Tet3 and DNA Replication Mediate Demethylation of Both the Maternal and Paternal Genomes in Mouse Zygotes

Abstract

SUMMARY With the exception of imprinted genes and certain repeats, DNA methylation is globally erased during pre-implantation development. Recent studies have suggested that Tet3-mediated oxidation of 5-methylcytosine (5mC) and DNA replication-dependent dilution both contribute to global paternal DNA demethylation, but demethylation of the maternal genome occurs via replication. Here we present genome-scale DNA methylation maps for both the paternal and maternal genomes of Tet3-depleted and/or DNA replication-inhibited zygotes. In both genomes, we found that inhibition of DNA replication blocks DNA demethylation independently from Tet3 function, and that Tet3 facilitates DNA demethylation by coupling with DNA replication. For both genomes, our data indicate that replication-dependent dilution is the major contributor to demethylation, but Tet3 plays an important role, particularly at certain loci. Our study thus defines the respective functions of Tet3 and DNA replication in paternal DNA demethylation and reveals an unexpected contribution of Tet3 to demethylation of the maternal genome.

Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

“…The genome-wide DNA demethylation observed in early embryos appears to be a default pathway independent of TET function or oxi-mC modification: both the 5hmC-modified paternal genome and the unmodified maternal genome are passively demethylated at similar rates during the robust cell divisions that occur in the early embryo (69). Even in zygotes, demethylation of both maternal and paternal pronuclei requires DNA replication, a process only partially dependent on Tet3 (14,15). For each observed outcome, the relative contributions of oxi-mC production and DNA demethylation may be distinguished by comparing cells deficient in DNA methyltransferases, which would be depleted for both 5mC and oxi-mC, with cells deficient in TET proteins, which lack only oxi-mC.…”

“…RRBS measures the sum of 5mC and 5hmC (vs. C, 5fC, and 5caC) at a fraction of cytosines in the genome, and the data show that loss of 5mC+5hmC in both maternal and paternal pronuclei occurs primarily through a passive, replication-dependent process (14,15). Despite the high expression of Tet3 in oocytes and zygotes (11), Tet3-deficient zygotes display only a marginal increase in 5mC+5hmC in either paternal or maternal pronuclei (14), suggesting the redundant involvement of other TET proteins.…”

“…In PGCs, Tet1 and Tet2 contribute to DNA demethylation through a replication-dependent mechanism (9). In fertilized zygotes, Tet3 was originally thought to oxidize 5mC preferentially in the paternally inherited genome (11)(12)(13); more recently, however, reduced-representation bisulfite sequencing (RRBS) has been used to suggest that demethylation of the maternal genome is also catalyzed by Tet3 (14). RRBS measures the sum of 5mC and 5hmC (vs. C, 5fC, and 5caC) at a fraction of cytosines in the genome, and the data show that loss of 5mC+5hmC in both maternal and paternal pronuclei occurs primarily through a passive, replication-dependent process (14,15).…”

“…In fertilized zygotes, Tet3 was originally thought to oxidize 5mC preferentially in the paternally inherited genome (11)(12)(13); more recently, however, reduced-representation bisulfite sequencing (RRBS) has been used to suggest that demethylation of the maternal genome is also catalyzed by Tet3 (14). RRBS measures the sum of 5mC and 5hmC (vs. C, 5fC, and 5caC) at a fraction of cytosines in the genome, and the data show that loss of 5mC+5hmC in both maternal and paternal pronuclei occurs primarily through a passive, replication-dependent process (14,15). Despite the high expression of Tet3 in oocytes and zygotes (11), Tet3-deficient zygotes display only a marginal increase in 5mC+5hmC in either paternal or maternal pronuclei (14), suggesting the redundant involvement of other TET proteins.…”

“…Specifically, adult Tet1 and Tet2 KO mice are viable and fertile, displaying relatively mild behavioral and hematopoietic phenotypes, respectively (17)(18)(19), whereas Tet3 KO mice die perinatally for unknown reasons (11). Tet3-deficient early embryos show no difference in gene expression compared with WT embryos (14), suggesting Significance Development of preimplantation embryos entails global DNA demethylation on the zygotic genome. The original thought was that TET-deficient embryos would be unlikely to survive early embryogenesis because they would be unable to mediate genome-wide demethylation in the zygote and preimplantation embryo.…”