Epigenetic regulation encapsulates a wide range of mechanisms that can result in heritable changes in gene expression. From physical localization of genes within the nucleus to post-translational modifications of DNA and histones, epigenetic mechanisms can profoundly influence gene expression and alter cellular lineage development and function. The site-specific methylation and demethylation of CpG motifs in DNA by DNA methyltransferases (DNMTs) and the TET family of proteins is perhaps the best studied epigenetic mechanism that directly regulates gene expression (5). Although DNA methylation has been implicated in lymphoid cell responses (6–8), this review will focus on the posttranslational methylation of histones.

Regulation of gene expression by histone modifying enzymes is an important mechanism that regulates cellular development and differentiation. Histones can be modified posttranslationally by phosphorylation, acetylation, ubiquitination, sumoylation, and methylation (9). In particular, methylation of histone lysine residues is an important regulator of gene expression. Mixed lineage leukemia-1 (MLL1)-dependent H3K4me3 (10) and enhancer of zeste homolog-2 (EZH2)-dependent H3K27me3 (11) are the best known modifications and are associated with gene expression and repression, respectively (12–14). Other histone-methylation sites have been shown to be critical as well, including H3K9, with G9a-dependent H3K9me2 and Suv39h1/2-mediated H3K9me3 playing important roles in cell differentiation and function (15–18). Of these, H3K9me2 has been shown to modify euchromatin and dynamically regulate gene expression in differentiating cells.

In embryonic stem (ES) cells, it has been proposed that H3K9me2 marks increase across the genome as cells differentiate and acquire lineage specificity (19) although this is contentious (20). Specifically, H3K9me2 is found enriched at lineage non-specific genes, suggesting that acquisition of H3K9me2 is critical for gene silencing during differentiation (21). However, there is very little known about the role of G9a in cells of the immune system. In lymphoid cells such as T cells and ILCs, it is clear that G9a-dependent H3K9me2 is critical for cellular differentiation and function, although the mechanisms differ from ES cells. In this review, we focus on the role of the histone lysine methyltransferase G9a in lymphoid cell responses in health and disease.

G9a (Ehmt2) was first identified as a gene located in the major histocompatibility complex (MHC) locus in mice and human leukocyte antigen (HLA) locus in humans and was also called HLA-B-associated transcript 8 (BAT8) (22–25). The Ehmt2 gene is located in the ~700 kb (mouse)/~1.1 Mb (human) MHC/HLA Class III region that contains over 60 genes (26) including cytokines (TNF-α and TNF-β), complement proteins C2 and C4, heat shock proteins (HSP70), and enzymes (steroid 21-hydroxylase Cyp21). The Ehmt2 gene is made up of 28 exons that code for a 1,263 amino acid protein. A splice form lacking exon 10 that codes for 34 amino acids has also been identified, although the functional significance is still unknown (24).

The related protein G9a-like protein (GLP, EHMT1) whose gene is not located in the MHC/HLA locus forms a heterodimer with G9a in vivo and is critically required for the H3K9me2 methylation activity (27). Genetic deletion of either protein results in a significant reduction in H3K9me2, suggesting that both subunits are essential to the enzymatic activity (28). Mutation of the active sites has shown that the methyltransferase activity of G9a plays a larger role in H3K9me2 methylation in vivo (29). However, global gene expression analysis of neurons of mice with targeted deletions of either G9a or GLP identified differences that may be due to differential requirement of each subunit in gene-specific expression (30). Further, loss of GLP is associated with Kleefstra Syndrome, a rare genetic disease that is characterized by intellectual disability and other social and physical impairments (31). There has been no analysis of the function of immune cells in Kleefstra Syndrome patients. Thus, although GLP may play a specific role in regulation of gene expression, it remains to be directly tested.

