Transcriptional activation of transposable elements in mouse zygotes is independent of Tet3-mediated 5-methylcytosine oxidation

Abstract

The methylation state of the paternal genome is rapidly reprogrammed shortly after fertilization. Recent studies have revealed that loss of 5-methylcytosine (5mC) in zygotes correlates with appearance of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). This process is mediated by Tet3 and the 5mC oxidation products generated in zygotes are gradually lost during preimplantation development through a replicationdependent dilution process. Despite these findings, the biological significance of Tet3-mediated oxidation of 5mC to 5hmC/5fC/5caC in zygotes is unknown. DNA methylation plays an important role in silencing gene expression including the repression of transposable elements (TEs). Given that the activation of TEs during preimplantation development correlates with loss of DNA methylation, it is believed that paternal DNA demethylation may have an important role in TE activation. Here we examined this hypothesis and found that Tet3-mediated 5mC oxidation does not have a significant contribution to TE activation. We show that the expression of LINE-1 (long interspersed nucleotide element 1) and ERVL (endogenous retroviruses class III) are activated from both paternal and maternal genomes in zygotes. Inhibition of 5mC oxidation by siRNA-mediated depletion of Tet3 affected neither TE activation, nor global transcription in zygotes. Thus, our study provides the first evidence demonstrating that activation of both TEs and global transcription in zygotes are independent of Tet3-mediated 5mC oxidation.

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

“…Immunostaining with an antibody against the C-terminus of Tet3 protein (Gu et al, 2011) confirmed lack of Tet3 protein in the CKO zygotes (Figure 1A). Similar to previous reports (Gu et al, 2011; Inoue et al, 2012), Tet3 is predominantly localized in the paternal pronucleus. Interestingly, weaker but detectable Tet3 staining signals were also observed in the maternal pronucleus, and this signal was absent in CKO zygotes (Figure 1A).…”

“…This loss of 5mC is explained by rapid conversion of 5mC to 5hmC and its further oxidation products catalyzed by the maternally stored Tet3 proteins (Gu et al, 2011; Inoue et al, 2012; Wossidlo et al, 2011). Immunostaining studies indicated that 5mC derived oxidation products are gradually lost during preimplantation development through a DNA replication-dependent manner (Inoue et al, 2011; Inoue and Zhang, 2011).…”

LINEs in mice: features, families, and potential roles in early development

“…3). Furthermore, removal of Tet3, which takes part in the active demethylation of DNA in zygotes via oxidation of 5mC to 5hmC, does not lead to changes in L1 transcription (Inoue et al 2012). Thus, the temporal pattern of L1 transcriptional activation and subsequent silencing cannot be explained through changes in DNA methylation or in H3K9me3 after fertilization and prior to implantation, implying that additional pathways are involved in the process.…”

Haploinsufficiency, but Not Defective Paternal 5mC Oxidation, Accounts for the Developmental Defects of Maternal Tet3 Knockouts

“…This suggests that Tet3 protein retained in the recipient cytoplasm is not sufficient to trigger massive 5mC oxidation in the reconstructed embryos. This is plausible because Tet3 mainly localizes to the paternal pronuclei that have been removed from the recipients (Gu et al, 2011;Inoue et al, 2012). Additionally, since Tet3 is no longer localized to nuclei after the first mitosis (Gu et al, 2011), the time window during which the remaining Tet3 can function is very limited due to the quick entry of the embryo into the first mitosis (within 2 hr after fusion).…”