TET Enzymes and 5hmC in Adaptive and Innate Immune Systems

Abstract

DNA methylation is an abundant and stable epigenetic modification that allows inheritance of information from parental to daughter cells. At active genomic regions, DNA methylation can be reversed by TET (Ten-eleven translocation) enzymes, which are responsible for fine-tuning methylation patterns. TET enzymes oxidize the methyl group of 5-methylcytosine (5mC) to yield 5-hydroxymethylcytosine (5hmC) and other oxidized methylcytosines, facilitating both passive and active demethylation. Increasing evidence has demonstrated the essential functions of TET enzymes in regulating gene expression, promoting cell differentiation, and suppressing tumor formation. In this review, we will focus on recent discoveries of the functions of TET enzymes in the development and function of lymphoid and myeloid cells. How TET activity can be modulated by metabolites, including vitamin C and 2-hydroxyglutarate, and its potential application in shaping the course of immune response will be discussed.

Keywords: 5 hydroxymethylcytosine; 5hmC; DNA modification; epigenetics (methylation/demethylation); gene regulation and expression; ten eleven translocation (TET).

Figures

Figure 1

TET-mediated DNA modifications and demethylation. (A) Unmodified cytosine (C) is methylated by DNA methyltransferases (DNMTs) at the 5 position to become 5-methylcytosine (5mC). TET proteins oxidize 5mC into 5-hydroxymethylcytosine (5hmC), a stable epigenetic mark, and subsequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TET can demethylate DNA via replication-dependent (passive) or replication-independent (active) mechanisms. (B) Left, passive DNA demethylation. DNMT1/UHRF1 complex recognizes 5mC at the hemi-methylated CpG motif during DNA replication and methylates the unmodified cytosine on the newly synthesized DNA strand (left; pink strand). However, the oxidized methylcytosines 5hmC, 5fC, and 5caC (together, oxi-mC) are not recognized by DNMT1/UHRF1, resulting in unmodified cytosine on the new DNA strand. Further DNA replication in the presence of continuing TET activity will result in progressive dilution of 5mC in the daughter cells. Right panel, active DNA demethylation. While 5hmC is stable and persists in the genome, 5fC and 5caC can be recognized and excised by thymine DNA glycosylase (TDG), and the resulting abasic sites are repaired as unmodified C by base excision repair (BER). Other mechanisms (e.g., decarboxylation of 5caC) have been suggested but have not yet been proven to exist. (C) The approximate abundance of unmodified and modified cytosines in the haploid human/mouse genome. About 5% of cytosine is methylated (5mC); in most cells, the vast majority of 5mC is present at CG dinucleotides although it is low at CpG islands. 5hmC amounts to about 1-10% of 5mC (estimated at 10% here as in embryonic stem cells), while the levels of 5fC and 5caC are each about an order of magnitude lower than the previous oxidative modification. It is not known whether the low levels of 5fC and 5caC are due to features of TET enzymes that cause them to arrest at 5hmC, or to their continuing removal by TDG or other mechanisms.

Figure 2

Gene regulation by TET proteins. (A) Enzymatic activity of TET. TET proteins, with the co-factors Fe2+ and α-ketoglutarate (αKG), use oxygen to oxidize 5mC into 5hmC, generating CO2 and succinate as by-products. The enzymatic activity of TET can be modulated by additional factors. For instance, vitamin C (ascorbate) can enhance TET activity, potentially via reduction of the iron ion. On the other hand, the “oncometabolite” 2-hydroxyglutarate (2-HG), generated in acute myeloid leukemia and glioblastoma by recurrent dominant-active mutants of isocitrate dehydrogenase 1 or 2 (IDH1/2), inhibits TET activity. Furthermore, lack of oxygen in hypoxia also inhibits TET function. (B) Model of TET-mediated enhancer regulation. Prior to the commissioning of an enhancer, pioneer transcription factor (indicated as TF1) binds to nucleosomal DNA and recruits TET which oxidizes the surrounding 5mC into 5hmC (and/or other oxi-mCs), facilitating DNA demethylation. TET proteins and, TF1 promote enhancer accessibility by recruiting nucleosome remodeling complexes, thus allowing binding of secondary transcription factors (indicated here as TF2) that are otherwise inhibited by DNA methylation or the presence of nucleosomes.

Figure 3

Regulation of lymphoid development and function by TET proteins in the mouse. (A–G) List of known TET functions in lymphoid cells. The interacting transcription factors and the phenotypes found in TET-deficient mice are shown in the right columns. (A) The use of Mb1-cre Tet2/3-deficient mice showed that Tet2 and Tet3 regulate the pro-B to pre-B cell transition, in part by enhancing the rearrangement of immunoglobulin light chains (22, 37). (B) Acute deletion of Tet2/3 using CreERT2 in B cells resulted in decreased Aicda expression and thus class switch recombination (28). (C) Deletion of Tet2 using Vav-Cre and Cd19-Cre resulted in hyperplasia of germinal center B cells. Vav-Cre-driven Tet2 deletion resulted in decreased plasma cell differentiation (38). (D)Cd4-cre Tet2/3-deficient mice exhibited skewed differentiation toward iNKT17 cells, partly due to decreased expression of Tbx21 and Zbtb7b expression, and a massive T-cell-receptor-dependent expansion of affected T cells (33). (E) Tet proteins facilitate the in vitro differentiation of naïve CD4 T cells to iTreg cells by demethylating Foxp3 enhancer CNS2, a process enhanced by the presence of vitamin C. All three TET proteins have a role in stabilizing the expression of Foxp3 in Treg cells in vivo (39, 40). (F) CD4 T cells from Cd2-cre Tet2-deficient mice showed impaired Th1, Th2, and Th17 differentiation and cytokine production (41). (G) Increased differentiation of CD8 memory cells from Cd4-cre Tet2-deficient mice in response to lymphocytic choriomeningitis virus infection (42).

Figure 4

The role of TET2 in myeloid differentiation and function. (A) TET2 regulates myeloid cell differentiation. TET2, together with the thymine DNA glycosylase (TDG), facilitates active DNA demethylation and promotes lineage-specific gene expression during the differentiation of osteoclasts, macrophages, and dendritic cells from human monocytes. In mice, TET2 is required for the differentiation of mast cells in vitro and in vivo (43, 44). (B) TET2 regulates the function of myeloid cells. In mouse and human macrophages, TET2 repressed expression of the pro-inflammatory cytokines IL-1β and IL-6 (–48). TET2 was shown to associate with Iκbζ, bind to the Il6 promoter, recruit HDAC2, and repress Il6 expression (46). Tet2-deficient macrophages also expressed a high level of Socs3 mRNA, which in normal mice was suggested to be demethylated by TET2 and subsequently degraded by ADAR1 (44). In plasmacytoid dendritic cells, CXXC5 recruited TET2 to an intragenic CpG island in Irf7, facilitating the demethylation and maintaining basal expression. As a result, loss of Cxxc5, and to a lesser extent Tet2, resulted in decreased levels of IRF7, decreased type I interferon (IFN-I) production, and decreased anti-viral responses (49).