Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine

Abstract

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.

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number

“…Furthermore, a study of the DNA methylation pattern in human 3PN zygotes established that active demethylation of the paternal PN occurs post-fertilization in both c-IVF and ICSI [32]. In addition, recent studies have shown that the demethylation of paternal PN is induced by conversion from 5mC to 5hmC by ten-eleven translocation methylcytosine dioxygenases (TETs) [9][10][11]. Among TET proteins, TET3 is intensely expressed in oocytes and zygotes.…”

“…Recently, it was found that 5-methylcytosine (5mC) in the paternal genome is specifically converted to 5-hydroxymethylcytosine (5hmC) during the pronuclear stage [9][10][11]. Although these three studies were conducted in animals, we applied a similar approach in this study, using epigenetic divergence to determine the origin of mPN and fPN in abnormal human zygotes with 1PN and 3PN.…”

Mouse Tet1 Protein can Oxidize 5mC to 5hmC and 5caC on Single-stranded DNA

“…[5,6] Further biological studies and genome-wide profiling of 5hmC have revealed significant roles played by this modified base in stem cell differentiation, leukemia, epigenetic reprogramming, neurodegenerative diseases, hypoxia, and angiogenesis. [7][8][9][10][11][12][13][14][15] These findings have fostered wide speculation that 5hmC modification is an important epigenetic modification after 5mC. [16] In collaboration with two groups, we contributed to the discovery that 5hmC could be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (Figure 1).…”

“…For example, Tet1 maintains the stem cell properties of embryonic stem cells, [6,8] Tet2 significantly affects leukemia, [9][10][11][12][13] and Tet3 plays a key role in epigenetic reprogramming in zygote development. [14,15] Here we present that the active domain of the mouse Tet1 can not only oxidize 5mC in duplex DNA, but also 5mC in single-stranded DNA (ssDNA), indicating that Tet proteins can potentially be involved in DNA replication or/and transcription, as well as directly oxidizing the corresponding modifications on RNA.…”

Challenges and perspectives to enhance cattle production via in vitro techniques: focus on epigenetics and cell-secreted vesicles

“…However, the demethylation pattern is different in each parental chromatin, while maternal genome is slowly and passive, the paternal is fast and active (MAYER et al, 2000;OSWALD et al, 2000). For a long time the active process was unknown, however in 2011 studies showed that the responsibility for such process is the formation of 5-hydroxymethylcytosine (5-hmc), a cytosine variation, in which a -OH is added to the methylated cytosine by a protein from the Tet family; Tet 1, 2 or 3 (IQBAL et al, 2011;WOSSIDLO et al, 2011). Tet 1 and 2 have been related to embryonic stem cells pluripotency and depletion of Tet1 implicates in lineage differentiation difficulties (COSTA et al, 2013).…”