G9a is a 1,263 amino acid protein with several distinct domains (Figure 1). G9a does not contain a DNA-binding domain and must rely on cofactors for its localization to specific genes. Functionally, the C-terminal SET domain contains the lysine methyltransferase activity that defines the major function of this family of proteins. The SET domain of G9a is able to mono- and dimethylate H3K9 but is less efficient in mediating trimethylation (32). Consistent with this, deletion of G9a leads to a global reduction in H3K9me2 while H3K9me3 is largely unaffected (18, 28). Unlike other members of the SET domain family, G9a also has unique domains that provide additional functions. First, G9a has a series of eight 33-amino acid repeats that have homology to the ankyrin repeat domain of Drosophila Notch (25). This region was further shown to act as a domain that could specifically bind to dimethylated lysine residues, providing a protein that can not only generate a specific posttranslational modification but also bind to that modification (33). Interestingly, although the ankyrin repeats of G9a have an affinity for H3K9me2, GLP binds to H3K9me1 with higher affinity (33) and mice that carry a knock in of G9a with non-functional ankyrin repeats develop normally while mice with a mutated GLP have severe developmental defects resulting in perinatal lethality (34). These results further demonstrate that G9a and GLP have some non-overlapping roles in vivo. G9a also has a stretch of 25 glutamic acid residues as well as a Cysteine-rich region, whose functions remain unknown.

Although G9a has predominantly been studied in the context of gene repression via its methyltransferase activity on histones, it is clear that G9a also has a role in gene activation under certain conditions (35–37), which is methyltransferase-independent (discussed below). This function has been mapped to the N-terminus of the protein as the first 280 amino acids are sufficient to promote gene expression by acting as a scaffold to recruit transcriptional coactivators such as CARM1 and p300 (36, 38). Thus, G9a is a complex protein that is involved in gene repression and activation through distinct mechanisms.

G9a is the enzyme that is responsible for the dimethylation of H3K9, a hallmark of silenced euchromatin (18, 28, 39–41). H3K9me2 acts as a binding site for heterochromatin protein 1 (HP1) that recruits transcriptional repressors to prevent gene activation (42). Although H3K9me2 is the main product of G9a-dependent methylation, the G9a/GLP complex has also been described to methylate H1 (43, 44) and contributes to the methylation of H3K27 (39, 45). In addition, G9a has been shown to have activity against several non-histone proteins including itself (46) though the most well-studied aspect of G9a biology is the H3K9me2-dependent repression of gene expression. From genetic and biochemical studies, it is clear that G9a-dependent H3K9me2 is associated with genomic regions that are expressed at low levels (21) but the mechanisms that regulate the dynamic methylation patterns mediated by G9a still remain unclear. Indeed, as G9a lacks a domain that would promote direct interaction with DNA or chromatin, G9a has to rely on the DNA-binding capacity of its interaction partners.

In ES cells, G9a-dependent H3K9me2 is linked to de novo DNA methylation (47, 48). DNA methylation of endogenous retroelements, and a subset of non-repetitive sequences including CpG-rich promoters, is reduced in G9a-deficient cells, and Dnmt3a recruitment to retrotransposons is decreased in these cells. However, the interaction between G9a, H3K9me2, and DNMTs was absent in differentiated cells (48), suggesting that functional G9a–DNMT interactions are not maintained past development. Related to this function of G9a, the sustained silencing of pluripotency-associated genes in G9a-deficient ES cells is impaired and results in the reversal from the differentiated into a pluripotent state in a significant fraction of cells (49, 50). This effect has been exploited in the generation of induced pluripotent stem cells as inclusion of a G9a-specific chemical inhibitor BIX-01298 can replace viral transduction of Sox2 in fibroblasts (51).

Taken together, a general theme of G9a playing a role in the epigenetic silencing of cell-type inappropriate genes has emerged from studies in ES cells (52, 53). However, the role of G9a in immune cell function is less well understood.

In addition to its role as a histone lysine methyltransferase, several studies have shown that G9a is also able to methylate a wide range of non-histone targets, including G9a itself (46), CDYL1, WIZ and ACINUS, C/EBPβ, CSB, histone deacetylase (HDAC)1, mAM, KLF12, SIRT1, Reptin, MyoD, p21, and p53 (54). Although the precise role of posttranslational methylation on protein function remains unclear, methylation of non-histone proteins may affect protein stability, protein–protein interactions, subcellular localization, or function (55). Nevertheless, the precise physiological role of G9a-dependent methylation of non-histone proteins remains unclear and will not be discussed in depth in this review.

