Cytosine methylation is critical for gene imprinting and cell lineage specification, as discussed above. The reverse of this process – the removal of the methyl group – allows cells to newly express previously repressed genes or to recover their totipotent potential. Until recently, this process of cytosine demethylation was thought to be a passive process in which replication without the action of maintenance DNMTs dilutes mC from DNA. However, mounting evidence suggests that replication-independent, “active” (enzymatic) demethylation occurs globally in totipotent cells (85
) and also in a locus-specific fashion within somatic cells (87
). Active cytosine demethylation, therefore, has now been recognized as a crucial molecular process and is yet another example of the role of cytosine in modulating genomic potential.
Cytosine demethylation is relevant even at the earliest stages of mammalian development. Upon penetrating the zona pelucida, the paternal pronucleus is rapidly demethylated (85
). Remarkably, the maternal pronucleus sits in the same cytoplasm and is exclusively demethylated via
passive demethylation; the mechanism for such asymmetric demethylation remains unclear. Beyond the zygote and blastula stages, a subset of cells are induced to travel to the gonadal ridge and become primordial germ cells (PGCs). Although PGC genomes are widely methylated at the time they are designated, they are globally demethylated by the time they arrive at the gonadal ridge several days later (92
). Given that maintenance DNMTs are expressed in PGCs, such global demethylation is assumed to require active demethylation.
Several examples of locus-specific active demethylation suggest that this process is likewise important in the normal functioning of somatic cells. Fast methylation and demethylation cycling at the estrogen receptor promoter provide a notable example of locus-specific active demethylation (88
). Other studies in CD8+ T-cells illustrated that expression of IL-2 can be induced via
replication-independent demethylation, suggesting a role for active demethylation in sustained immune responses (90
). Finally, even neural plasticity is impacted by active demethylation as evidenced by changes at the promoter for brain-derived neurotrophic factor (91
Although active demethylation is increasingly accepted as an important physiological process, its molecular basis remains controversial. Several DNA glycosylases have been described in Arabidopsis
that can excise mC specifically; however, mammals appear to lack this activity (93
). In the past several years, a wealth of new evidence has implicated several of the key cytosine modifying enzymes we have reviewed, particularly the AID/APOBEC deaminases, TET oxidases, and DNA glycosylases (94
). Two major types of models have emerged: a deamination-initiated pathway (97
) and several variants of an oxidation-initiated pathway (17
Integrated Model for Cytosine Demethylation
In the deamination-initiated pathway, mC is first deaminated by an AID/APOBEC family member to yield thymine. The BER pathway subsequently recognizes the T:G mismatch and reverts the lesion to an unmodified cytosine. In support of the role AID/APOBEC enzymes may play in demethylation, AID-deficient PGCs were found to be more methylated than wild-type PGCs in a mouse model (99
). In zebrafish embryos, coexpression of multiple AID/APOBEC members along with MBD4 caused global demethylation of the genome (100
). AID was also shown to contribute to demethylation at key pluripotency loci such as the Nanog and Oct4 promoters in a heterokaryon system used to generate stem cells (101
). Recent evidence that a TDG knockout is embryonic lethal supports the deamination-initiated pathway (38
), although not to the exclusion of the oxidation-initiated pathway, as we note below.
Several factors suggest that the deamination-initiated pathway is insufficient to fully explain demethylation, although this mechanism may indeed be an important accessory pathway towards that end. Deletion of AID is not embryonic lethal, as would be expected if this were the sole pathway for active demethylation (99
). It is also hard to reconcile a prominent, genome-wide activity for AID with its known properties at the molecular level. While AID has indeed been shown to act outside of the Ig locus, this occurs several orders of magnitude less frequently than within the Ig locus (51
). Furthermore, AID/APOBEC enzymes preferentially act on single-stranded DNA in particular sequence contexts (22
), but most methylated, silenced loci are likely to be double-stranded in CpG contexts. In addition, although deaminases have been suggested to deaminate mC (24
), such activity on mC is diminished relative to activity on cytosine (22
). Therefore, the deamination-initiated pathway, although likely relevant in some instances, may not represent the major mechanism for demethylation.
