide in the CTLA4 promoter. Methylation of this site prevented the binding of the transcription factor, NFAT, which in turn led to a reduction in the transcriptional activity of the CTLA4 gene. [Bernard, 2014; Cribbs et al. 2014]. Subsequently, we showed that in a cohort of RA patients treated with methotrexate (MTX), demethylation of the upstream enhancer in the FOXP3 locus in RA patients is correlated with decreased Dnmt1 expression and increased expression of Foxp3 protein levels [Kennedy et al. 2014; Cribbs et al. 2015]. A consequence of this is that CTLA-4 protein expression normalizes and Tregs are subsequently able to suppress effector T-cell responses (Figure 2). Overall, this suggests that MTX can induce DNA methylation changes in the FOXP3 locus that regulate the expression of Foxp3 in established RA disease, leading to increased Treg function and reduced disease activity.

Figure 2.

Methotrexate (MTX) treatment restores regulatory T cell (Treg) function in rheumatoid arthritis (RA) patients. In MTX naïve RA Tregs, a newly identified differentially methylated region (DMR) is methylated and this is associated with normal Foxp3 expression but reduced CTLA-4 expression. In patients treated with MTX, a reduction in DNMT1 expression correlates with reduced methylation of the DMR in Tregs. This leads to increased Foxp3 expression and a consequential increase in CTLA-4 expression that restores suppressive function. Overall, this suggests that MTX can induce epigenetic modifications in RA Tregs that lead to normal suppressive function and highlight a newly identified mechanism of action for MTX.

Therapeutic implications of altered DNA methylation patterns in RA

In malignant cells, global DNA hypomethylation is a commonly observed feature [Feinberg and Vogelstein, 1983], and the most important DNA methylation changes occur in CpG rich promoter regions. Despite the global hypomethylation pattern observed, around 5–10% of normally unmethylated CpG island promoters often display abnormal methylation in cancer genomes [Dawson and Kouzarides, 2012], affecting a set of critical tumour suppressors that are aberrantly silenced [Herman et al. 1994]. This has led to the implementation of strategies designed to reduce the overall DNA methylation in these regions, primarily through targeting DNMT activity. Currently, 5-azacytidine and decitabine are two FDA-approved therapies for use in myelodysplastic syndrome, a treatment that targets changes in DNA methylation patterns. Several studies have shown that 5-azacytidine and decitabine therapy can significantly extend survival time and improve quality of life, even in patients with late-stage disease [Kantarjian et al. 2006; Fenaux et al. 2009]. However, it is unlikely that those DNMT inhibitors will be of any significant benefit in RA since treatment of RASF with 5-azacytidine leads to increased activation, suggesting that hypomethylating treatments may result in increased disease [Karouzakis et al. 2009]. Moreover, 5-azacytidine and another DNMT inhibitor, procainamide, can induce self-reactivity in CD4⁺ T cells and are both sufficient to induce lupus-like disease [Richardson, 1986; Richardson et al. 1986; Quddus et al. 1993]. However, the recently described DNA methylation inhibitor, zebularine, has been used in combination with IFN-γ in vitro to synergistically induce the immunomodulatory indoleamine 2,3-dioxygenase (IDO) pathway [Zhou et al. 2002; Xue et al. 2012]. This suggests that in order for DNMT inhibitors to be successful and provide superior outcomes over established therapies they need to be given in the right disease context. Likewise, DNMT inhibitors may be more successful when supplied in combination with other epigenetic therapies such as HDAC inhibitors [Oppermann, 2013], a finding also reported in oncology [Gore, 2011].

Another promising therapeutic approach that may have a benefit for RA and is indirectly related to DNA methylation was suggested from the results of Karouzakis and colleagues. The authors found that increased expression of S-adenosyl methionine decarboxylase (AMD), spermidine/spermine N1-acetyltransferase (SSAT-1) and polyamine-modulated factor binding protein 1 (PMFBP-1) led to reduced levels of the methyl-donor SAM, which was associated with hypomethylation in RASF [Karouzakis et al. 2012]. It was suggested that the deficiency of SAM in RASFs could be overcome with dietary supplementation with SAM or L-methionine [Klein and Gay, 2013]. However, the authors suggest that the high levels of SSAT-1 expression would make this an unattractive therapeutic option, since the high levels of SSAT-1 expression would constantly consume the SAM. More recently, it has been shown that diminazene aceturate (DA), an inhibitor of SSAT-1, can restore both the levels of DNA methylation and the expression of DNMT1, leading to reduced RASF invasive function [Neidhart et al. 2014]. These results open up the possibility of using pharmacological therapy to restore the DNA methylation patterns in RASF, and subsequent efforts are being made to develop a more specific inhibitor of SSAT-1 [Onuora, 2014].

