Impaired DNA methylation and its mechanisms in CD4+T cells of systemic lupus erythematosus

Journal of Autoimmunity xxx (2013) 1e8

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Review

Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus Yiqun Zhang a, Ming Zhao a, Amr H. Sawalha b, Bruce Richardson b, Qianjin Lu a, c, * a

Department of Dermatology, Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Medical Epigenetics, Changsha, Hunan 410011, PR China Department of Medicine, University of Michigan, Ann Arbor, MI, USA c Key Laboratory of Diabetes Immunology, Ministry of Education, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 December 2012 Accepted 2 January 2013

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens. Although the exact cause of SLE is still unknown, the influence of environment, which is largely reflected by the epigenetic mechanisms, with DNA methylation changes in particular, are generally considered as key players in the pathogenesis of SLE. As an important post-translational modification, DNA methylation mainly suppresses the expression of relevant genes. Accumulating evidence has indicated that abnormal DNA hypomethylation in T cells is an important epigenetic hallmark in SLE. Apart from those classic methylation-sensitive autoimmunityrelated genes in lupus, such as CD11a (ITGAL), Perforin (PRF1), CD70 (TNFSF7), CD40 ligand (TNFSF5) and PP2Aca, the genome-wide methylation pattern has also been explored recently, providing us a more and more full-scale picture of the abnormal status of DNA methylation in SLE. On the other hand, certain miRNAs, RFX1, defective ERK pathway signaling, Gadd45a and DNA hydroxymethylation have been proposed as potential mechanisms leading to DNA hypomethylation in lupus. In this review, we summarize current understanding of T cell DNA methylation changes and the consequently altered gene expressions in lupus, and how they contribute to the development of SLE. Possible mechanisms underlying these aberrancies are also discussed based on the reported literature and our own findings. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: DNA methylation T cells Systemic lupus erythematosus

1. Introduction Systemic lupus erythematosus (SLE) is a female-predominant heterogeneous systemic autoimmune disease characterized by the production of a variety of antinuclear autoantibodies and multiorgan involvement. Although the etiology of SLE remains unclear, studies have shown that epigenetic factors, especially abnormal DNA methylation patterns, play essential roles in the development of the disease [1]. Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence [2]. The mechanisms of epigenetic regulation include DNA methylation, histone modification and chromatin remodeling, and microRNA interference. As the most prevalent and bestdescribed epigenetic modification, DNA methylation changes are thought to be closely related to the pathogenesis of SLE. Our work

* Corresponding author. Second Xiangya Hospital, Central South University, #139 Renmin Middle Rd, Changsha, Hunan 410011, PR China. Tel.: þ86 731 85295860; fax: þ86 731 85533525. E-mail address: [email protected] (Q. Lu).

has shown that aberrant DNA hypomethylation in some specific genes of CD4þT cells can result in generation of autoreactive T cells and autoantibody production [3e6]. In this review, we give a thorough summary of the abnormal DNA hypomethylation mechanisms in SLE CD4þT cells and discuss how they are involved in the pathogenesis of this disease. 2. DNA methylation DNA methylation typically refers to the biochemical process which involves the addition of a methyl group to the 50 position of the cytosine pyrimidine ring, mediated by DNA methyltransferases (DNMTs), predominantly at CpG dinucleotides; however, it has been reported that in human embryonic stem cells about 25% of 5methylcytosine residues (5 mC) occur in a non-CG contexts [7]. As by far the only known epigenetic mark of DNA itself in mammals [8], DNA methylation is established by three DNMTs e DNMT3A, DNMT3B and DNMT1 e in two kinds of enzymatic activities e de novo methylation and maintenance methylation. New DNA methylation patterns are initially established by DNMT3A and DNMT3B [9,10], known as de novo methyltransferases, in unmethylated CpG sites.