Apart from its ability to methylate substrates, G9a has also been shown to have methyltransferase-independent activities through the N-terminal domain of the G9a protein (35, 36, 38). Work from the Stallcup group was the first to show that in contrast to expectations, G9a was a strong coactivator of nuclear hormone receptor activity including androgen, estrogen, and glucocorticoid receptors by associating with the transcriptional coactivators GRIP1, CARM1, and p300 (35), and in the case of estrogen, with the receptor itself (36). G9a has also been shown to positively regulate gene expression at the β-globin gene locus (37). Regulation of the β-globin gene locus is a well-characterized system to examine the role of locus control regions (LCRs) in tissue- and stage-specific expression of genes (56). In red blood cells, the β-globin locus is regulated partially through the addition and removal of histone modifications including H3K4me3 and H3K9me2 (57). Knockdown of G9a in adult erythroid progenitor cells led to the heightened mis-expression of fetal β-globin as well as a significant reduction in adult β-globin gene expression (37), further demonstrating inhibitory and activating effects of G9a. Similar to studies with nuclear hormone receptors, the activation of gene expression by G9a was independent of methyltransferase activity. Importantly, the β-globin gene locus is regulated in a similar manner to the type 2 response Il4-Il5-Il13 locus (58) and may provide a common mechanistic link to regulate gene expression. Thus, in addition to its repressive functions, it is clear that G9a can positively influence gene expression at select genetic loci.

The vast majority of studies on the function of G9a have been carried out in ES cells and very little is known about the role of G9a in innate and adaptive immune cells. However, the ability of immune cells to respond to the external environment and differentiate into functionally distinct cell lineages is reminiscent of the cellular plasticity of ES cells and suggests that epigenetic mechanisms may be a conserved regulatory mechanism in these cell types.

In innate immune cells such as macrophages, G9a-dependent H3K9me2 has been associated with gene repression during endotoxin tolerance (59–61). Macrophages that are chronically stimulated with lipopolysaccharide (LPS) become unresponsive to further LPS stimulation through the acquisition of H3K9me2 at repressed genetic loci (61). In tolerized macrophages, G9a has been shown to interact with the TF ATF7 as well as several members of the NF-κB family including RelB, RelA, c-Rel, and NF-κB1 (59–61). It is proposed that G9a is recruited to specific loci by these factors to deposit H3K9me2, leading to gene repression. However, a direct role for G9a in macrophages during endotoxin tolerance has not been tested. Nevertheless, these studies identify a role for G9a in gene silencing during cellular responses to inflammatory signals. Consistent with this role in promoting tolerance, G9a has been also shown to limit JAK/STAT signaling in Drosophila following viral infections (62). In the absence of G9a, viral infection leads to increased lethality in flies but is not due to increased pathogen burden but due to heightened expression of JAK/STAT-dependent target genes. Thus, G9a is an important regulator of innate inflammatory gene expression.

G9a has also been implicated in several aspects of T cell biology. Although genome-wide studies mapping the binding of G9a or the deposition of H3K9me2 in immune cells has not been carried out due to technical reasons, a descriptive genome-wide analysis of H3K9me2 marks in resting human lymphocytes using ChIP-on-chip methods demonstrated that this epigenetic mark is enriched on genes that are associated with several specific pathways including T cell receptor signaling, IL-4 signaling, and GATA3 transcription (63). Furthermore, lymphocytes isolated from patients with type I diabetes displayed a distinct H3K9me2 profile, with genetic regions that had increased (CXCL3, CTLA-4, SLC17A4) and reduced (RARA, CAMK4, TNF) levels of H3K9me2 (64). Thus, G9a-mediated H3K9me2-dependent regulation of T cell responses may be associated with T cell function as well as development of inflammatory diseases such as diabetes.

More recently, cell lineage-specific deletion of G9a has been used to delineate the role of G9a in immune cells. Three independent strains of mice with a “floxed” G9a allele (G9a^fl/fl mice) have been generated (16, 65, 66). Crossing G9a^fl/fl mice with Cd4-Cre mice or Lck-Cre mice results in a T cell-specific deletion of G9a (G9a^ΔT mice). G9a^ΔT mice are born and develop normally and have no discernable defects in the generation of T cells in the thymus, spleen, or lymph nodes (16, 67), suggesting that unlike ES cells G9a is dispensable for cellular development of peripheral naive T cells. However, upon activation of T cells in vitro or in vivo, G9a was shown to play a critical role in regulating the function of Th cells. Consistent with the ability of G9a to promote and repress gene expression, G9a-deficient Th cells had a failure to produce certain cytokines while overproducing others (15, 16). Under distinct differentiating conditions, G9a was differentially required to activate or repress specific gene programs.