Despite the aforementioned advantages and disadvantages of targeting DNA methylation pathways for treating RA, there is indeed evidence to suggest that current therapies can actually modulate their anti-inflammatory effects through modification of DNA methylation. MTX, a therapy used successfully in high doses in oncology and at low dose in RA for several decades, is one example of a routinely used drug having epigenome modifying effects. For example, it has been known for a number of years that in human tumours, administration of MTZ induces profound DNA hypermethylation [Nyce, 1989]. Similarly in RA, MTX was found to reverse global DNA hypomethylation [Kim et al. 1996]. However, the mechanism underpinning this phenomenon is currently unknown and certainly warrants further investigation. Intriguingly, this raises the possibility that other drugs used to treat RA may also act long-term by altering and reversing causative epigenetic changes underlying chronic disease.

DNA methylation as a biomarker for RA

Despite the challenges associated with DNA methylation as a therapeutic target, its biggest potential impact may come from its use as a biomarker to determine response to therapy. An important issue that still needs to be addressed in epigenetic research is whether any identified disease-associated epigenome change precedes disease onset, or is a result of the ongoing disease process. Identification of aberrant DNA methylation changes before disease onset may lead to a better understanding of the risk factors that contribute to disease development and thus result in the identification of biomarkers for disease. However, this is currently very challenging to achieve because of the limited sample availability in predisease states.

A more realistic approach to using DNA methylation as a biomarker would be to identify responders and nonresponders to therapy. There have been a number of successful therapies for RA, however despite this, a proportion of patients fail to respond to conventional therapy. Ideally, it would be useful to identify this fraction of non-responders earlier, in order to provide better treatment regimes that are tailored towards the individual patient. It is becoming clear that RA patients display a differentially methylated genome when compared to healthy individuals. This raises the possibility that measuring DNA methylation patterns of responders and nonresponders may lead to the use of DNA methylation as a predictive biomarker for treatment response. This approach has been validated in colorectal cancer, where circulating methylated DNA in peripheral blood can be measured using a simple polymerase chain reaction (PCR) assay [Lofton-Day et al. 2008; Lange et al. 2012]. More recently, reverse transcriptase (RT)-PCR assays have been developed to quantify the number of Foxp3⁺ cells within tissue samples [Wieczorek et al. 2009]. Collectively, these techniques have revealed the potential for DNA methylation to be used as a biomarker for disease status and suggest that it may be useful for identifying patient response to therapy.

Conclusion and future direction

In recent years, a major advancement has taken place in understanding the role of DNA methylation in the pathogenesis of RA. It is hoped that the progress made in identifying epigenetic mechanisms occurring in cancer can also be exploited in inflammatory disease. The substantial clinical progress obtained by introducing highly effective biological treatments for autoimmune diseases will also lead to a better understanding of epigenetic factors that contribute or control the phenotype and clinical responses.

However, a consideration regards to the complexity of DNA methylation regulation in different cell types needs to be made. The main thrust of epigenetic research in RA is currently focused on synovial fibroblasts, not just because they are an important cell type that contributes to RA pathogenesis, but because they can be easily grown in sufficient quantities, thereby facilitating epigenome investigations that frequently rely on large numbers of cells to be analysed. However, if we are to completely understand the contribution of epigenetic modifications to disease then we need novel approaches that allow us to investigate all populations of cells from RA patients. Therefore, it is imperative to employ strategies that use cell sorting techniques to isolate cells to homogeneity and employ technologies that allow analysis of epigenetic modifications with small cell numbers. Technically, this has been hampered by the requirement for large quantities of DNA to perform techniques such as DNA sequencing and chromatin immunoprecipitation (CHIP) technology. However, the progress made in miniaturizing these technologies, and importantly the reduction in cost, will allow more precise and detailed genome wide analysis of epigenetic modifications in all cell populations in RA [Shankaranarayanan et al. 2011] and will constitute a further important step towards personalized medicine. One other area of epigenetics that still needs to be explored is the relationship in our understanding between the specific environmental factors and the influence on DNA methylation profiles [Javierre et al. 2011], which could provide the ultimate link between environmental factors and manifestation of nongenetic, but epigenomic effects leading to disease. It is hoped that in the near future in combination with technological advances some of these questions will be adequately addressed.