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Please cite this article in press as: Zhang Y, et al., Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.01.005

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Y. Zhang et al. / Journal of Autoimmunity xxx (2013) 1e8

During cell division the pattern is effectively preserved by DNMT1, the maintenance methyltransferase, which prefers hemimethylated DNA as its substrate and is localized to replication foci through its interaction with the ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) [11,12]. In addition, maintenance DNA methylation can occur outside of the replication fork, when DNMT1 is recruited to hemimethylated DNA by methyl-CpG-binding protein 2 (MeCP2) during DNA replication [13]. Both de novo and maintenance methylation are crucial for embryonic development [9,14], their deletion can lead to embryonic lethality or postnatal deaths. As the main sites where DNA methylations occur, CpG dinucleotides, nearly 70e80% of which methylated in human DNA [15], are unevenly distributed throughout the genome. Particularly, certain regions of DNA are found to be rich in CpG and are termed CpG islands (GCIs). Most, but not all [16], GCIs are unmethylated and cover the transcription start sites. In fact, approximately 70% of annotated gene promoters, including most housekeeping genes, some tissue-specific genes and developmental genes, are associated with CGIs, which is considered to be the most common promoter type in the vertebrate genome [17,18]. DNA methylation was reported to correlate with gene repression for over three decades ago [19]. Methylation of CGI promoters can eventually lead to transcriptional silencing, although the condition rarely happens under normal circumstances. Presently, the mechanisms by which DNA methylation represses gene expression are incompletely understood, but it is generally believed that DNA methylation may perform this role either by directly interfering with the binding of specific transcription factors to the gene [20e23], or indirectly binding with methyl-CpG-binding proteins (MBDs), such as MBD2 and MeCP2 [24], which can initiate their associated repressive chromatin remodeling activities after further recruiting additional proteins like histone deacetylases. Being a stable and highly heritable epigenetic mark, DNA methylation is crucial for many cellular processes, including embryonic development, transcription, chromatin structure, X chromosome inactivation, genomic imprinting and chromosome stability [25]. Disturbance in the DNA methylation machinery is related to many human diseases, such as autoimmune diseases, cancers and imprinting disorders. 3. Aberrant T cell DNA methylation in SLE 3.1. Methylation sensitive genes in lupus T cells The relationship between abnormal DNA methylation status and SLE was first discovered by Dr. Richardson’s group more than twenty years ago when they found that T cells from active lupus patients had a decreased global DNA methylation level (15e20% reduction) [26]. Actually, the group had discovered earlier that 5azacytidine (5-azaC), an inhibitor of DNA methylation, could induce autoreactivity in cloned CD4þT cells and autoimmune syndrome [27]. The link between DNA hypomethylation, T cell autoreactivity and SLE was further supported by the finding that the percentage of 5 mC inversely correlates with the disease activity of lupus patients [28]. Since DNA methylation can influence gene expressions, those genes affected by DNA methylation changes in SLE were identified to gain more understanding of the pathogenesis of the disease, and they were referred to as methylationsensitive genes in lupus T cells. 3.1.1. CD11a (ITGAL) CD11a, along with CD18, forms the leukocyte functionassociated antigen-1 (LFA-1, integrin aLb2, CD11a/CD18), which plays a central role in cellular adhesion and costimulatory signaling [29]. It was initially discovered that CD11a expression was increased in both 5-azaC-treated normal T cells and a T cell subset

