Dissecting complex epigenetic alterations in human lupus
© BioMed Central Ltd 2013
Published: 29 January 2013
Systemic lupus erythematosus is a chronic relapsing autoimmune disease that primarilyafflicts women, and both a genetic predisposition and appropriate environmentalexposures are required for lupus to develop and flare. The genetic requirement isevidenced by an increased concordance in identical twins and by the validation of atleast 35 single-nucleotide polymorphisms predisposing patients to lupus. Genes alone,though, are not enough. The concordance of lupus in identical twins is oftenincomplete, and when concordant, the age of onset is usually different. Lupus is alsonot present at birth, but once the disease develops, it typically follows a chronicrelapsing course. Thus, genes alone are insufficient to cause human lupus, andadditional factors encountered in the environment and over time are required toinitiate the disease and subsequent flares. The nature of the environmentalcontribution, though, and the mechanisms by which environmental agents modify theimmune response to cause lupus onset and flares in genetically predisposed peoplehave been controversial. Reports that the lupus-inducing drugs procainamide andhydralazine are epigenetic modifiers, that epigenetically modified T cells aresufficient to cause lupus-like autoimmunity in animal models, and that patients withactive lupus have epigenetic changes similar to those caused by procainamide andhydralazine have prompted a growing interest in how epigenetic alterations contributeto this disease. Understanding how epigenetic mechanisms modify T cells to contributeto lupus requires an understanding of how epigenetic mechanisms regulate geneexpression. The roles of DNA methylation, histone modifications, and microRNAs inlupus pathogenesis will be reviewed here.
Epigenetics and gene expression
DNA methylation refers to the methylation of cytosines in CpG pairs and silences genesby stabilizing chromatin in the tightly packaged, transcriptionally repressiveconfiguration. DNA methylation patterns are established during development and serve inpart to silence genes which would be inappropriate or detrimental to the function of anygiven cell but for which a cell might have transcription factors that would otherwisedrive their expression. Different cell types have different functions, determined by therepertoire of genes they express, so each cell type has a distinct pattern of methylatedand unmethylated genes.
Once established, the methylation patterns are replicated each time a cell divides byDNA methyltransferase 1 (Dnmt1). As cells enter S phase, Dnmt1 levels increase. Dnmt1binds the DNA replication fork and reads CpG pairs. Where deoxycytosine (dC) in theparent strand is methylated, Dnmt1 transfers the methyl group from S-adenosylmethionine(SAM) to the corresponding dC in the daughter strand to form deoxymethylcytosine,replicating the methylation patterns and producing S-adenosylhomocysteine (SAH), aninhibitor of transmethylation reactions . Importantly, this reaction is sensitive to environmental agents and drugsthat decrease Dnmt1 activity, decrease SAM, or increase SAH, preventing methylation ofnewly synthesized DNA in the daughter cells and causing inappropriate gene expression [1–3]. Furthermore, the errors can be replicated during subsequent rounds of celldivision and accumulate over time, causing an age-dependent decrease in DNA methylationand increase in aberrant T-cell gene expression . These age-dependent changes are evidenced by a report that lymphocyte DNAmethylation patterns are the same in identical twins at 3 years of age but different at50. DNA methylation patterns also diverged more when twins had different lifestyles orspent less of their lives together, compared to twins who had similar lifestyles orspent more of their lives together .
T-cell DNA methylation and gene expression
T lymphocytes are particularly dependent on DNA methylation to suppress inappropriategene expression. T cells differentiate into multiple subsets throughout life but haveoverlapping sets of transcription factors and use DNA methylation to silence genesinappropriate for specific subsets. Like other cells, resting T cells expressrelatively little Dnmt1, but as T cells enter mitosis, signals through theextracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)pathways upregulate Dnmt1 to replicate the methylation patterns; and decreasing Dnmt1enzymatic activity with inhibitors like 5-azacytidine (5-azaC) or procainamide, orpreventing Dnmt1 upregulation with signaling inhibitors like hydralazine, preventsthe methylation of the newly synthesized DNA, activating normally silenced genes andaltering effector functions in the daughter cells . For example, inhibiting DNA methylation induces interferon-gamma(IFNγ) in Th2 cells, FoxP3 in CD4+CD25− T cells , and others .