Footnotes

Funding

Research in our laboratories is supported through funding from Arthritis Research UK (program grant number 20522), the Oxford NIHR Biomedical Research Unit, the Kennedy Trust for Rheumatology Research, the Rosetrees Trust, Sarcoma (UK), Bone Cancer Research Trust, Medical Research Council, GlaxoSmithKline, UCB and Bayer Healthcare. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from Abbvie, Bayer Healthcare, Boehringer Ingelheim, the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Eli Lilly and Company, Genome Canada, GlaxoSmithKline, the Ontario Ministry of Economic Development and Innovation, Janssen, the Novartis Research Foundation, Pfizer, Takeda, and the Wellcome Trust.

Conflict of interest statement

The authors declare that there is no conflict of interest.

References

Aho

Koskenvuo

Tuominen

Kaprio

(1986) Occurrence of rheumatoid arthritis in a nationwide series of twins. J Rheumatol 13: 899–902.

Bednarik

Duckett

Kim

Perez

Griffis

Guenthner

. (1991) DNA CPG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs. New Biol 3: 969–976.

Bernard

(2014) Rheumatoid arthritis: who knows why regulatory T cells are defective in Ra … IDO. Nat Rev Rheumatol 10: 381.

Bestor

Laudano

Mattaliano

Ingram

(1988) Cloning and sequencing of a CDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. J Mol Biol 203: 971–983.

Bestor

Ingram

V.M.

(1983) Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA. Proc Natl Acad Sci U S A 80: 5559–5563.

Bhattacharya

Ramchandani

Cervoni

Szyf

(1999) A mammalian protein with specific demethylase activity for MCPG DNA. Nature 397: 579–583.

Bird

(2007) Perceptions of epigenetics. Nature 447: 396–398.

Boyes

Bird

(1991) DNA methylation inhibits transcription indirectly via a methyl-CPG binding protein. Cell 64: 1123–1134.

Brennan

Maini

Feldmann

(1992) TNF alpha - a pivotal role in rheumatoid arthritis? Br J Rheumatol 31: 293–298.

10.

Campanero

Armstrong

Flemington

(2000) CPG methylation as a mechanism for the regulation of E2F activity. Proc Natl Acad Sci U S A 97: 6481–6486.

11.

Chen

(2004) Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol 60: 55–89.

12.

Chiang

Gordon

Tal

Zeng

Doctor

Pardhasaradhi

. (1996) S-adenosylmethionine and methylation. FASEB J 10: 471–480.

13.

Christman

(2002) 5-azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21: 5483–5495.

14.

Cohen

Crome

Macdonald

Dai

Mager

Levings

(2011) Human Th1 and Th17 cells exhibit epigenetic stability at signature cytokine and transcription factor loci. J Immunol 187: 5615–5626.

15.

Costenbader

Karlson

(2006) Epstein–Barr virus and rheumatoid arthritis: is there a link? Arthritis Res Ther 8: 204–210.

16.

Cribbs

Kennedy

Penn

Amjadi

Green

Read

. (2015) Methotrexate restores regulatory T cell function through demethylation of the Foxp3 upstream enhancer in patients with rheumatoid arthritis. Arthritis Rheumatol, in press.

17.

Cribbs

Kennedy

Penn

Read

Amjadi

Green

. (2014) Treg cell function in rheumatoid arthritis is compromised by CTLA-4 promoter methylation resulting in a failure to activate the indoleamine 2,3-dioxygenase pathway. Arthritis Rheumatol 66: 2344–2354.

18.

Cutolo

Villaggio

Craviotto

Pizzorni

Seriolo

Sulli

(2002) Sex hormones and rheumatoid arthritis. Autoimmun Rev 1: 284–289.

19.

Dawlaty

Breiling

Raddatz

Barrasa

Cheng

. (2013) Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev Cell 24: 310–323.

20.

Dawson

Kouzarides

(2012) Cancer epigenetics: from mechanism to therapy. Cell 150: 12–27.

21.

De La Rica

Urquiza

Gomez-Cabrero

Islam

Lopez-Bigas

Tegner

. (2013) Identification of novel markers in rheumatoid arthritis through integrated analysis of DNA methylation and microrna expression. J Autoimmun 41: 6–16.