from active SLE patients [30]. The CD11a overexpression in T cell clones, whether resulting from treatment with DNA methylation inhibitors or transfection with CD18 cDNA, correlated with the development of T cell autoreactivity in vitro [31]. Adoptive transfer of CD11a-overexpressed, autoreactive T cells could produce a lupuslike disease in mice [32], similar to T cells treated with 5azacytidine or procainamide [33]. These results indicate that T cell hypomethylation may contribute to autoimmunity such as SLE through, at least in part, overexpression of CD11a. After investigating the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus, Lu et al. [4] demonstrated that specific sequences flanking the promoter of ITGAL, the gene encoding CD11a, was hypomethylated in T cells from patients with active lupus and in T cells treated with 5-azaC and procainamide, which lead to elevated CD11a expression and thus probably contributes to the development of lupus. In agreement with this, Luo et al. [34] found that CD4þT-cell DNA from patients with subacute cutaneous lupus erythematosus (SCLE), a less severe form of lupus, was also hypomethylated, and that CD11a mRNA expression, inversely correlated with DNA methylation, was significantly increased in these cells. 3.1.2. Perforin (PRF1) Perforin, encoded by the PRF1 gene and mainly expressed by CD8þT cells and NK cells, is a cytolytic protein which can form a pore in its target cell’s plasma membrane, promoting immunemediated cell lysis [35]. CD4þT cells from patients with active, but not inactive, lupus were observed to abnormally overexpress perforin [3]. The overexpression is related to demethylation of the enhancer 30 -flanking sequence, the same regulatory element that suppresses perforin transcription in primary CD4þT cells and was found to be demethylated by DNA methylation inhibitors [36]. DNA demethylation of the perforin promoter region and consequently perforin overexpression were also seen in SCLE CD4þT cells [37]. Additionally, the perforin inhibitor concanamycin A could block autologous monocyte killing by CD4þ lupus T cells, suggesting that aberrant perforin expression may contribute to the pathogenesis of lupus by monocyte killing [3]. In fact, Denny et al. have shown that increased monocyte/macrophage apoptosis induces autoantibody formation and organ damage in SLE [38]. It should be noted that the elevated perforin expression, directly proportional to disease activity of lupus, is seen in both CD4þ and CD8þT cells [39], but the methylation of the PRF1 gene seems to be regulated differently in these two subsets since the above specific regulatory element is not significantly hypomethylated in CD8þT cells [3]. 3.1.3. CD70 (TNFSF7) CD70, another methylation-sensitive T cell gene, is the cellular ligand for the tumor necrosis factor (TNF) receptor family member CD27 on B cells. Encoded by gene TNFSF7, CD70 is transiently expressed on the surface of activated lymphocytes. The costimulatory signals transmitted via CD27-CD70 interactions play key roles in regulating B cell activation and IgG synthesis [40]. Oelke et al. [6] reported an increase in CD70 expression, resulting in autologous B cell stimulation and IgG production, in both lupus CD4þT cells and CD4þT cells treated with 2 DNMT inhibitors (5-azacytidine and procainamide) or 3 ERK pathway inhibitors known to decrease DNMT expression (U0126, PD98059, and hydralazine). Adding anti-CD70 inhibited the excessive IgG synthesis. Lu et al. [41] further identified a genetic element that suppresses CD70 expression when methylated, and which was also found to be hypomethylated in lupus T cells and in T cells treated with DNMT and ERK pathway inhibitors. These results are strongly supported by the findings that similar defective DNA methylation and CD70 overexpression in CD4þT cells also exist in 16-week-old

Please cite this article in press as: Zhang Y, et al., Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.01.005

Y. Zhang et al. / Journal of Autoimmunity xxx (2013) 1e8

autoimmunity-established MRL/lpr lupus-prone mice [42] and in patients with SCLE [43]. All these studies indicate that CD70 overexpression, resulting in B cell overstimulation, is another functional consequence of relevant DNA hypomethylation in T cells which contributes to the pathogenesis of SLE. 3.1.4. CD40 ligand (TNFSF5) CD40 ligand (CD40L), also called CD154, is a member of the TNF superfamily and is primarily expressed on activated CD4þT cells. The interaction of CD40L on T cells with CD40 on antigen presenting cells or B cells plays an essential role in immune regulation depending on the target cell type [44,45]. Similar to CD70, CD40L is also a B cell costimulatory molecule but is encoded on the X chromosome, not an autosome, by the gene TNFSF5, which makes CD40L a special methylation-sensitive gene with a potential to explain the gender differences in SLE. Given the well known facts that one X chromosome in females is inactivated for dosage compensation, primarily by epigenetic mechanism including DNA methylation, to get equalized gene expression between the sexes [46], and that women are 9 times more likely to develop SLE than men [47], Lu et al. [5] explored the role of DNA methylation in silencing CD40L on the inactive X chromosome and its contribution to the female predominance of SLE. Bisulfite sequencing revealed that CD40L is unmethylated in normal men, while one copy of this X-chromosome gene is methylated and the other is unmethylated in normal women. With 5-azaC treatment demethylating CD40L in vitro, the expression of CD40L doubled in CD4þT cells from normal women, but not men. Likewise, CD40L is hypomethylated and overexpressed in CD4þT cells from female but not male lupus patients. These studies demonstrate that overexpression of CD40L, partially as a result of demethylated regulatory sequences on the inactive X chromosome in T cells, contributes to the development of SLE in females. More importantly, these results have pointed out the significant relationship between abnormal CD40L methylation status and the striking female predisposition of SLE. Indeed, the high level of CD40L in lupus patients has been shown to promote overproduction of autoantibodies through TeB cell interaction [5]. Our further studies demonstrated that CD40L-transfected T cells and female lupus T cells induced autologous B cell activation and subsequent IgG production [48]. With regard to the sex-specific difference in the development of lupus, Sawalha et al. [49] reported that both T cell DNA demethylation and genetic risk interact to influence the severity of lupus flares. Men actually require greater degree of T cell DNA demethylation to achieve the lupus flare at the same level as women. 3.1.5. Other methylation-sensitive genes In addition to the above relatively well-characterized methylation-sensitive genes, some other genes, such as PP2Aca, IL-4, IL-6 and the KIR gene family are also emerging as instigators of SLE due to abnormal methylation patterns. Tsokos et al. [50] showed evidence that PP2Aca, the a isoform of the catalytic subunit of protein phosphatase 2A (PP2Ac), displayed significantly increased expression levels in lupus T cells as a result of decreased DNA methylation in its promoter. Specifically, p-CREB, the transcriptional enhancer which only binds to demethylated CRE motif in PP2Aca promoter, exhibited increased binding in lupus T cells following reduced DNMT1 transcripts and low methylation status of the corresponding promoter region. More importantly, methylation intensity of the PP2Aca promoter correlated inversely with disease activity of SLE. Mi and Zeng [51] found in T cells from SLE patients higher levels of IL-4 and IL-6 transcripts as well as demethylation of CpG islands in the relevant promoters in T cells from SLE patients,, both of which were closely related to the disease severity. Similar phenomena were observed in 5-azaC-treated T cells.