Inhibiting DNA methylation also makes CD4+ T cells autoreactive to selfclass II major histocompatibility complex (MHC) molecules. The autoreactivity is dueto LFA-1 (lymphocyte function-associated antigen 1) (CD11a/CD18) overexpression, andincreasing LFA-1 by transfection makes T cells similarly autoreactive . Importantly, CD4+ T cells similarly responding to class II MHCmolecules cause a lupus-like disease in the chronic graft-versus-host disease model , suggesting that demethylated, autoreactive T cells may cause a similarlupus-like disease. This was confirmed by treating mouse CD4+ T cells with5-azaC and then injecting the cells into genetically identical mice. The recipientsdeveloped antidsDNA antibodies and an immune complex glomerulonephritis closelyresembling human lupus nephritis .
The observation that 5-azaC, a drug that inhibits DNA methylation, can cause alupus-like disease suggested that drugs which cause a lupus-like disease may be DNAmethylation inhibitors. Subsequent studies demonstrated that CD4+ T cellstreated with procainamide or hydralazine, which cause antinuclear antibodies (ANAs)in a majority of people and a lupus-like disease in genetically predisposed people,are also DNA methylation inhibitors  and that, when injected into genetically identical mice, mouse T cellstreated with these drugs also caused a lupus-like disease . Procainamide was found to inhibit Dnmt1 enzymatic activity, whereashydralazine decreases Dnmt1 levels in dividing cells by inhibiting ERK pathwaysignaling .
Functional studies demonstrated that experimentally demethylated, autoreactiveCD4+ T cells are cytotoxic and kill autologous or syngeneic macrophages(Mφ), causing release of apoptotic nuclear material as well as impairing itsclearance, since Mφ clear apoptotic debris . Others have reported that injecting apoptotic cells into mice orimpairing their clearance through genetic manipulation is sufficient to causeanti-DNA antibodies and a lupus-like disease , suggesting that the Mφ apoptosis caused by demethylated T cellscontributes to anti-DNA antibody development. The demethylated CD4+ Tcells also overstimulate B-cell antibody production through demethylation andoverexpression of B-cell co-stimulatory genes, including CD70,CD40L, IFNγ, and others [1, 12, 13]. When injected into mice, the demethylated cells accumulate in the spleen,where they kill Mφ and stimulate B cells, and removing the spleen prior toinjection prevents disease development .
T-cell DNA demethylates in patients with active lupus
These studies demonstrate that epigenetically altered CD4+ T cells aresufficient to cause a lupus-like disease in mice. Since the two drugs that mostcommonly cause drug-induced lupus are DNA methylation inhibitors, these studies alsosuggest that epigenetically altered CD4+ T cells may similarly causeidiopathic lupus. This was initially tested by comparing overall DNA methylationlevels in T cells from patients with active lupus, inactive lupus, or other rheumaticand inflammatory diseases and healthy controls. Patients with active lupus had lowermethylcytosine levels than the other groups . Interestingly, T-cell DNA methylation levels also decline with age , suggesting an explanation for the development of ANAs with age inotherwise healthy older people .
The decrease in lupus T-cell Dnmt1 levels was traced to decreased ERK pathwaysignaling, which prevents Dnmt1 upregulation during mitosis . A role for decreased signaling in lupus pathogenesis was confirmed byexperiments demonstrating that inhibiting ERK pathway signaling with MEK inhibitorsalso inhibited DNA methylation in mouse CD4+ T cells, and injecting thetreated cells into syngeneic mice also caused a lupus-like disease . More recently, a double-transgenic mouse strain was generated in whichexpression of a dominant negative MEK (dnMEK) could be selectively induced in T cellsby adding doxycycline to their drinking water. Activating the dnMEK inhibited T-cellDNA methylation and caused anti-DNA antibodies and an 'interferon signature' in mice,similarly to what was observed in patients with lupus . Interestingly, no kidney disease was seen in these transgenic mice on aBL6 background. However, crossing the double-transgenic BL6 strain with SJL mice,which have lupus genes, resulted in the development of an immune complexglomerulonephritis as well as anti-DNA antibodies when doxycycline was given . Thus, impaired T-cell ERK pathway signaling is sufficient to causelupus-like autoantibodies in nonlupus-prone mice, but renal disease also requireslupus genes.
