Enzymatic DNA oxidation: mechanisms and biological significance

Abstract

DNA methylation at cytosines (5mC) is a major epigenetic modification involved in the regulation of multiple biological processes in mammals. How methylation is reversed was until recently poorly understood. The family of dioxygenases commonly known as Ten-eleven translocation (Tet) proteins are responsible for the oxidation of 5mC into three new forms, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Current models link Tet-mediated 5mC oxidation with active DNA demethylation. The higher oxidation products (5fC and 5caC) are recognized and excised by the DNA glycosylase TDG via the base excision repair pathway. Like DNA methyltransferases, Tet enzymes are important for embryonic development. We will examine the mechanism and biological significance of Tet-mediated 5mC oxidation in the context of pronuclear DNA demethylation in mouse early embryos. In contrast to its role in active demethylation in the germ cells and early embryo, a number of lines of evidence suggest that the intragenic 5hmC present in brain may act as a stable mark instead. This short review explores mechanistic aspects of TET oxidation activity, the impact Tet enzymes have on epigenome organization and their contribution to the regulation of early embryonic and neuronal development.

Fig. 1.. Cycle of DNA methylation and demethylation. Active demethylation is achieved by iterative oxidation of the methyl group of 5mC by Tet dioxygenases and restoration of unmodified cytosines (C). The latter is thought to occur by either replication-dependent dilution (not shown) or TDG glycosylase-initiated base excision repair. Of note, TDG can recognize and excise both 5fC and 5caC. An alternative direct mechanism is feasible (grey arrow), but an enzyme responsible for 5caC decarboxylation remains to be identified.

Fig. 2.. Two scenarios of DNA demethylation via Tet-mediated 5mC oxidation. The first concerns erasure of regional methylation in association with timely reactivation of methylated genes as demonstrated for pluripotency loci in the paternal genome in mouse post-fertilization early embryos (see main text). The second deals with the removal of excessive methylation by pruning methyl groups stochastically placed due to an inaccurate methylation machinery. This might be a mechanism to keep CpG islands free of 5mC in the mammalian genome.

Fig. 3.. Possible scenario for TET-mediated events during neural development. In progenitor cells promoter CpG islands may be maintained free of 5mC by the action of a TET protein, likely targeted to this region by its CXXC domain, while the gene body and distal enhancer elements are relatively heavily methylated. During differentiation a transient wave of TET1 activity frees enhancers of 5mC by oxidation to 5hmC then further to 5fC and 5caC, allowing interaction with the proximal promoter and driving transcription. TET2 association with the transcription unit drives the production of 5hmC and also chromatin alterations through its recruitment of O-linked N-acetylglucosamine transferase (OGT). Gene bodies are characterised by a high level of 5hmC, particularly at exons and near splice junctions, a moderate level of 5mC and the absence of substantial 5fC or 5caC.