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Killer-cell immunoglobulin-like receptors (KIRs) are a family of cell surface proteins expressed on NK cells and a small portion of T cells, serving as killing function regulators via interacting with MHC class I molecules. Albeit a highly polymorphic gene family, KIR genes share >91% sequence similarity in their promoters [52], and many of them, such as KIR2DL2 and KIR2DL4, were found to be activated by inhibiting DNA methylation [53], suggesting a crucial role of DNA methylation on regulating KIR expressions. It was demonstrated that KIR genes, normally suppressed primarily by DNA methylation in CD4þT cells [53], were overexpressed in lupus T cells in proportion to disease activity [54]. Moreover, abnormal KIR expression in lupus T cells was linked to enhanced production of IFN-g and macrophage killing [54]. 3.2. Genome-wide methylation pattern in lupus T cells Most of the aforementioned classic methylation-sensitive genes relevant to SLE pathogenesis were identified by using DNA methylation inhibitors and oligonucleotide expression arrays. Recently, however, high-throughput approaches enabled us to gain a glimpse of genome-wide DNA methylation profiles in lupus T cells. Using DNA samples extracted from whole white blood cells, Javierre et al. [55] compared over 1500 CG sites in 807 gene promoters in 30 monozygotic twins discordant for SLE, rheumatoid arthritis and dermatomyositis and found 49 differentially methylated genes only in the 5 pairs of SLE-discordant monozygotic twins. These 49 genes are implicated in defense response, cell activation, immune response, cell proliferation or cytokine production as a result of gene ontology analysis and 8 genes e IFNGR2, MMP14, LCN2, CSF3R, PECAM1, CD9, AIM2, and PDX1 e were confirmed and revealed significant methylation difference and functional relevance to SLE. Most of these genes showed diminished methylation levels, and global loss of 5-mC was observed in SLE twins consistent with previous findings [26,28]. More recently, Jeffries et al. [56] performed a genome-wide study of DNA methylation in lupus CD4þT cells. 27,578 CG sites in promoter regions of 14,495 genes from 12 matched SLE patients and controls were interrogated and 341 CG loci with significant methylation differences were further identified. It turned out that hypomethylated genes were preponderant in all differentially methylated loci, having twice the number of hypermethylated genes. Among the hypomethylated genes, 11 were overrepresented in the category of ‘development of connective tissue’. Hypomethylated genes included matrix metalloproteinase 9 (MMP-9), platelet-derived growth factor receptor a (PDGFRa), BST2 (Tetherin) and CD9. MMP-9 has been demonstrated to be associated with cartilage destruction in rheumatoid arthritis and Sjögren’s syndrome as an important part of connective tissue basement membrane [57,58]. Studies have also shown higher levels of MMP-9 in lupus patients [59,60] and its close link to neuropsychiatric lupus [61,62] and lupus nephritis [63]. However, it should be noted that the opposite alterations of MMP-9 in lupus were reported too [64,65], which is in concert with a newfound protective role of MMP-9 in SLE [66]. PDGFRa autoantibodies exist in 46% of lupus patients and contribute to the development of autoimmune hemolytic anemia [67]. BST2, to some extent, inhibits virus infection by impeding the release of viral particles via tethering them to the cell surface [68]. Of particular interest, CD9, the T cell co-activator, was also hypomethylated in the lupus discordant twins mentioned above [55]. These newly identified hypomethylated genes in SLE CD4þT cells have yielded us novel insight into elucidating the etiology of SLE. Additionally, hypermethylated genes, such as FOLH1 and GGH involved in ‘folate biosynthesis’, and the transcription factor RUNX3 were as well revealed in this genome-wide study. Proteineprotein interaction maps surprisingly identified HNF4,