Evidence that hydralazine inhibits ERK pathway signaling, that ERK pathway signalingis impaired in T cells from patients with active lupus, and that inhibiting ERKpathway signaling causes a lupus-like disease in adoptive transfer and transgenicmouse models  prompted studies identifying the signaling molecule(s) inactivated byhydralazine and inactivated in CD4+ T cells from patients with activelupus. The ERK pathway defect was traced to PKCδ, which fails to respond todirect stimulation with phorbol myristate acetate in both the idiopathic lupus andhydralazine-induced models . Importantly, PKCδ 'knockout' mice develop lupus , demonstrating a critical role for PKCδ in lupus-likeautoimmunity.
More recent studies demonstrate that PKCδ is inactivated by oxidative damage inlupus T cells. Lupus onset and flares are associated with environmental agents thatcause oxidative stress, such as ultraviolet light exposure, acute infections, silicaexposure, and smoking , and all cause oxidative stress . Furthermore, lupus flares are characterized by biomarkers of oxidativestress such as protein nitration, caused by superoxide(O2−) combining with nitric oxide (NO), anintracellular signaling molecule, to form peroxynitrite (ONOO−) . T-cell PKCδ is nitrated in patients with active lupus, and thenitrated fraction is catalytically inactive , providing a direct link between environmental agents associated withlupus and epigenetic changes in T cells.
Lupus T-cell epigenomics
The observation that experimentally demethylated CD4+ T cells overexpressLFA-1 due to ITGAL (CD11a) demethylation, making them autoreactive , raised the possibility that other genes may similarly demethylate and beinappropriately overexpressed by T cells from patients with active lupus. Additionalgenes were sought by treating normal human CD4+ T cells with 5-azaC andcomparing gene expression with mRNA expression arrays. These experiments identifiedCD70 (TNFSF7), perforin (PRF1), and the KIR gene family asgenes primarily regulated by DNA methylation in CD4+ T cells. CD70 isexpressed on some but not all CD4+ T cells and promotes B-cell antibodyproduction . Perforin is a cytotoxic molecule expressed in killer cells and lysestarget cells by forming a pore in their cytoplasmic membrane . The KIR genes normally are expressed by natural killer (NK) cells but notT cells and encode proteins that recognize I MHC molecules. Stimulatory Kir proteinsmediate cytotoxic and inflammatory responses. Aberrant expression of KIR genes bydemethylated T cells makes them likely candidates for contributing to autoimmuneresponses .
The DNA sequences demethylated to activate these genes were identified by bisulfitesequencing. Bisulfite converts cytosine to uracil but does not affect methylcytosine,and the change can be detected by DNA sequencing. Bisulfite sequencing revealed thatregulatory sequences upstream of the CD11a, CD70, KIR, and perforin genes alldemethylate in 5-azaC- treated CD4+ T cells, and methylation of theseregions in transfection experiments silenced these genes [1, 25].
Subsequent experiments compared expression and methylation of the same genes inCD4+ T cells from patients with either inactive or active lupus. Aspredicted, CD11a, perforin, and the KIR genes were overexpressed in patients withactive but not inactive lupus, and the same sequences demethylated in proportion todisease activity and gene overexpression in these patients [1, 24]. The exception was CD70, which remains demethylated once itdemethylates . Others have reported that protein phosphatase 2A, a signaling molecule,is also demethylated and overexpressed in T cells from patients with active lupus . Very recently, Jeffries and colleagues  used microarrays to survey hypomethylated and hypermethylated genes inCD4+ T cells from patients active lupus and identified 236hypomethylated and 105 hypermethylated sites, confirming widespread methylationchanges throughout the genome.