The discovery of genomic hmC raised the possibility of oxidation-first pathways to demethylation (18
). Despite the ongoing controversy, several observations bolster support for an oxidation-initiated mechanism. The striking prevalence of hmC in promoters suggests that TET oxidation of mC is likely to be an important step in demethylation (74
). TET knockdown in ES cells may decrease expression at loci involved in pluripotency, including Nanog (73
), and promoters undergoing active demethylation have also demonstrated a physiological association with TET (45
). Finally, TET has also been shown to have a preference for binding at CpG nucleotides, where methylation is most relevant in humans (73
The route from hmC to cytosine is still under debate, but several potential pathways are worthy of consideration. These pathways can be characterized as deamination-coupled, BER-coupled or direct-reversion mechanisms. As yet, an enzyme capable of direct removal of the hydroxymethyl group from the 5-position of the base (dehydroxymethylation) has not been discovered; however, this is a mechanistically feasible reaction . Alternatively, hmC could be deaminated by AID/APOBEC enzymes to yield hmU, subsequently removed by an enzyme such as SMUG or TDG (41
). In this system, suggested to be active in neurons, overexpression of AID decreased endogenous hmC levels and both TET and AID contributed to demethylation at several neuron-specific promoters, although overall levels of demethylation were low (45
). However, this proposed model relies on assumptions about the ability of AID/APOBEC enzymes to efficiently deaminate hmC. This activity has not yet been established, nor has sequencing revealed the presence of hmU as a detectable demethylation intermediate, although efficient removal of hmU from the genome may explain the latter point.
A more recent model for efficient demethylation integrates several observations into a more appealing mechanism involving iterative oxidation directly coupled to BER. In several recent reports, the higher oxidation products of hmC, 5-formylcytosine (fC) and 5-carboxylcytosine (caC), were detected in the genome of ES cells (20
). Furthermore, it was shown that fC and caC directly result from iterative oxidation of mC by TETs (20
). Based on the precedent of a related enzyme in pyrimidine salvage, Zhang and colleagues have proposed that an undiscovered decarboxylase could catalyze the regeneration of cytosine from caC (20
). While the search for such an activity could be justified, support for a much more appealing model comes from He et. al.
who revisit the dependence of demethylation on BER (103
). These authors looked for DNA glycosylase activity against the higher oxidation products of mC. They found that the BER enzyme TDG recognizes and excises the highly oxidized caC nucleobase (21
). Notably, no such activity was detected with MDB4. In line with their proposal, knockdown of TDG leads to an accumulation of caC in the genome of ES cells, while conversely TDG overexpression decreases caC content. An independent report from Maiti and Drohat has also subsequently confirmed that TDG excises fC and caC, while leaving hmC untouched (104
). This proposed mechanism is consistent with the observation that TDG deficiency is embryonic lethal and leads to perturbed methylation patterns in embryogenesis (38
). While it has been assumed previously that a role for TDG in demethylation implicates a deamination-mediated pathway, this need not be the case; TDG can directly excise cytosine bases with weakened N
-glycosidic bonds, as would likely be the case for fC and caC.
Although the field itself is rapidly evolving, we propose that these apparently disparate studies invoking deamination, oxidation and BER can be integrated into a more coherent model () (105
). A gathering body of evidence supports important roles for the various TET isoforms in physiological niches where DNA demethylation is thought to be relevant. Though much remains to be resolved, disrupting expression leads to perturbed demethylation of paternal paternal pronuclei and embryonic demise in the case of TET3 (106
), dysregulation of hematopoiesis in the case of TET2 (107
) and diminished embryonic growth of viable offspring in the case TET1 (109
). These genetic findings couple with the biochemical studies to make a case for the TET enzymes as major regulators of DNA demethylation. We therefore suggest that an iterative oxidation-initiated/BER-coupled pathway could be a major route to demethylation, but that deaminase enzymes could serve an important accessory role to accelerate demethylation in certain physiological settings. This could occur because deamination would generate a uracil-related base, rather than a cytosine-related base, and the relevant BER enzymes are more efficient in excision of the products of deamination. This paradigm could explain the apparent contribution of deamination in heterokaryon systems (101
), neurons (45
), or settings where AID/APOBEC enzymes are overexpressed (45
). Together, a model invoking both major and accessory pathways accounts for the observations that TET, AID/APOBEC enzymes and BER enzymes all appear to contribute to demethylation, but that a predominant pathway is required in the setting of embryogenesis, where demethylation is critical to proper development and differentiation.
While the current evidence suggests that an iterative oxidation/TDG-coupled pathway plays a major role in cytosine demethylation, the model is far from resolved and several major gaps remain in our understanding (105
). For instance, hmC accumulates to higher levels than fC and caC; what controls the extent of oxidative modification by TET? Next, although Xu and colleagues (21
) propose a model where caC is the intermediate just prior to BER, Maiti and Drohat observe that fC is a better substrate for TDG than caC (104
). What is the final oxidation intermediate prior to BER? Further, if BER is involved in lesion recognition, the process of reversion to cytosine would generate a basic sites and DNA nicks. Given the high load of lesions that would result from DNA cytosine methylation in CpG islands, how is genomic instability averted? There are also fundamental questions that remain regarding the proposed deamination-mediated, accessory pathway. For example, the biochemical plausibility of cytosine analogs as substrates for deamination by AID/APOBEC enzymes remains largely unassessed. Addressing these open questions will be essential to the ongoing debate over the mechanism of demethylation.