22.

De Pablo

Dietrich

Mcalindon

. (2008) Association of periodontal disease and tooth loss with rheumatoid arthritis in the US population. J Rheumatol 35: 70–76.

23.

Deaton

Bird

(2011) CPG islands and the regulation of transcription. Genes Dev 25: 1010–1022.

24.

Delhommeau

Dupont

Della Valle

James

Trannoy

Masse

. (2009) Mutation in Tet2 in myeloid cancers. N Engl J Med 360: 2289–2301.

25.

Di Fiore

Palena

Felsani

Palitti

Caruso

Lavia

. (1999) Cytosine methylation transforms an E2F site in the retinoblastoma gene promoter into a binding site for the general repressor methylcytosine-binding protein 2 (MECP2). Nucleic Acids Res 27: 2852–2859.

26.

Eden

Cedar

(1994) Role of DNA methylation in the regulation of transcription. Curr Opin Genet Dev 4: 255–259.

27.

Elliott

Maini

Feldmann

Long-Fox

Charles

Katsikis

. (1993) Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha. Arthritis Rheum 36: 1681–1690.

28.

ENCODE Project Consortium, Bernstein

Birney

Dunham

Green

Gunter

. (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489: 57–74.

29.

Ernst

Kheradpour

Mikkelsen

Shoresh

Ward

Epstein

. (2011) Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473: 43–49.

30.

Esteve

Chin

Smallwood

Feehery

Gangisetty

Karpf

. (2006) Direct interaction between DNMT1 and G9A coordinates DNA and histone methylation during replication. Genes Dev 20: 3089–3103.

31.

Feinberg

Vogelstein

(1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301: 89–92.

32.

Feldmann

Brennan

Maini

(1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14: 397–440.

33.

Fenaux

Mufti

Hellstrom-Lindberg

Santini

Finelli

Giagounidis

. (2009) Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol 10: 223–232.

34.

Gore

(2011) New ways to use DNA methyltransferase inhibitors for the treatment of myelodysplastic syndrome. Hematol Am Soc Hematol Educ Program 2011: 550–555.

35.

Hansen

Wijmenga

Luo

Stanek

Canfield

Weemaes

. (1999) The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci U S A 96: 14412–14417.

36.

Hashimshony

Zhang

Keshet

Bustin

Cedar

(2003) The role of DNA methylation in setting up chromatin structure during development. Nat Genet 34: 187–192.

37.

Chen

Zhu

(2011) Regulation and function of DNA methylation in plants and animals. Cell Res 21: 442–465.

38.

Hellman

Chess

(2007) Gene body-specific methylation on the active X chromosome. Science 315: 1141–1143.

39.

Herman

Latif

Weng

Lerman

Zbar

Liu

. (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 91: 9700–9704.

40.

Heyn

Ferreira

Moran

Pisano

Gomez

. (2012) Distinct DNA methylomes of newborns and centenarians. Proc Natl Acad Sci U S A 109: 10522–10527.

41.

Hirst

Marra

(2009) Epigenetics and human disease. Int J Biochem Cell Biol 41: 136–146.

42.

Ishida

Kobayashi

Ito

Komatsu

Yokoyama

Okada

. (2012) Interleukin-6 gene promoter methylation in rheumatoid arthritis and chronic periodontitis. J Periodontol 83: 917–925.

43.

Janson

Linton

Bergman

Marits

Eberhardson

Piehl

. (2011) Profiling of CD4+ T cells with epigenetic immune lineage analysis. J Immunol 186: 92–102.

44.

Javierre

Hernando

Ballestar

(2011) Environmental triggers and epigenetic deregulation in autoimmune disease. Discov Med 12: 535–545.

45.

Jenuwein

Allis

(2001) Translating the histone code. Science 293: 1074–1080.

46.

Jin

Tao

Peng

Soo

Ying

. (2008) DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function. Hum Mol Genet 17: 690–709.

47.

Johansson

Tumber

Che

Cain

Nowak

Gileadi

. (2014) The roles of Jumonji-type oxygenases in human disease. Epigenomics 6: 89–120.

48.

Kantarjian

Issa

Rosenfeld

Bennett

Albitar

Dipersio

. (2006) Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer 106: 1794–1803.

49.

Karouzakis

Gay

Neidhart

(2012) Increased recycling of polyamines is associated with global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum 64: 1809–1817.