Th1 cells that produce IFN-γ are critical for immunity against intracellular pathogens including bacteria, viruses, and protozoan parasites (68). Strikingly, the absence of G9a in T cells has no effect on the development or magnitude of Th1 cell responses in vitro or in vivo (16). There was no difference in the frequency of Th1 cells that developed following activation of G9a-deficient T cells or wild type T cells in the presence of a G9a-specific inhibitor under Th1 cell-promoting conditions (16). Thus, from our understanding so far, G9a is dispensable for Th1 cell responses.

Immunity to infection with parasitic helminths such as the whipworm Trichuris muris is associated with a polarized type 2 cytokine response, with the production of IL-4, IL-5, and IL-13 by Th cells leading to mucus production, intestinal epithelial cell turnover, and worm expulsion (69). Infection of G9a^ΔT mice with Trichuris resulted in significantly reduced frequencies of protective Th2 cells, heightened frequencies of non-protective Th1 cells, and susceptibility to infection (16). This was consistent with a reduction in the production of type II cytokines by Th cells after in vitro activation. A role for G9a in repressing promiscuous type I cytokine gene expression in Th2 cells was only observed temporarily, as H3K9me2 is replaced by H3K27me3 at the Ifng locus during differentiation (70). Thus, G9a is required for activation of the type II cytokine gene program. G9a-deficient Th2 cells express comparable levels of the major Th2 cell transcriptional factors including GATA3 and STAT6 but fail to produce type II cytokines, suggesting that G9a is a central component of the transcriptional machinery for type II cytokines. As the Il4-Il5-Il13 locus is similar in genetic structure to the β-globin locus, it is perhaps not surprising that the ability of G9a to transactivate the type II cytokine gene locus was also independent of its methyltransferase activity (16). Taken together, these results identify a role for G9a as a critical component of the Th2 cell regulatory machinery.

In contrast to Th2 cells, G9a plays an important role in limiting Th17 and Treg cell differentiation that is dependent upon its methyltransferase activity (15). Activation of G9a-deficient Th cells under Th17- or Treg cell-promoting conditions resulted in a significant increase in the frequencies of IL-17A-producing and FOXP3-expressing cells, respectively (15). Inhibition of G9a methyltransferase activity with the small-molecule inhibitors BIX-01294 or UNC0638 also resulted in enhanced Th17 and Treg cell differentiation. Unlike the proposed role for G9a in ES cells, H3K9me2 is not deposited at lineage-promiscuous genes to control lineage differentiation in Th cells. Instead, G9a-dependent H3K9me2 is found at high levels in naive undifferentiated Th cells and is rapidly lost at lineage-specific and -non-specific loci after T cell activation (15). The loss of H3K9me2 alone is insufficient to promote gene expression, providing an explanation for the lack of a phenotype in the steady state. Thus, in naive T cells, H3K9me2 and G9a act as an additional layer of negative regulation to maintain cells in a naive state. Mechanistically, loss of G9a-dependent H3K9me2 results in an increase in the accessibility of the chromatin to transactivating factors, which leads to heightened responsiveness to external signals such as cytokines. In the case of Th17 and Treg cells, G9a-deficient Th cells are ~40 times more sensitive to TGF-β (15). Taken together, these results suggest that G9a, through the deposition of H3K9me2 at a wide variety of immune genes, is specifically important in naive T cells to repress gene expression, possibly by limiting accessibility of TFs and coactivators to specific genetic loci.

Unlike naive T cells, G9a has been proposed to repress expression of CD25 and CD27, cell surface receptors that are associated with cell activation and proliferation, in memory T cells during viral infection (71). Through a specific interaction with the transcriptional repressor Blimp-1 (PRDM1), G9a (but not EZH2) is recruited to specific genetic loci resulting in gene repression. In the absence of Blimp-1, reduced levels of H3K9me2, H3K9me3, and H3K27me3 is observed, suggesting that G9a is critical for the silencing of genes during memory T cell differentiation and development. Whether G9a is required for memory T cell generation has not been examined directly; however, this is an active area of research, which will shed further light on the role of G9a in T cell memory development. Thus, in the absence of G9a, other repressive modifications such as Suv39h1/2-dependent H3K9me3 and EZH2-dependent H3K27me3 may compensate. Although, the details remain unclear, the identification of the epigenetic mechanisms in memory T cell development and function will be critical for the optimal design of vaccines as well as the development of therapeutic strategies to target dysregulated memory T cell responses in inflammatory diseases.

