Twenty-five years ago it was shown in vitro, that a methylated rat a-actin gene promoter transfected in L8 myoblasts is demethylated in the absence of DNA replication (Paroush et al., 1990). This process occurred in two steps: within a few hours on one DNA strand and after a >48-h delay on the complementary strand. Genetic analysis revealed the existence of ds-acting elements required for demethylation. The recognition of such sites early in the cell differentiation process probably leads to the demethylation required to activate gene transcription.

A transient DNA demethylation in Friend erythroleukemia cells induced to differentiation was accompanied by incorporation of deoxy[5-3H]cytidine, but not of deoxy[6-3H]adenosine, into preexistent DNA chains (Razin et al., 1986). Thus, demethylation of DNA seemed to be achieved by an enzymatic mechanism whereby 5mC was replaced by cytosine.

Incubation of hemimethylated oligonucleotide DNA substrates with nuclear extracts of chicken embryo promoted active demethylation of 5mCpGs by a nucleotide excision repair mechanism (Jost, 1993). The first step of demethylation was the formation of specific 5' nick at the 5mC residue. Nicks also occurred on symmetrically methylated CpGs, but they resulted in breakage of the oligonucleotides with no repair. Nicks were strictly 5mCpG specific and did not occur on CpG, 5mCpC, 5mCpT, 5mCpA, or 6mApT. Purification of the demethylation activity revealed it to be a combination of 5mC-DNA glycosylase and apyrimidinic endonuclease (Jost, Siegmann, Sun, & Leung, 1995). The purified 5mC-DNA glycosylase also possessed a mismatch-specific thymine- DNA glycosylase (TDG) activity. It had a >6-fold preference for hemimethylated DNA substrates over symmetrically methylated substrates. Activity of the purified 5mC-DNA glycosylase was abolished by treatment of either proteinase K or ribonuclease A, suggesting it to belong to a protein- and RNA-enriched complex (Fremont et al., 1997). Indeed, RNA molecules, necessary for enzymatic activity, were found in purified 5mC- DNA glycosylase. Their cloning and sequencing revealed variable sequences of 200600 nt unrelated to known RNAs or to each other, potentially reflecting interactions with long noncoding RNAs (Jost, Fremont, Siegmann, & Hofsteenge, 1997). The common feature of all clones analyzed was a high CpG density. On average, they have one CpG per each 14 nt and a CpG/GpC ratio ofl.1. It was shown that at least 4 nt sequences complementary to target methylated sites, mCpG and two adjacent nucleotides at each side, are required for efficient targeting in the demethylation reaction. Of the 16 possible NpCpGpN sequences, between 75% and 100% were present in each. The longest clone (618 nt) contained all of them and thus could serve as a universal targeting sequence. The different RNAs tightly linked to 5mC-DNA glycosylase were then suggested to represent transcripts from CpG islands, which should remain unmethylated (Jost et al., 1997).

A methylated DNA binding protein MBD2b, produced by in vitro translation of cloned cDNA, was found to transform 5mC residues in labeled substrate to C residues (Bhattacharya, Ramchandani, Cervoni, & Szyf, 1999). When demethylated DNA was subjected to CpG methyltransferase M.SssI, it was completely remethylated. The demeth- ylase seemed to transform methylated cytosines in DNA to cytosines without disrupting the integrity of DNA chains. As the cleavage of a carbon-carbon bond requires high energy, direct demethylation of 5mC has been widely believed to be thermodynamically very unfavorable. In addition, formaldehyde has been shown to be released in demeth- ylation reaction, indicating that the loss of the methyl group occurs due to oxidative demethylation of 5mC via the 5-hydroxymethylcytosine (5hmC) intermediate (Hamm et al., 2008). Unfortunately, direct demethylation of DNA by MBD2 could not be reproduced by others, who found MBD2 to participate in transcription repression through the recruitment of histone deacetylase (HDAC) to MeCP1 complex in HeLa cells (Hendrich, Guy, Ramsahoye, Wilson, & Bird, 2001; Ng et al., 1999).

Three proteins of the Gadd45 family are widely known to be involved in numerous biological processes, such as DNA damage responses, cell cycle control, senescence, apoptosis, and nucleotide excision repair (Salvador, Brown-Clay, & Fornace, 2013). A screen of Xenopus expression cDNA library for sequences able to reactivate the transcription of a methylation-silenced luciferase reporter gene resulted in isolation of Gadd45a (Barreto et al., 2007). Its reactivating activity seemed to be sequence and cell type independent. In human embryonic kidney 293 cells Gadd45a transfection led to a reduction of 5mC from 2.1% to 0.9% both in dividing and serum-starved nonproliferating cells, demonstrating active demethylation. An endonuclease XPG responsible for the 3' incision during nucleotide excision repair and a DNA helicase were required for Gadd45a-mediated DNA demethylation. Thus, Gadd45a acted by promoting repair of methylated DNA sequences. In zebrafish embryos 5mC removal in vivo proceeds via coupled activities of activation-induced deaminase (AID) that converts 5mC to T and of Mbd4, a G:T mismatch-specific TDG containing a methyl-CG-binding domain, whereas Gadd45 serves as a nonenzymatic supporting factor (Rai et al., 2008). In mice, TDG KO is embryonically lethal (Cortellino et al., 2011), and TDG is involved in protection of CpG islands from hypermethylation and in active demethylation of tissue- specific developmentally and hormonally regulated promoters. In these roles TDG interacts with AID and Gadd45a.

A new twist in the DNA demethylation saga has begun with the discovery of the modified DNA base 5hmC in animal DNA (Kriaucionis & Heintz, 2009; Tahiliani et al., 2009). 5hmC is enriched exclusively in the brain, with higher abundance in the cortex and brainstem (Kriaucionis & Heintz, 2009). Three 2-oxoglutarate- and Fe(II)-dependent oxygenase human enzymes, TET1, TET2, and TET3, and their homologues in other animals, produce 5hmC through hydroxylation of the methyl group of 5mC (Tahiliani et al., 2009). Some 5mCs in mammalian cells are oxidized by TET proteins to 5hmC, which can be either deaminated to 5-hydroxymethyluracil (5hmU) by AID/APOBEC deaminases or further oxidized to 5-formylcytosine (5fC) and then to 5-carboxylcytosine (5caC). The 5hmU and 5caC are removed by the DNA glycosylase TDG, and the gap is refilled by unmethylated C through the BER pathway (Gong & Zhu, 2011; He et al., 2011). Also, Gadd45a has been demonstrated to directly interact with TDG and stimulate the removal of 5fC and 5caC from DNA (Li et al., 2015). KO ofboth Gadd45a and Gadd45b in mouse ESCs led to hypermethylation of genomic loci, most of which are targets for TDG and show 5fC enrichment in TDG-deficient cells. Thus, the DNA demethylation effects of Gadd45a could be mediated by TDG activity. These findings illustrate the increasing complexities of active DNA demethylation pathways in animals and, particularly, within the brain.

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