Please cite this article in press as: Zhang Y, et al., Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.01.005

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a new transcription factor, as a crucial controller of the network for hypo- and hypermethylated genes. Further, 5 genes, RAB22A, STX1B2, LGALS3BP, DNASE1L1 and PREX1 were found to correlate with lupus disease activity which rendered themselves as possible biomarkers for SLE. These methylation patterns in lupus CD4þT cells, either in some specific genes or at a genome-wide level (Table 1), give us an increasingly panoramic view of the abnormal status of DNA methylation in SLE. Interestingly, Youinou et al. recently propose that there is a link between altered patterns of epigenetic changes in lupus and auto-antibody production [69], which reinforces the idea that epigenetic mechanisms are responsible for the development of lupus. However, it should not escape our attention that the mass of data which genome-wide studies provide us do not live up to their potential unless a more clear functional consequences of these methylation changes in the pathogenesis of SLE are identified. 4. Possible mechanisms of DNA demethylation in lupus T cells 4.1. miRNAs and DNA hypomethylation in lupus miRNAs are short w23 nucleotide noncoding RNAs which act primarily as post-transcriptional regulators, mostly suppressors, through binding to target mRNAs [74]. It is well acknowledged that miRNAs have a widespread impact on expression of protein-coding genes, with approximately over one third of human genes as their conserved targets [75]. Not surprisingly, critical associations between miRNAs and DNA methylation patterns in lupus CD4þT cells have also been reported. Pan et al. [76] demonstrated that expression of miR-21 and miR-148a increased in both lupus patients and lupus-prone MRL/lpr mice, which may inhibit DNMT1 expression and thus contribute to demethylation of lupus-relevant methylation-sensitive genes such as CD70 and CD11a. However, the way that miR-21 and miR-148a downregulate DNMT1 expression varies. miR-21 indirectly alters DNMT1 expression by targeting RASGRP1, an important gene in autoimmunity [77] which mediates the Ras-MAPK pathway upstream of DNMT1. On the other hand, miR-148a directly targets DNMT1 and represses its transcription. The decreased level of DNMT1 expression and DNA hypomethylation in CD4þT cells from lupus patients could be abrogated