In addition to T cells, G9a also plays a critical role in the development and differentiation of ILCs (72). Mice with a hematopoietic cell-specific deletion of G9a (generated by crossing G9a^fl/fl mice with Vav-Cre mice) have reduced numbers of group 2 ILCs (ILC2s) and increased frequencies of ILC3s in all tissues examined. Although the small number of ILC2s present in lung tissue are phenotypically normal, they are dysfunctional, failing to produce effector cytokines IL-5 and IL-13 following stimulation with the activating cytokine IL-33 or following intranasal administration of the protease allergens papain or house dust mite antigen (72). Genome-wide expression analysis of bone marrow-derived ILC2 precursors identified a global shift in expression from ILC2-specific transcripts to ILC3-specific genes, placing G9a and H3K9me2 as a central regulator of the ILC2/ILC3 lineage choice.

In contrast to T cells and ILCs, G9a appears to play a minor role in B cell development and function (52, 67). An early study using a V–D–J minilocus suggested that G9a inhibited germline transcription and recombination (73). However, mice with a B cell-specific deletion of G9a (Mb1-Cre) fail to show any overt phenotypes, including displaying normal V–D–J recombination (67). The absence of G9a did result in a skewed usage of the κ light chain over the λ light chain, as well as a slight reduction in IL-4- and LPS-induced proliferation and differentiation into plasma cells. This reduction in plasma cell differentiation is consistent with a study showing that G9a directly interacts with Blimp-1, a critical plasma cell differentiation factor (74). Thus, G9a is dispensable for normal B cell development and has little effect on most functions of B cells. However, it is possible that G9a has a subtle role in specific aspects of B cell biology that remain to be determined.

Together, these results demonstrate that G9a is an important regulator of immune cell function. However, the precise mechanisms are yet to be defined. In the following sections, immune functions regulated by proteins known to interact with G9a will be discussed and potential mechanisms for G9a-dependent regulation will be proposed.

From the studies outlined above, it is clear that G9a is a central regulatory node in the establishment of the epigenetic landscape of cells and is critical for shaping cellular identity. However, how G9a mediates its function is poorly understood. As G9a lacks a DNA-binding domain, it is dependent upon additional cofactors for its localization at specific genetic loci. Several classes of G9a-interacting proteins have been identified (Figure 2). Strikingly, many of the G9a cofactors have important roles in immune cell development and function, potentially offering a mechanistic explanation for the phenotypes associated with loss of G9a in lymphoid cells.

Currently, there are several chemical probes that specifically target the methyltransferase activity of G9a. BIX-01294 was first identified in a high-throughput screen (125) and was shown to bind to the SET domain of G9a and GLP in the peptide-binding site, preventing methylation (126). BIX-01294 was subsequently optimized through structure–activity relationships to generate UNC0224, UNC0321, and E72 that showed increased activity and specificity (127, 128). The further development of UNC0638 and UNC0642 resulted in a potent, specific, stable, and cell-permeable inhibitor of G9a (129). However, UNC0642 has poor pharmacokinetics for in vivo use. More recently, an additional inhibitor of G9a that is unrelated to UNC0642, called A-366, was discovered through an independent high-throughput screen (130, 131). Treatment of mice with A-366 showed no overt toxicity and was able to reduce the growth of tumor xenografts (131). Thus, although A-366 has not been tested in the context of inflammatory disease, these results suggest that inhibition of G9a could prove to be a significant therapeutic strategy to modulate immune responses.

It is clear that G9a is a central control point in lymphoid cell development, differentiation, and function. Acting through its diverse binding partners, G9a can repress and activate gene programs associated with a wide variety of immune responses. As G9a has been shown to be amenable to drug inhibition, blocking the function of G9a may provide a new therapeutic modality to modulate a wide variety of inflammatory diseases. For example, both Blimp-1 and G9a are required to limit Th17 cell development in a cell-intrinsic manner (15, 123). However, the increased frequency of Th17 cells does not result in enhanced pathogenicity of inflammatory diseases such as EAE or intestinal inflammation (15, 124), demonstrating that reducing the activity of Blimp-1 or G9a, or by inhibiting their interaction, may be a viable method to reduce the development of pathogenic Th17 cells. Future studies will define the precise role of G9a in immune cell development, differentiation, and function and determine the relevance of G9a as a drug target to treat inflammatory disease.

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.