Together, these results demonstrate that inhibiting T-cell DNA methylation, eitherwith drugs in vitro or environmental agents in patients with lupus, causesthe same epigenetic changes in DNA methylation patterns and overexpression of thesame genes. Since similar demethylated T cells cause lupus-like autoimmunity in mice,these studies indicate that T-cell DNA demethylation is likely fundamental to lupusonset and flares.
Histone modifications also regulate gene expression. Histone acetyltransferases (HATs)and histone deacetylases (HDACs) regulate gene expression by adding or removing acetylgroups on lysine residues in the histone proteins . Acetylation neutralizes the positive charge of lysines, weakeningelectrostatic DNA-histone interactions and increasing DNA accessibility for geneexpression . Conversely, deacetylation strengthens DNA-histone interactions, decreasingDNA accessibility and subsequent gene expression . Promoters of actively transcribed genes are also characterized bymethylation of lysine 4 on histone H3 (H3K4) and of lysine 36 on H3 (H3K36) . In contrast, inactive genes are methylated at H3K27 and permanently silencedgenes are frequently methylated at H3K9 . A large number of other histone modifications serve a number of regulatoryand other functions (reviewed in ).
Lupus T-cell histone modifications
Histone acetylation may also contribute to lupus pathogenesis. Hu and colleagues  showed that SLE CD4+ T cells have decreased overall acetylationof histones H3 and H4, and the degree of H3 deacetylation correlated inversely withSLE disease activity. This raises the possibility that decreased histone acetylationcontributes to lupus pathogenesis by promoting silencing of some genes. However, thegenes affected are unclear.
Histone methylation patterns are also altered in CD4+ lupus T cells . Hu and colleagues  also reported hypomethylation at lysine 9 of histone H3 (H3K9) in SLECD4+ T cells, compared with healthy controls. mRNA levels of thehistone methyltransferases SUV39H2 and EZH2 were also decreased in CD4+ SLE T cells . Zhao and colleagues  subsequently reported that a different histone methyltransferase, SUV39H1,is recruited to the CD11a and CD70 promoters. This is mediated by RFX1, and RFX1 isdownregulated in lupus CD4+ T cells . RFX1 recruitment to SUV39H1 causes both increased H3K9 methylation anddecreased CD11a and CD70 expression . In a second report, Zhou and colleagues  showed increased H3K4 methylation at the CD70 promoter in lupus CD4+ T cells. Zhang and colleagues  showed that hematopoietic progenitor kinase 1 (HPK1) levels are decreasedin lupus CD4+ T cells. Furthermore, blocking HPK1 expression increasedT-cell proliferation, cytokine secretion, and B-cell co-stimulatory functions. Thesechanges reversed when HPK1 was overexpressed. The decreased HPK1 expression in lupusCD4+ T cells is due to decreased H3K27 methylation, and the decreasedmethylation is due to decreased binding of the enzyme jumonji domain containing 3(JMJD3), which methylates H3K27 .
MiRNAs are 18- to 22-nucleotide non-coding RNA molecules that regulate gene expressionby degrading mRNA or blocking protein translation. An miRNA binds to a target sequencein the 3' untranslated region of its target mRNA, leading to either degradation ortranslational silencing by other mechanisms. Multiple miRNAs can bind to and blockexpression of a target mRNA. Additionally, a single miRNA can bind to and block theexpression of multiple target mRNAs. These events are possible because the sequencesthat mediate binding between an miRNA and mRNA are short and thus recognizable by manymiRNAs .
Interestingly, histone modifications and DNA methylation also affect miRNAs in lupusCD4+ T cells. Ding and colleagues  showed that miR-142-3p and -5p are decreased in lupus CD4+ Tcells. This causes overexpression of SLAM-associated protein, IL-10, and CD84. H3K27methylation levels were increased in the putative miR-142 regulatory regions. The threeCpG pairs closest to the miR-142 transcription start site were also hypermethylated inlupus CD4+ T cells, but average methylation of 11 CpG pairs across themiR-142 regulatory region was not different in lupus CD4+ T cells .