by inhibition of miR-21 and miR-148a expression. Our work [78,79] has identified another miRNA, miR-126, which was upregulated in CD4þT cells from patients with SLE and was able to directly inhibit DNMT1 expression via binding to its 30 -untranslated region. Overexpression of miR-126 in CD4þT cells from healthy people gave rise to DNA demethylation and elevated levels of CD11a and CD70, resulting in overreactive T and B cells. More recently, miR-29b has also been reported to promote DNA hypomethylation of lupus CD4þT cells by indirectly targeting DNMT1 [80]. 4.2. RFX1 and DNA hypomethylation in lupus RFX1 (Regulatory factor X 1) is a member of the RFX family, a group of transcriptional activators that recognize the X-box in promoters of MHC class II gene via strongly conserved DNA-binding domains [81]. Either in an active or a repressive way, RFX1 has been implicated in the transcriptional regulation of many cellular and viral genes [82]. Our group has found [83] that both the activity and expression of RFX1 are significantly decreased in SLE CD4þT cells. Evidence has indicated that RFX1 binds to the promoter region of CD11a as well as CD70, and then recruits the transcriptional repressor complex formed by DNMT1 and HDAC1 (Histone deacetylase 1), which suppresses the expression of CD11a and CD70. Similarly, RFX1 downregulation in normal CD4þT cells leads to DNA hypomethylation and histone H3 hyperacetylation of CD11a and CD70 promoter, which consequently increase the expression of CD11a and CD70 and therefore promote autoreactivity of CD4þT cells. The opposite situations appear when overexpressing RFX1 in SLE CD4þT cells. Results of our further investigation [84] indicate that RFX1 also recruits histone methyltransferase SUV39H1 to CD11a and CD70 promoter regions, contributing to H3K9 trimethylation, which is another epigenetic mechanism crucial for shaping repressive chromatin structure. 4.3. Defective ERK pathway signaling and DNA hypomethylation in lupus Impaired extracellular signal-regulated kinase (ERK) pathway signaling in T cells has been implicated in the pathogenesis of lupus by Dr. Richardson’s group [85] since almost 15 years ago when they

Table 1 Impaired DNA methylation in lupus CD4þT cells. Genes/molecules

Methylation status

Specific methylation-sensitive genes CD11a (ITGAL) Y Perforin (PRF1) Y CD70 (TNFSF7) Y CD40L (TNFSF5) Y

Expression level

Function

Contribution to SLE pathogenesis

Ref.

[ [ [ [

Cellular adhesion Cytolysis Costimulaiton Costimulation

T cell autoreactivity Monocyte/macrophage killing B cell overstimulation B cell overstimulation, predisposes women to SLE Production of IFN-g; Macrophage killing Promotes B cell differentiation and antibody production Stimulates B cell proliferation, differentiation and antibody production Suppresses IL-2 production

[4,30e32] [3,36] [6,41] [5,48]

Contributes to proteinuria and deposition of immune complexes in LN; Clearance of autoepitopes in B cell; Promoting lymphocytes migration ND

[56,59,66,71e73]

? T cell autoreacitivity ND

[55,56] [56]

KIRs IL-4

Y Y

[ [

Discrimination of non-self from self Th2 growth and differentiation factor

IL-6

Y

[

B cell growth and differentiation factor

PP2Aca

Y

[

Cell cycle progression and signal transduction

[/Y

Degradation of the extracellular matrix proteins

Inhibits virus particles release from cell membrance Costimulation Cell growth and division

Genome-wide studies MMP9 Y

BST2

Y

[

CD9 PDGFRa

Y Y

[ [

[53,54,70] [51] [51] [50]

[56,68]

Y: decreased; [: increased; LN: lupus nephritis; ND: Not determined.

Please cite this article in press as: Zhang Y, et al., Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.01.005

