Although indirect evidence has been accumulating for decades, recent advances discussed here now support the hypothesis that DNA demethylation and methylation may be bidirectional and dynamically regulated throughout early and late development and in certain adult tissues, especially the brain (Guo et al., 2011
; Miller and Sweatt, 2007
). Much remains to be learned, including which loci are targeted for demethylation and how the process is spatially and temporally regulated in diverse cell types and stages of development. The long held notion that epigenetic marks, such as DNA methylation, are generally maintained in stable differentiated states is likely true. Nonetheless, nuclear reprogramming shows that marked perturbations are possible (Jullien et al., 2011
; Yamanaka and Blau, 2010
). As is the case for the regulation of gene expression by transcription factors (Blau and Baltimore, 1991
; Jacob and Monod, 1961
; Ptashne, 2009
), the regulation of DNA methylation may also be continuous and dictated by a balance of enzymes and targeting factors.
As shown in , our current understanding of the DNA methylation and demethylation circuitry entails members of the following enzyme families with roles in either passive or active DNA demethylation: (1) the DNMT family of three methyltransferases responsible for the de novo generation and maintenance of 5mC. DNA demethylation can occur passively by a dilution or inactivation of DNMTs; (2) the TET family of three 5mC hydroxylases, which generate 5hmC (and further oxidized intermediates) from 5mC; (3) the AID/APOBEC family of deaminases, which initiate an active process of demethylation either by deaminating 5mC or 5hmC generated by the TET family; (4) the family of BER glycosylases that initiate DNA repair culminating in the replacement of methylated cytosines with unmethylated cytosines. We have designated these enzymes as the DNA methylation ‘editors’ that are responsible for the regulation of the DNA methylome associated with a particular cell fate. It remains to be determined if active DNA demethylation in different scenarios always requires a representative member from each of these families. In other words, does the entire TET-AID/APOBEC-BER pathway operate broadly or is only a subset thereof required to achieve active DNA demethylation in different cell contexts.
A dynamic interplay of regulators is an interesting theme that has emerged in parallel with approaches for reprogramming nuclei that have demonstrated the remarkable plasticity of cellular fates. When the balance of transcription factors that recognize DNA sequence is perturbed by either nuclear transfer, cell fusion, or defined factors (i.e., in generating iPSCs), it leads to a dramatic shift in cell fate. A provocative, yet perhaps over-simplistic view of how cell fate is controlled and maintained is provided by an analogy with a sailboat. Transcription factors comprise the rudder that determines the direction of the differentiated state (i.e., whether it is muscle or liver). Threshold concentrations of these factors, achieved by feedback loops, continuously regulate the differentiated states. The ‘editors’ of DNA methylation described in this review (and possibly the methylation/acetylation of the histones) are also probably regulated, both actively and continuously, at specific loci, but these factors serve as the keel, preventing the cell from responding to minor changes in wind or current. However, a blast of ectopic transcription factors can overwhelm the rudder and reset it as well as the DNA methylation regulators. This occurs in cellular reprogramming, either following nuclear transfer into oocytes, cell fusion in heterokaryons, or in induced pluripotency (iPSCs). In the first two cases, the somatic nucleus encounters an overwhelming abundance of pre-existing proteins, whereas in iPSCs, this protein abundance is progressive, as it derives from the overexpression of four genes (reviewed in Yamanaka and Blau, 2010
The recent discoveries that TET and AID/APOBEC enzymes are active regulators of DNA demethylation highlight the hypothesis that even apparently stable states are continuously regulated. Thus, the stable differentiated state is governed by regulatory pathways that are surprisingly perturbable. This raises the intriguing question of how cellular plasticity is kept in check to maintain cellular fate. A future goal and major challenge is to understand how this inherent cell plasticity can be first enlisted to reprogram cells and then regulated to derive stable differentiated cell types. Indeed, understanding the mechanisms that govern this dichotomy is critical for successfully applying cellular reprogramming to regenerative medicine.