Epigenetics is the study of potentially heritable changes in gene expression without alteration of the nucleotide sequence. Epigenetic modifications exert their effects at the level of transcription or translation (Fig. 1). These epigenetic phenomena are dynamic and potentially modifiable, making them attractive therapeutic targets (1–3). Technological advances have enabled cost-effective, population-based epigenome-wide association studies to be conducted (Fig. 2), and online epigenetics resources have been created to aid researchers. These include the International Human Epigenome Consortium (www.ihec-epigenomes.org), the National Institute for Health RoadMap Epigenomics Project (www.roadmapepigenomics.org/), and the Human Epigenome Atlas (www.genboree.org/epigenomeatlas/index.rhtml).
Several clinical risk factors are known to adversely impact transplant outcomes, including donor age, organ ischemic times, recipient comorbidity, degree of human leukocyte antigen mismatch, and immunologic and nonimmunologic gene polymorphisms (4–8). Chronic allograft dysfunction results from both immunologic and nonimmunologic insults, and efforts to minimize these should abrogate ongoing graft injury. This model fails to consider the epigenetic effects of environmental stressors, which can persist long after the removal of the injurious process. Transplant recipients have typically experienced significant chronic illness, and it is conceivable that their epigenomes have been altered by the pathologic cellular milieu or drugs prescribed during periods of ill health. Understanding the contribution of epigenetics is one of the fundamental components of the “omics” approach to complex disease analysis (Fig. 1). Epigenetic modifications and genetic variation can act independently to influence gene expression but may also contribute synergistically to regulate gene expression. Integrating genetic and epigenetic datasets can provide powerful tools to identify risk factors for complex disease (9, 10), and this integration of biologic data could improve clinical risk prediction for long-term transplantation outcomes.
The concept of inducible and heritable phenotypic modification was first proposed by Lamarck (11)in 1809 when he hypothesized that organisms undergo physiologic changes in response to environmental stimuli that persist after cell division. More recently, evidence to support this Lamarckian view of inheritance has emerged from studies of individuals experiencing intrauterine malnutrition (12). Follow-up of children born after the Dutch winter hunger famine of 1944 to 1945 confirmed they had a higher body mass index and increased risk of diabetes and cardiovascular events in adulthood compared with their unexposed siblings; these complex disease phenotypes have persisted in their descendants (13). Epigenetic modifications are an important mechanistic link between remote environmental stress and longer term changes to the program of gene expression.
Major epigenetic mechanisms that impact gene expression include DNA methylation, histone modifications, and RNA interference. Experiments to address the effect of such modifications on transplantation outcomes are still in the early stages, but recent technological advances have enhanced the ability to generate robust and reproducible epigenetic data.
DNA methylation is an important mechanism that can silence or repress gene expression. DNA methylation is critical for fine control of multiple cellular functions, including differentiation, genomic imprinting, and X chromosome inactivation (14, 15). DNA methylation primarily involves the covalent binding of a methyl group to the cytosine pyrimidine ring in cytosine-phosphate diester-guanine (CpG) islands (16). CpG islands are high-density clusters of CpG dinucleotides; these are associated with gene promoters and are conserved across species (2, 17, 18). Methylation of CpG islands inhibits gene expression by directly impeding the binding of transcription factors (1, 19–22) or by attracting methyl CpG-binding proteins and histone-modifying enzymes, resulting in the formation of condensed chromatin (2, 18, 19).
Methylation-sensitive restriction endonucleases distinguish between methylated and unmethylated CpG islands, and the density of DNA methylation can be measured by immunoprecipitation. However, these do not provide information about the methylation status of specific CpG dinucleotides (2, 15, 23). Bisulfite treatment induces deamination of unmethylated cytosine to uracil with preservation of methylated cytosine. These methylated cytosine sites can subsequently be analyzed by mass spectrometry or DNA sequencing (1, 2, 23). Commercial arrays have recently been developed that facilitate analysis of the entire methylome (23).
Histone proteins are essential to chromatin structure. There are multiple epigenetic modifications that can occur within the amino acid tails of histones 2a, 2b, 3, and 4. These affect the affinity of the histone molecules for DNA, thereby altering the structure of chromatin and determining the accessibility of DNA to transcription factors (15, 20). Histone modifications include acetylation, phosphorylation, methylation, and ubiquitination (17). Acetylation typically results in transcriptional activation, whereas the effects of the other modifications are variable and depend on the site of action of acetyltransferases, histone deacetylases (HDAC), and histone methylases (17).