Lupus T-cell miRNAs
Yu and colleagues  provided the first report of a possible role for miRNAs in autoimmunediseases. Sanroque mice, which have a defect in Roquin, develop a lupus-like syndromeattributed to increased ICOS (inducible co-stimulatory molecule) expression on Tcells. A sequence in the ICOS 3' untranslated region is required for Roquin to blockICOS expression, and this sequence is recognized by miR-101 . MiR-101 does not completely explain Roquin's ability to block ICOSexpression, although other miRNAs could also be responsible.
Multiple miRNAs are differentially expressed in CD4+ T cells from patientswith lupus compared with healthy donors, and two recent reports link miRNAs and DNAmethylation. MiRNAs-21, -126, and -148a are upregulated in CD4+ lupus Tcells and decrease Dnmt1 expression [42, 43]. MiRNAs-126 and -148a decrease Dnmt1 directly. MiR-21 decreases RASGRP1,in the JNK signaling pathway, which leads to decreased Dnmt1 expression. BlockingmiR-21, 126, or 148a in lupus CD4+ T cells increases Dnmt1 levels anddecreases CD70 and CD11a levels. MiR-21 is also increased in CD4+ lupus Tcells , and blocking miR-21 expression decreases levels of themethylation-sensitive gene CD40L. Blocking miR-21 also decreases proliferation, IL-10expression, and B-cell maturation. Overexpressing miR-21 has opposing effects,including increased CD40L expression. A therapeutic potential for miRNAs is shown inthese cases since blocking their expression reverses lupus-like phenotypes.
Effects of miRNAs in total CD3+ T cells and PBMCs from patients with lupushave also been studied. For example, miR-31 and IL-2 levels are decreased in lupus Tcells. This effect is mediated by RhoA, which blocks IL-2 expression. MiR-31decreases RhoA, which increases IL-2 . MiR-125a is also decreased, and RANTES (regulated and normal T cellexpressed and secreted) increased, in lupus T cells, and these effects are mediatedby interactions between miR-125a and KLF13, a regulator of RANTES expression . Again, suppressing these miRNAs in lupus T cells reversed the defects inIL-2 and RANTES expression. MiR-146a is also downregulated in lupus PBMCs. MiR-146anegatively regulates IFN-α/β expression by targeting STAT-1 and IRF-5,suggesting a link between miR-146a and lupus pathogenesis .
Mouse models corroborate these findings. MiR-21 is increased in CD3+ Tcells from lupus-prone B6.Sle123 mice, and blocking miR-21 expression decreasessplenomegaly . Suppressing miR-21 also increases PDCD4 expression. PDCD4 is decreased inCD4+ T cells from patients with lupus, and miR-21 suppresses PDCD4 whentransfected into normal CD4+ T cells .
Genetic/epigenetic interactions in lupus
As discussed above, lupus develops when genetically predisposed people encounterenvironmental agents that initiate disease onset and flares, and the environment appearsto trigger lupus onset and flares at least in part by modifying T-cell DNA methylation.The relationship between T-cell DNA demethylation and genetic predisposition is complexbecause both can vary. T-cell DNA demethylates in lupus, and the degree of DNAdemethylation is directly related to lupus flare severity . Genetic predisposition to lupus approximates a continuous variable. So far,38 lupus single-nucleotide polymorphisms (SNPs), each with its own relative risk or oddsratio for lupus, have been identified . These genes assort largely independently, so any given person can inherit 0to 38 distinct lupus genes, with 0, 1, or 2 copies of each. It is reasonable to proposethat those with a higher total genetic risk for lupus may have more problems with lupusthan those with a lower genetic risk.
Genetic/epigenetic interactions and age of lupus onset
The variable nature of T-cell DNA demethylation and genetic risk suggests that thedegree of T-cell DNA demethylation may interact with total genetic predisposition toinitiate lupus onset and flares in any given person. One example of this interactionis suggested by a study relating age of lupus onset to genetic risk. T-cell DNAdemethylates with age , and T-cell DNA demethylation contributes to lupus flares . The age of lupus onset is also variable . Typing for 19 risk alleles, this study confirmed that those developinglupus early in life had a greater genetic risk than those developing lupus later, andsome differences between ethnic groups were observed . Typing for 22 lupus SNPs and adjusting each for their relative risk forlupus, Criswell and colleagues  found a similar relationship between age of lupus onset and total lupusgenetic risk in Caucasians. Since young people have higher T-cell DNA methylationlevels than older individuals, these studies suggest that young people with high DNAmethylation levels may require more lupus genes to develop lupus but that those witha lesser genetic risk require a greater environmental impact on their T-cellepigenome. This also suggests that those with an even lower total genetic risk mayonly develop an ANA with age.