Y. Zhang et al. / Journal of Autoimmunity xxx (2013) 1e8

reported that DNMT in human T cells could be reduced by inhibiting ras-mitogen-activated protein kinase (MAPK) pathway signaling, suggesting the pathway as a regulator of T cell DNMT. In accordance with this, decreased DNMT levels as well as ERK pathway signaling were also observed in T cells from patients with active lupus. Importantly, the reduction in ERK signaling correlates with disease activity. Inhibition of ERK pathway signaling with a MEK1 inhibitor decreased the expression and activity of DNMT, and consequently led to DNA hypomethylation, all of which were similar to what was seen in lupus T cells [86]. The contribution of deficient ERK pathway to the development of lupus through causing reduced DNMT activity and DNA hypomethylation in lupus patients indicated by these studies was further supported by the finding that inhibiting T cell ERK pathway with an MEK inhibitor was able to induce autoreactivity in vitro and lupus-like autoimmunity in mice by promoting DNA hypomethylation and overexpression of methylation-sensitive genes [87]. Moreover, hydralazine, a DNMT inhibitor, similarly appeared to have the capacity for inhibiting ERK pathway signaling, thereby indirectly decreasing DNMT expression and finally inducing autoimmune phenomena as noted above [87]. Using a transgenic mouse with an inducible defective T cell ERK pathway signaling in the presence of doxycycline, Sawalha et al. [88] demonstrated in vivo that reduced ERK pathway signaling in T cells led to overexpression of CD11a and CD70 by means of decreasing DNMT expression. Besides, anti-dsDNA antibodies and boosted expression of a few interferon-regulated genes in the spleen similar to peripheral blood cells in human lupus were also observed in this transgenic mouse [88]. Furthermore, in an autoimmune-susceptible genetic background, this mouse develops clinical features of lupuslike disease such as immune-complex glomerulonephritis. Of interest, only female but not male mice develop disease, an observation linked to T cell CD40L hypomethylation and overexpression in female but not male mice. Similar to humans, CD40L is also located on the X-chromosome in mice [89]. Since it is clearly established that DNA methylation patterns are regulated, at least in part, by ERK pathway signaling, whose defect seems to be one of the mechanisms underlying autoimmunity in idiopathic and hydralazine-induced lupus, Gorelik et al. [90] gave a particular focus on the reasons for the impaired ERK pathway signaling in lupus and identified defective protein kinase C (PKC) d phosphorylation as a culprit for the decreased ERK signaling in both lupus T cells and hydralazine-treated T cells, which is in agreement with the observation that mice lacking PKCd presented with circulating autoantibodies, autoimmune glomerulonephritis and lymphocyte infiltration in several organs [91]. Interestingly, PKCd is inactivated by oxidative damage in lupus T cells, and oxidative stress is associated with lupus flares [92]. 4.4. Gadd45a and DNA hypomethylation in lupus The growth arrest and DNA damage-induced 45a (Gadd45a) protein is a controversial global demethylator [93,94]. Our studies [95] revealed that CD4þT cells from patients with SLE displayed increased level of Gadd45a proportional to decreased global methylation. Normal CD4þT cells, either transfected with Gadd45a or exposed to ultraviolet radiation, exhibited reduced methylation in promoters of CD11a and CD70, as well as reduced global methylation, leading to development of autoreactivity and overstimulated production of autoantibodies. Specifically decreasing Gadd45a expression with siRNA inhibited autoimmunity by reversing the above-mentioned pathological processes [95]. Together, our findings identified Gadd45a as an instigator of autoimmunity in SLE possibly by demethylating in CD4þT cells, though more intensive studies are necessary to elucidate the capacity of Gadd45a for demethylation.

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Fig. 1. Mechanisms underlying DNA demethylation in lupus T cells. Elevated level of miR-126 leads to decreased DNMT1 expression in lupus CD4þT cells. Diminished activity and amount of the transcription factor RFX1 recruits less DNMT1 to the promoter regions of certain methylation-sensitive genes. Defective ERK pathway signaling contributes to reduced DNMT1 expression levels of in CD4þ lupus T cells. Gadd45a likely serves as a DNA demethylator in lupus. 5 hmC, which is generated by TET from 5 hC, may be an intermediate in several potential DNA demethylation pathways.

4.5. DNA hydroxymethylation and DNA hypomethylation in lupus The recent discovery of DNA hydroxymethylation [96] where 5 mC can be further oxidated into 5-hydroxymethylcytosine (5 hmC) by Ten-eleven translocation (TET) family proteins lends an exciting prospect for the ever slow-progressing research on DNA demethylation mechanisms. Rapidly accumulated studies have reported that 5 hmC and TET proteins play important roles in dynamically regulating DNA methylation in embryonic development, leukemia and the brain by participating in several potential pathways for DNA demethylation [97e100]. In view of the possibility of 5 hmC as a demethylation intermediate, we investigated whether 5 hmC and TET proteins are involved in the development of SLE, a disease characterized by abnormal DNA hypomethylation in T cells. We observed high levels of TET1, TET2 and 5 hmC, probably generated by the former two enzymes, in CD4þT cells from SLE patients compared to healthy individuals, and we propose that DNA hydroxymethylation may be responsible for aberrant DNA methylation contributing to the pathogenesis of SLE (unpublished data). 5. Conclusion and perspectives Looking back on the more than 20 years of investigations into the involvement of disturbed DNA methylation patterns in the pathogenesis of SLE since the initial discovery that SLE T cells displayed globally hypomethylation and reduced DNMT levels, it has been well recognized that DNA hypomethylation in T cells contributes to the onset and development of drug-induced and idiopathic lupus. An array of genes, sensitive to DNA methylation status in their promoter regions, are overexpressed in T cells treated with DNA methylation inhibitors or from lupus patients, whose expression levels importantly parallel SLE disease activity. Excessive expressions of these genes in CD4þT cells, with CD11a causing autoreactivity, perforin promoting monocyte/macrophage killing, and CD70 as well as CD40L overstimulating autoantibodies productions, functionally promote the development of human lupus. Further, with the development of high-throughout methods, genome-wide DNA methylation patterns in lupus T cells are being determined, which not only offer novel insights into SLE pathogenesis but also calls for further investigation in those newly revealed molecules. On the other hand, along the way in unraveling mechanisms underlying abnormal DNA hypomethylation in lupus CD4þT cells, miRNAs including miR-126, miR-21 and miR-148a, transcription