Chromatin immunoprecipitation is used to quantify histone modifications. Chromatin is precipitated using antibodies to specific histone modifications and the affected DNA region analyzed by microarray or sequencing (1, 24).
RNA can reduce gene expression after transcription by repressing translation of messenger RNA (mRNA) or increasing mRNA degradation (25, 26). Advances in transcriptomic profiling have revealed relatively large numbers of noncoding RNA sequences transcribed from the human genome; micro-RNA (miRNA) form part of this group. These small, noncoding RNA molecules can regulate gene expression and epigenetic phenomena, resulting in changes in levels of thousands of proteins. Sequencing, microarrays, or quantitative real-time polymerase chain reaction may be used to analyze miRNA.
The discovery and continued characterization of miRNAs, RNA molecules composed of approximately 22 nucleotides (27), has revolutionized the understanding of gene regulation, cell differentiation, proliferation, and apoptosis. A complex epigenetic network exists whereby even miRNA expression is under epigenetic control (28). Several miRNAs have been associated with allograft rejection (29). Lorenzen et al. (30) recently reported alteration in miRNAs in the urine of renal transplant recipients with acute rejection, suggesting that miRNAs may prove to be useful noninvasive biomarkers in transplantation.
EPIGENETICS AND DISEASE
There is increasing evidence that epigenetic phenomena play a major role in chromosomal defects (31–33) and common complex diseases (1, 34, 35).
DNA methylation, histone modification, and RNA interference have been implicated in oncogenesis. Aberrant epigenetic control causes transcriptional silencing of tumor suppressor genes, activation of oncogenes, and increased chromosomal instability (14, 32, 36); recognition of this has led to diagnostic, prognostic, and therapeutic advances. Epigenetic biomarkers have been developed for gastrointestinal, breast, and prostate cancers (36–38). In hemato-oncology, demethylating agents have proven to be effective treatments (39, 40). The oncogenic phenotype of lung and breast cancer cells has been successfully modified by RNA interference in vitro, but this has not yet been translated into clinical practice (41).
Altered epigenomes have been demonstrated in chronic diseases, including cardiovascular disease, chronic obstructive pulmonary disease, depression, schizophrenia, chronic kidney disease (CKD), and obesity (1, 34, 35, 42–44). Prospective epigenetic studies are required to establish the causal role of these epigenetic modifications in complex disease.
CKD is associated with accumulation of uremic toxins and enhanced oxidative stress, and these have the potential to modify the epigenetic signature. In patients with CKD, global hypermethylation is independently associated with cardiovascular mortality (44). Recently, epigenetic hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, has been reported to be associated with persistent fibroblast activation and renal fibrogenesis (45). This kidney fibrosis is ameliorated in animal models that are heterozygous for the gene encoding the methyltransferase enzyme DNMT1 (45). Both epigenetic modifications occurring in the recipient’s “uremic” environment and those resulting from allograft ischemia-reperfusion injury could potentially change gene expression and affect renal allograft outcomes.
EPIGENETICS AND TRANSPLANTATION
The epigenetic signature of transplanted organs may be attributable to both the recipient and donor environments; the recipient epigenome has a broader role in the regulation of immune function (Table 1).
The cumulative injurious burden of nonimmunologic insults contributes to the development of chronic allograft dysfunction. Recipients who receive a human leukocyte antigen–identical transplant can develop interstitial fibrosis as a result of nonimmunologic injury (46).
Prolonged ischemia preoperatively is associated with delayed graft function, acute rejection, chronic allograft dysfunction, and reduced transplant survival (47). Reperfusion of the ischemic graft stimulates the release of oxygen and hydroxyl free radicals, which damage DNA, proteins, lipids, and plasma membranes, and these free radicals have the potential to alter the epigenome. In a rodent model of renal transplantation, it was demonstrated that prolonged cold ischemia of rat kidneys caused hypomethylation of the complement factor 3 gene (48, 49). Loss of transcriptional repression of this gene provides a plausible explanation for the accentuated immunologic injury, which often follows protracted ischemia of the allograft. Of interest, myocardial hypoxia induces HDAC enzymes, but the area of experimental cardiac ischemic injury can be substantially reduced by treatment with HDAC inhibitors. These acetylase antagonists inhibit cell death and reduce vascular permeability in the injured myocardium (50). H3 acetylation follows acute renal ischemia in mice, with resulting activation of inflammatory genes, enhanced cytokine expression, and fibrosis (51). If these epigenetic modifications contribute to allograft dysfunction, there may be an opportunity for therapeutic pharmacologic intervention.