50.

Karouzakis

Gay

Michel

Gay

Neidhart

(2009) DNA hypomethylation in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum 60: 3613–3622.

51.

Kass

Landsberger

Wolffe

(1997) DNA methylation directs a time-dependent repression of transcription initiation. Curr Biol 7: 157–165.

52.

Kennedy

Schmidt

Cribbs

Penn

Amjadi

Syed

. (2014) A novel upstream enhancer of FOXP3, sensitive to methylation-induced silencing, exhibits dysregulated methylation in rheumatoid arthritis Treg cells. Eur J Immunol 44: 2968–2978.

53.

Kim

Logan

Mason

Roubenoff

(1996) DNA hypomethylation in inflammatory arthritis: reversal with methotrexate. J Lab Clin Med 128: 165–172.

54.

Klein

Gay

(2013) Epigenetic modifications in rheumatoid arthritis, a review. Curr Opin Pharmacol 13: 420–425.

55.

Kohli

Zhang

(2013) TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502: 472–479.

56.

Kriaucionis

Heintz

(2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324: 929–930.

57.

Lange

Campan

Hinoue

Schmitz

Van Der Meulen-De Jong

Slingerland

. (2012) Genome-scale discovery of DNA-methylation biomarkers for blood-based detection of colorectal cancer. PLoS One 7: e50266.

58.

Langemeijer

Kuiper

Berends

Knops

Aslanyan

Massop

. (2009) Acquired mutations in TET2 are common in myelodysplastic syndromes. Nat Genet 41: 838–842.

59.

Lawson

Brown

Bejarano

Douglas

Burgoyne

Greenstein

. (2006) Early rheumatoid arthritis is associated with a deficit in the CD4+CD25 high regulatory T cell population in peripheral blood. Rheumatology (Oxford) 45: 1210–1217.

60.

Ley

Ding

Walter

Mclellan

Lamprecht

Larson

. (2010) DNMT3A mutations in acute myeloid leukemia. N Engl J Med 363: 2424–2433.

61.

(2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3: 662–673.

62.

Bestor

Jaenisch

(1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915–926.

63.

Liao

Liang

Xie

Zhao

Zuo

. (2012) CD40L demethylation in CD4(+) T cells from women with rheumatoid arthritis. Clin Immunol 145: 13–18.

64.

Liu

Fang

Lin

. (2011a) Global DNA methylation, DNMT1, and mbd2 in patients with rheumatoid arthritis. Immunol Lett 135: 96–99.

65.

Liu

Lin

. (2011b) Global DNA methylation, DNMT1, and MBD2 in patients with systemic lupus erythematosus. Lupus 20: 131–136.

66.

Liu

Aryee

Padyukov

Fallin

Hesselberg

Runarsson

. (2013) Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis. Nat Biotechnol 31: 142–147.

67.

Lofton-Day

Model

Devos

Tetzner

Distler

Schuster

. (2008) DNA methylation biomarkers for blood-based colorectal cancer screening. Clin Chem 54: 414–423.

68.

Lund

Narva

Lahesmaa

(2012) Genetic and epigenetic stability of human pluripotent stem cells. Nat Rev Genet 13: 732–744.

69.

Lundberg

Wegner

Yucel-Lindberg

Venables

(2010) Periodontitis in RA - the citrullinated enolase connection. Nat Rev Rheumatol 6: 727–730.

70.

Malottki

Barton

Tsourapas

Uthman

Liu

Routh

. (2011) Adalimumab, etanercept, infliximab, rituximab and abatacept for the treatment of rheumatoid arthritis after the failure of a tumour necrosis factor inhibitor: a systematic review and economic evaluation. Health Technol Assess 15: 1–278.

71.

Martens

Goronzy

Schaid

Weyand

(1997) Expansion of unusual CD4+ T cells in severe rheumatoid arthritis. Arthritis Rheum 40: 1106–1114.

72.

Merlino

Curtis

Mikuls

Cerhan

Criswell

Saag

. (2004) Vitamin D intake is inversely associated with rheumatoid arthritis: results from the Iowa Women’s Health Study. Arthritis Rheum 50: 72–77.

73.

Momparler

(2003) Cancer epigenetics. Oncogene 22: 6479–6483.

74.

Monk

Boubelik

Lehnert

(1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99: 371–382.

75.

