Iqbal K, Jin SG, Pfeifer GP, Szabo PE. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci USA 108: 3642-3647

Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2011; 108(9):3642-7. DOI: 10.1073/pnas.1014033108
Source: PubMed


Genome-wide erasure of DNA cytosine-5 methylation has been reported to occur along the paternal pronucleus in fertilized oocytes in an apparently replication-independent manner, but the mechanism of this reprogramming process has remained enigmatic. Recently, considerable amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5-methylcytosine (5mC) by TET proteins, have been detected in certain mammalian tissues. 5hmC has been proposed as a potential intermediate in active DNA demethylation. Here, we show that in advanced pronuclear-stage zygotes the paternal pronucleus contains substantial amounts of 5hmC but lacks 5mC. The converse is true for the maternal pronucleus, which retains 5mC but shows little or no 5hmC signal. Importantly, 5hmC persists into mitotic one-cell, two-cell, and later cleavage-stage embryos, suggesting that 5mC oxidation is not followed immediately by genome-wide removal of 5hmC through excision repair pathways or other mechanisms. This conclusion is supported by bisulfite sequencing data, which shows only limited conversion of modified cytosines to cytosines at several gene loci. It is likely that 5mC oxidation is carried out by the Tet3 oxidase. Tet3, but not Tet1 or Tet2, was expressed at high levels in oocytes and zygotes, with rapidly declining levels at the two-cell stage. Our results show that 5mC oxidation is part of the early life cycle of mammals.

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    • " ' Doherty et al . , 2012 ) . Following fertilization there is a global cascade of DNA demethylation during the early stages of embryogenesis , whereby the paternal genome is rapidly demethylated in the zygote and the maternal genome is passively demethy - lated in a replication - dependent manner ( Dean et al . , 2001 ; Yang et al . , 2007 ; Iqbal et al . , 2011 ) . More recently , it has been hypothesized that both the maternal and paternal genomes undergo global active demethylation and replication - mediated passive demethylation ( Gkountela and Clark , 2014 ; Guo et al . , 2014 ) . Irrespective of the mechanisms con - trolling these genome - wide reprogramming events in the pre - implantati"
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    • "Thus, consecutive passive dilutions during preimplantation development might compensate for the loss of maternal Tet3, leading to hypomethylation of the paternal genome. Alternatively, Tet1 and Tet2 might also contribute to DNA demethylation, as both begin to be expressed after the two-cell stage (Iqbal et al., 2011). It is also possible that a Tet-independent demethylation pathway is involved in the observed compensation (Wang et al., 2014). "
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    ABSTRACT: DNA methylation, the addition of a methyl group to the fifth position of cytosine (5-methylcytosine, 5mC), plays important roles in gene silencing and genome stability, and is essential for mammalian development (Smith and Meissner, 2013). DNA methylation is established by the de novo DNA methyltransferases DNMT3A and DNMT3B, and is maintained by DNMT1. Although the DNA methylation pattern is faithfully maintained throughout generations in somatic cells, it is globally erased during preimplantation development (Saitou et al., 2012 and Sasaki and Matsui, 2008). After fertilization, both paternal and maternal genomes become hypomethylated and reach their lowest levels at the blastocyst stage even though the hypomethylated status is established differentially between the parental genomes. Maternal 5mC is mostly diluted in a DNA replication-dependent manner (Rougier et al., 1998), likely because of the limited availability of DNMT1 in early embryos (Hirasawa et al., 2008). In contrast, the paternal genome is subjected to global active demethylation (Mayer et al., 2000 and Oswald et al., 2000). We and others have found that Tet3 oxidizes paternal 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine in mouse zygotes (Gu et al., 2011, Inoue et al., 2011 and Wossidlo et al., 2011), and that the 5mC oxidation products are gradually lost during preimplantation development through DNA replication-dependent passive dilution (Inoue et al., 2011 and Inoue and Zhang, 2011). Recent genome-scale analyses have also revealed that a large proportion of paternal 5mCs undergo passive dilution without oxidation (Guo et al., 2014a and Shen et al., 2014).
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    • "Active DNA demethylation has been described to occur in the paternal genome soon after fertilization occurs [27]. Work by several groups revealed that 5hmC accumulates in the paternal pronucleus at the beginning of S-phase (PN3), reaching a maximum in G2-phase (PN5), while 5mC concomitantly decreases [19] [31] [32] [60] [79]. Tet3 is highly transcribed in oocytes and zygotes and localizes to the paternal pronucleus. "
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