Mehta et al. (52) compared the promoter methylation status of the CALCA gene between renal transplant recipients and healthy controls. Promoter hypermethylation was increased in the transplanted group compared with healthy controls. CALCA promoter hypermethylation was greatest in recipients with acute tubular necrosis compared with those with acute rejection or normal graft function (52). The methylation status of CADM1 is an independent predictor of the risk of recurrence of hepatocellular carcinoma after liver transplantation (53). Methylation-based biomarkers may prove to be noninvasive diagnostic and prognostic tools in transplantation.
Epigenetic modifications may play a role in viral infection after transplantation. Epstein-Barr virus infection is the predominant risk factor for posttransplant lymphoproliferative disorder (54), and Epstein-Barr virus infection of B lymphocytes results in the expression of several viral genes (55). A small study of recipients with posttransplant lymphoproliferative disorder identified enhanced DNA methylation at the promoter regions of two genes in the majority of affected individuals (56). Cytomegalovirus (CMV) infection causes significant morbidity in solid organ transplant recipients (57). In vitro, CMV gene expression in infected cells can be silenced by DNA hypermethylation, and this gene silencing may be reversed by the addition of a DNA demethylase (58). Of further interest, the transition from acute to latent CMV infection is associated with histone modification in murine models (59). Recent evidence demonstrates that hepatocellular carcinoma secondary to hepatitis B infection exhibits specific DNA methylation profiles compared with liver cancers associated with hepatitis C virus, alcoholism, or aflatoxins (60). It is unclear whether these epigenetic modifications contribute to the development of hepatocellular carcinoma. The role of epigenetics in viral infections after transplantation is beginning to be explored and may both provide valuable insights into the pathogenesis of these infections and identify potential therapeutic targets.
Allograft rejection is a complex process involving interaction between the donor cells of the transplanted organ and the recipient immune system. It remains a persistent challenge in transplantation. Although the incidence of acute rejection could be reduced by increasing the immunosuppressive load, this is at the expense of a predisposition to serious infection and malignancy. All of the major immune cells involved in allograft rejection are influenced by epigenetic factors (Table 1).
The major histocompatibility complex (MHC) encodes glycoproteins, which present antigens to the immune system. MHC class I antigens are present on almost all nucleated cells, whereas class II are selectively expressed by antigen-presenting cells. The initial phase of rejection is the recognition of an alloantigen by T cells (61, 62). Direct allorecognition relies on T-cell detection of an alloantigen presented by a donor cell. Donor dendritic cells have a key role; they reside in the allograft but migrate when activated by inflammatory cytokines to allow T-cell activation (63). The indirect pathway involves the presentation of alloantigens by self MHC molecules (64). Concurrent T-cell receptor and costimulatory signaling propagate proliferation and differentiation of the T-cell lineage, cytokine production, and T-cell migration to the inflammatory site (61, 65).
MHC expression is fundamental to alloantigen recognition. Downregulation of MHC expression is a characteristic of gametes, embryonic cells, and neoplastic cells and confers them a degree of protection from the immune system (21). DNA methylation controls MHC class I and II expression; MHC class II expression is also affected by histone modifications. The histone methyltransferase CARM1 is critical to the constitutive expression of MHC II in B cells, and acetylation of the MHC II promoter enhances gene expression (66). In acute rejection, there is an increase in MHC II glycoproteins within the allograft (67).
Epigenetic mechanisms control T-cell activation through the production of interleukin-2 (IL-2). In inactive T cells, the IL-2 promoter is methylated and packaged within condensed chromatin, making it inaccessible to transcription factors. Following simultaneous T-cell receptor and costimulatory signaling, DNA demethylation and chromatin remodeling allow upregulation of IL-2 (19). Histone deacetylation also determines T-cell activation; the HDAC inhibitor FR276457 prolongs graft survival in animal models of kidney and heart transplantation (68, 69). The costimulatory signal between dendritic cells and T cells has been successfully blocked by RNA interference in mice (70). This attenuated the T-cell response to alloantigens and prolonged allograft survival (70).