Nakano

Boyle

Firestein

(2013a) Regulation of DNA methylation in rheumatoid arthritis synoviocytes. J Immunol 190: 1297–1303.

76.

Nakano

Whitaker

Boyle

Wang

Firestein

(2013b) DNA methylome signature in rheumatoid arthritis. Ann Rheum Dis 72: 110–117.

77.

Neidhart

Karouzakis

Jungel

Gay

(2014) Inhibition of spermidine/spermine N1-acetyltransferase activity: a new therapeutic concept in rheumatoid arthritis. Arthritis Rheumatol 66: 1723–1733.

78.

Nile

Read

Akil

Duff

Wilson

(2008) Methylation status of a single CPG site in the IL6 promoter is related to IL6 messenger RNA levels and rheumatoid arthritis. Arthritis Rheum 58: 2686–2693.

79.

Nyce

(1989) Drug-induced DNA hypermethylation and drug resistance in human tumors. Cancer Res 49: 5829–5836.

80.

Okano

Bell

Haber

(1999) DNA methyltransferases DNMT3A and DNMT3B are essential for de novo methylation and mammalian development. Cell 99: 247–257.

81.

Okano

Xie

(1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 19: 219–220.

82.

Okitsu

Hsieh

(2007) DNA methylation dictates histone H3K4 methylation. Mol Cell Biol 27: 2746–2757.

83.

Ollier

Thomson

(1992) Population genetics of rheumatoid arthritis. Rheum Dis Clin North Am 18: 741–759.

84.

Ono

Taki

Taketani

Taniwaki

Kobayashi

Hayashi

(2002) LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having T(10;11)(Q22;Q23). Cancer Res 62: 4075–4080.

85.

Onuora

(2014) Rheumatoid arthritis: SSAT1 inhibition slows synovial fibroblast invasion. Nat Rev Rheumatol 10: 259.

86.

Oppermann

(2013) Why is epigenetics important in understanding the pathogenesis of inflammatory musculoskeletal diseases? Arthritis Res Ther 15: 209–218.

87.

Orozco

Rueda

Martin

(2006) Genetic basis of rheumatoid arthritis. Biomed Pharmacother 60: 656–662.

88.

Pieper

Johansson

Snir

Linton

Rieck

Buckner

. (2014) Peripheral and site-specific CD4(+) CD28(Null) T cells from rheumatoid arthritis patients show distinct characteristics. Scand J Immunol 79: 149–155.

89.

Pradhan

Esteve

(2003) Mammalian DNA (cytosine-5) methyltransferases and their expression. Clin Immunol 109: 6–16.

90.

Prendergast

Ziff

(1991) Methylation-sensitive sequence-specific DNA binding by the C-Myc basic region. Science 251: 186–189.

91.

Quddus

Johnson

Gavalchin

Amento

Chrisp

Yung

. (1993) Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice. J Clin Invest 92: 38–53.

92.

Richardson

(1986) Effect of an inhibitor of DNA methylation on T cells. II. 5-Azacytidine induces self-reactivity in antigen-specific T4+ cells. Hum Immunol 17: 456–470.

93.

Richardson

Kahn

Lovett

Hudson

(1986) Effect of an inhibitor of DNA methylation on T cells. I. 5-Azacytidine induces T4 expression on T8+ T cells. J Immunol 137: 35–39.

94.

Richardson

Scheinbart

Strahler

Gross

Hanash

Johnson

(1990) Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum 33: 1665–1673.

95.

Rose

Klose

(2014) Understanding the relationship between DNA methylation and histone lysine methylation. Biochim Biophys Acta 12: 1362–1372.

96.

Rothbart

Strahl

(2014) Interpreting the language of histone and DNA modifications. Biochim Biophys Acta 1839: 627–643.

97.

Saag

Cerhan

Kolluri

Ohashi

Hunninghake

Schwartz

(1997) Cigarette smoking and rheumatoid arthritis severity. Ann Rheum Dis 56: 463–469.

98.

Schulz

Steinhoff

Florl

(2006) Methylation of endogenous human retroelements in health and disease. Curr Top Microbiol Immunol 310: 211–250.

99.

Shankaranarayanan

Mendoza-Parra

Walia

Wang

Trindade

. (2011) Single-tube linear DNA amplification (Linda) for robust chip-seq. Nat Methods 8: 565–567.

100.

Shen

Song

Zhang

(2014) Mechanism and function of oxidative reversal of DNA and RNA methylation. Annu Rev Biochem 83: 585–614.