Please cite this article in press as: Zhang Y, et al., Impaired DNA methylation and its mechanisms in CD4þT cells of systemic lupus erythematosus, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.01.005

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factor RFX1, ERK pathway signaling and the DNA demethylator Gadd45a have gradually been found and demonstrated to play essential roles by their own means. Notably, a large portion of their effects comes down to DNMT1 incompetence, which suggests passive demethylation as a key player in the pathogenic hypomethylation in SLE. However, with the discovery of TET protein and 5 hmC in mammals, extensive efforts in the future should be made to explore the possible involvement of active T cell demethylation in SLE (Fig. 1). Although multiple genes take part in determining the predisposition to SLE, environmental factors, largely reflected by epigenetic alterations, have been demonstrated to be crucial for the pathogenesis of autoimmune diseases such as SLE [101]. Indeed, genetically predisposed individuals do not seem to inevitably develop this complex disease unless they are exposed to some environmental factors, such as EpsteineBarr virus, certain drugs, and some other unknown agents. The fact that in many cases the degree of abnormal DNA methylation in T cells goes hand in hand with SLE disease activity manifests a hopeful lupus biomarker in DNA methylation changes. More importantly, in light of the reversibility of epigenetic modifications, specific epigenetic drugs aiming at the aberrant DNA hypomethylation in lupus T cells also hold great promise in fighting SLE. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30730083, No. 30972745, No. 8110119, No. 30901300 and No. 81220108017), the National Basic Research Program of China (973 Plan) (2009CB825605), the Programs of Science-Technology Commission of Hunan province (2011FJ2007, 2011TP4019-7, 2012WK3046 and 2012TT2015) and the Fundamental Research Funds for the Central Universities. References [1] Pan Y, Sawalha AH. Epigenetic regulation and the pathogenesis of systemic lupus erythematosus. Transl Res 2009;153:4e10. [2] Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science 1999;286:481e6. [3] Kaplan MJ, Lu Q, Wu A, Attwood J, Richardson B. Demethylation of promoter regulatory elements contributes to perforin overexpression in CD4þ lupus T cells. J Immunol 2004;172:3652e61. [4] Lu Q, Kaplan M, Ray D, Zacharek S, Gutsch D, Richardson B. Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus. Arthritis Rheum 2002;46:1282e91. [5] Lu Q, Wu A, Tesmer L, Ray D, Yousif N, Richardson B. Demethylation of CD40LG on the inactive X in T cells from women with lupus. J Immunol 2007; 179:6352e8. [6] Oelke K, Lu Q, Richardson D, Wu A, Deng C, Hanash S, et al. Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors. Arthritis Rheum 2004;50:1850e60. [7] Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009;462:315e22. [8] Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16:6e21. [9] Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247e57. [10] Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet 1998;19:219e20. [11] Bostick M, Kim JK, Esteve PO, Clark A, Pradhan S, Jacobsen SE. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 2007; 317:1760e4. [12] Sharif J, Muto M, Takebayashi S, Suetake I, Iwamatsu A, Endo TA, et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 2007;450:908e12. [13] Kimura H, Shiota K. Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1. J Biol Chem 2003;278: 4806e12. [14] Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992;69:915e26.

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