B lymphocytes also play a significant role in rejection because of the high affinity of the B-cell receptor for foreign antigens. After interaction with a complementary epitope, the B cell communicates with its cognate T cell. Direct signaling and T-cell cytokine production stimulate the maturation, activation, and differentiation of B cells into plasma cells and memory cells (71). Alloantibodies cause severe graft rejection by binding to the transplanted organ and initiating the inflammatory cascade (72). A role for epigenetic regulation of B cells has recently been discovered. DNA methylation, histone modification, and chromatin remodeling determine B-cell receptor commitment, phenotypic stability, and cell signaling, whereas miRNAs have been implicated in differentiation (73).
T regulatory cells (Tregs) are of fundamental importance in immune modulation and the maintenance of self-tolerance. Their main role is to suppress self-reactive T cells that have escaped negative selection in the thymus, but Tregs also attenuate allograft rejection (74, 75). Several studies have identified an association between chronic rejection and low Treg populations (76, 77).
Although epigenetic modifications are important in many immune cells, they are essential to the stability and function of the suppressive Tregs. FOXP3 is a transcription factor that is constitutively expressed by Tregs and has a crucial role in the modulation of the immune system and the maintenance of self-tolerance (78–81). Stable FOXP3 expression is unique to Tregs and depends on the hypomethylation of the Treg-specific demethylating region (Fig. 3) (79, 80). Exposure of T cells to demethylases and HDAC inhibitors in vitro enhances FOXP3 expression, increases the Treg population, and augments suppressive function (18, 80, 82). Demethylating agents have been used to induce tolerance in graft versus host disease. They attenuate the disease course by enhancing FOXP3 expression (40). FOXP3 itself acts as a transcriptional repressor by inducing histone deacetylation of the promoter regions of pivotal cytokine genes (83).
TARGETING THE EPIGENOME
Epigenetic modifications influence many of the processes implicated in acute and chronic allograft dysfunction. The challenge is to modify the epigenome for therapeutic benefit. This has already been achieved in oncology where demethylating agents form part of the armamentarium used to treat hematologic malignancies (39, 40) and may also have a role in solid organ neoplasia (84, 85).
Genetic variation may have a significant effect on the action of immunosuppressants. Thiopurine methyltransferase genotyping can be used in clinical practice to assess the risk of myelosuppression with azathioprine treatment (86), and mutations in the multidrug resistance 1 gene can also affect clinical response to azathioprine (87). Calcineurin inhibitors are metabolized by cytochrome P450 enzymes, and an association has been identified between the CYP3A5 genotype and tacrolimus dosing requirements (88). Mycophenolate mofetil action and the incidence of adverse events are affected by single-nucleotide polymorphisms in the inosine monophosphate dehydrogenase type I and II genes (89, 90), the solute carrier organic anion transporter family member 1B1 gene (91), the IL-12A gene, complement factor H gene, and the cytochrome P450 2C8 gene (92). It is likely that immunosuppressive regimens induce clinically significant epigenetic modification. Glucocorticoids inhibit natural killer cell activity in vitro by altering histone deacetylation, but their global effect on the epigenome is unknown (93). The epigenetic effects of other immunosuppressive agents still remain to be elucidated. A greater understanding of the epigenetic modifications involved in allograft rejection could lead to the development of pharmacologic agents that act selectively on epigenetic targets.
There is also potential for epigenetic therapies to maintain the integrity of the epigenome of the transplanted organ. DNA methylating agents could prevent the aberrant demethylation of gene promoters that occurs during the cold ischemic period and reduce the subsequent inflammatory response.
The major disadvantages of current epigenetic therapies are the lack of specificity and unknown long-term safety profiles. Demethylases and HDAC inhibitors induce epigenome-wide changes; this is associated with chromosomal instability, a feature of neoplastic cells (32). Although this may be an acceptable risk in individuals with life-threatening malignancy, it would be more difficult to justify exposing other patients to these drugs. Transplant recipients already have an increased cancer predisposition as a consequence of immunosuppressive therapy. The risk:benefit ratio for current epigenetic therapies in transplant recipients is unknown.
The nonspecific nature of epigenetic treatments could be overcome by harnessing RNA interference. These RNA molecules are specific to target mRNA, and this is an area of rapid development (29). Several promising miRNA therapies are undergoing preclinical testing, but there has not yet been a translation of miRNA therapy from bench to bedside.
Epigenetic modification plays a key role in biologic processes that contribute to allograft dysfunction. Detailed interrogation of the epigenome in transplant recipients and donors will provide further insight into the epigenetic modifications associated with allograft survival. Epigenetic research is a hot topic, which is rapidly generating data that not only improve understanding of biologic mechanisms but also offer scope for prognostic and therapeutic clinical utility in the short to medium term.
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