101.

Siegfried

Eden

Mendelsohn

Feng

Tsuberi

Cedar

(1999) DNA methylation represses transcription in vivo. Nat Genet 22: 203–206.

102.

Silman

Macgregor

Thomson

Holligan

Carthy

Farhan

. (1993) Twin concordance rates for rheumatoid arthritis: results from a nationwide study. Br J Rheumatol 32: 903–907.

103.

Smith

Meissner

(2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14: 204–220.

104.

Suzuki

Kochi

Okada

Yamamoto

(2011) Insight from genome-wide association studies in rheumatoid arthritis and multiple sclerosis. FEBS Lett 585: 3627–3632.

105.

Svendsen

Holm

Kyvik

Petersen

Junker

(2002) Relative importance of genetic effects in rheumatoid arthritis: historical cohort study of Danish nationwide twin population. BMJ 324: 264–266.

106.

Tahiliani

Koh

Shen

Pastor

Bandukwala

Brudno

. (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324: 930–935.

107.

Takami

Osawa

Miura

Komai

Taniguchi

Shiraishi

. (2006) Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells. Arthritis Rheum 54: 779–787.

108.

Waddington

(1942) The epigenotype. Endeavour: 18–20.

109.

Waddington

(2012) The epigenotype. 1942. Int J Epidemiol 41: 10–13.

110.

Wade

Gegonne

Jones

Ballestar

Aubry

Wolffe

(1999) MI-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet 23: 62–66.

111.

Whitaker

Shoemaker

Boyle

Hillman

Anderson

Wang

. (2013) An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype. Genome Med 5: 40.

112.

Wieczorek

Asemissen

Model

Turbachova

Floess

Liebenberg

. (2009) Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. Cancer Res 69: 599–608.

113.

Wilson

Makar

Shnyreva

Fitzpatrick

(2005) DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin Immunol 17: 105–119.

114.

Winchester

(1994) The molecular basis of susceptibility to rheumatoid arthritis. Adv Immunol 56: 389–466.

115.

Zhang

(2011) Mechanisms and functions of TET protein-mediated 5-methylcytosine oxidation. Genes Dev 25: 2436–2452.

116.

Zhang

(2010) Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11: 607–620.

117.

Bestor

Bourc’his

Hsieh

Tommerup

Bugge

. (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402: 187–191.

118.

Xue

Sjogren

Salford

Widegren

(2012) An epigenetic mechanism for high, synergistic expression of indoleamine 2,3-dioxygenase 1 (IDO1) by combined treatment with zebularine and IFN-gamma: potential therapeutic use in autoimmune diseases. Mol Immunol 51: 101–111.

119.

Yan

Pan

Shen

. (2011) Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nat Genet 43: 309–315.

120.

Yanagisawa

Ito

Yuasa

Maruyama

(2002) The human DNA methyltransferases DNMT3A and DNMT3B have two types of promoters with different CPG contents. Biochim Biophys Acta 1577: 457–465.

121.

Yang

Liu

Bai

Zhang

Liu

. (2013) Tumor development is associated with decrease of TET gene expression and 5-methylcytosine hydroxylation. Oncogene 32: 663–669.

122.

Yisraeli

Frank

Razin

Cedar

(1988) Effect of in vitro DNA methylation on beta-globin gene expression. Proc Natl Acad Sci U S A 85: 4638–4642.

123.

Yoder

Walsh

Bestor

(1997) Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 13: 335–340.

124.

Zhang

Zhao

Sawalha

Richardson

(2013) Impaired DNA methylation and its mechanisms in CD4(+)T cells of systemic lupus erythematosus. J Autoimmun 41: 92–99.

125.

Zhou

Cheng

Connolly

Dickman

Hurd

Hornby

(2002) Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. J Mol Biol 321: 591–599.

Towards an understanding of the role of DNA methylation in rheumatoid arthritis: therapeutic and diagnostic implications

Abstract

Keywords

Introduction

Genetic and nongenetic factors influencing rheumatoid arthritis onset

Epigenetics and chromatin modification

DNA methylation

DNA demethylation

DNA methylation and gene regulation

DNA methylation in RA

DNA methylation in RA fibroblasts

DNA methylation in RA T cells

Therapeutic implications of altered DNA methylation patterns in RA

DNA methylation as a biomarker for RA

Conclusion and future direction

Footnotes

Funding

Conflict of interest statement

References