Genetic/epigenetic interactions in women and men with lupus
The strongest genetic factor predisposing patients to lupus is female sex. Women havetwo × chromosomes whereas men have just one, and the second × in women isinactivated by mechanisms that include DNA methylation. This raises the possibilitythat the second × chromosome may demethylate in women with active lupus,allowing overexpression of X-linked immune genes in women but not in men. CD40L(CD40LG) is an X-linked B-cell co-stimulatory molecule previously reported to beoverexpressed on lupus lymphocytes, and murine lymphocytes overexpressing CD40Linduce lupus [52, 53]. Treating CD4+ T cells from healthy men and women with 5-azaCcaused CD40L overexpression on the female but not the male T cells, and bisulfitesequencing confirmed that women have one methylated and one unmethylated CD40LG geneand that 5-azaC demethylated the methylated gene. In contrast, men had only one,unmethylated gene, and 5-azaC had no further effect on CD40L gene methylation orexpression .
Similar experiments compared CD40L on CD4+ T cells from men and women withactive lupus. CD40L levels increased with disease activity on CD4+ T cellsfrom the women, and the degree of overexpression correlated with demethylation oftheir methylated CD40LG gene , similar to CD11a, CD70, perforin, and KIR [1, 24]. In contrast, no change in CD40L expression levels was seen in men matchedwith the women for disease activity, consistent with their one unmethylated gene.Controls included demonstrating that the men had an increase in CD70 (encoded onchromosome 19) expression equivalent to that in women with active lupus . These results indicate that genes on the female inactive × cande-methylate and be overexpressed in women with lupus, potentially contributing tothe female predisposition to this disease. This is supported by a report that menwith Klinefelter's syndrome (XXY) develop lupus at approximately the same rate aswomen but that women with Turner's syndrome (XO) do not develop lupus .
Since the genetic and environmental contributions to lupus are variable and women arepredisposed to lupus because their second × chromosome can demethylate, it isreasonable to propose that men with only one × chromosome might require agreater degree of T-cell DNA demethylation or a greater total genetic risk (or both)to develop a flare equal in severity to that of women. This was tested by comparingthe interaction between the degree of DNA methylation in KIR and perforin genes,total genetic risk, and SLEDAI (Systemic Lupus Erythematosus Disease Activity Index)in men and women with lupus. Interestingly, the men had a slightly higher totalgenetic risk than the women (P = 0.05). Comparing the level of KIR orperforin methylation with SLEDAI scores showed no significant differences between themen and women. However, when the level of DNA methylation was adjusted by the totalgenetic risk and plotted against the SLEDAI (SLEDAI = risk/methylation) for eachsubject, the men required a stronger genetic/epigenetic interaction to achieve aflare equal in severity to that in women for both KIR (P = 0.01) andperforin (P = 0.005) .
Epigenetic mechanisms, including DNA methylation, histone modifications, and miRNAs,play an essential role in regulating gene expression. Recent evidence indicates thatdysregulation of these mechanisms alters gene expression in immune cells, contributingto the development of lupus in genetically predisposed people. Characterizing theepigenetic alterations and the mechanisms causing them is likely to provide importantnew insights into mechanisms causing human lupus and suggest new approaches to thetreatment of this disease.
This article is part of the series on Epigenetics and rheumatic diseases,edited by Nan Shen. Other articles in this series can be found athttp://arthritis-research.com/series/epigenetic
DNA methyltransferase 1
hematopoietic progenitor kinase 1
c-Jun N-terminal kinase
lymphocytefunction-associated antigen 1
regulated and normal T cell expressed and secreted
Systemic Lupus Erythematosus Disease Activity Index
The authors thank Julie Olivero for assistance in preparing the manuscript.
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