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 replication-dependent 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.

Figure 1

Expression dynamics of TEs during preimplantation development. Shown are RT-qPCR results of the relative expression levels of the various TEs at different preimplantation developmental stages. The results are normalized to an external control and the values of MII-stage oocytes were set as 1. The relative value of transcripts in 1-cell-stage embryos compared with that of MII oocytes was indicated in each graph. Asterisk represents statistical significance (P < 0.01). The experiments were repeated for 3-4 times. A total of 30-55 embryos were used for each experiment in a given developmental stage. Error bars represent standard deviations.

Figure 2

Expression of TEs in parthenogenetic and androgenetic 1-cell embryos. (A) Experimental scheme for generating parthenogenetic and androgenetic 1-cell embryos. Androgenetic embryos were produced by in vitro fertilization of enucleated MII-stage oocytes. Parthenogenetic embryos were generated by artificial activation with strontium. Embryos were collected 11 h after insemination or activation. (B) Representative images of zygotes, parthenogenetic (Partheno) and androgenetic (Andro) embryos co-stained with Tet3 (red) and H3K9me2 (green) antibodies. H3K9me2 marks the maternal pronucleus. Scale bar = 20 μm. (C) Representative images of zygotes co-stained with 5hmC (red) and 5mC (green) antibodies. More than six embryos were examined in each group. (D) RT-qPCR analysis of the expression of TEs in parthenogenetic (Partheno) and androgenetic (Andro) 1-cell embryos. The relative expression level was normalized to an external control. The values of MII-stage oocytes were set as 1. The experiments were repeated for 3 times. Error bars indicate standard deviations. * represents statistical significance (P < 0.05).

Figure 3

Activation of TEs in androgenetic embryos is independent of Tet3. (A) Scheme for generating Tet3-depleted androgenetic embryos. GV-stage oocytes were injected with siRNA specific for Tet3 (siTet3) or control siRNA (siCont). After meiotic maturation, the maternal genome was removed. The enucleated oocytes were then fertilized in vitro and collected at 11 hpi. (B) Representative images of androgenetic 1-cell embryos co-stained with Tet3 antibody (red) or DAPI (blue). Quantification of Tet3 signal intensity is shown in the right panel. The value of siCont embryos was set as 1. Five embryos in each group were quantified. Error bar indicates standard deviation. ** represents statistical significance (P <0.01). Scale bar = 20 μm. (C) Representative images of androgenetic 1-cell embryos co-stained with 5mC (green) and 5hmC (red) antibodies. Quantification of 5hmC signal intensity is shown in the right panel. The value of siCont embryos was set as 1. Six to nine embryos were quantified. Error bar indicates standard deviation. ** represents statistical significance (P <0.01). (D, E) RT-qPCR analysis of the expression of TEs in androgenetic 1-cell (D) or 2-cell (E) embryos. The relative expression level is normalized to an external control. The expression level in MII-stage oocytes was set as 1. The experiments were repeated for 3 times. Error bars represent standard deviations.

Figure 4

Global transcriptional activity in zygotes is not significantly altered by Tet3 depletion. (A) Validation of BrUTP incorporation assay for measuring ongoing transcriptional activity. Representative images of zygotes co-stained with BrdU (green) and H3K9me2 (red) antibodies. DNA was stained with DAPI (blue). H3K9me2 marks the maternal pronucleus. Note that the BrUTP signal is lost in zygotes treated with α-amanitin (α-Am). Scale bar = 20 μm. (B) Representative images of zygotes co-stained with Tet3 (red) and H3K4me3 (green) antibodies. H3K4me3 marks the maternal pronucleus. Quantification of Tet3 signal intensity is shown in the right panel. The signal intensity of maternal pronucleus in siCont zygotes was set as 1. Six to nine zygotes were quantified. Error bar indicates standard deviation. ** represents statistical significance (P <0.01). (C) Representative images of zygotes co-stained with 5hmC (red) and 5mC (green) antibodies. Quantification of 5hmC signal intensity is shown in the right panel. The signal intensity of maternal pronucleus in siCont zygotes was set as 1. Fifteen to 19 zygotes were quantified. Error bar indicates standard deviation. ** represents statistical significance (P <0.01). (D) Representative images of zygotes co-stained with BrdU (green) and H3K9me2 (red) antibodies after incorporation of BrUTP. (E) Quantification of fluorescent signal intensity for BrUTP incorporation assay. The signal intensity of BrdU in maternal or paternal pronucleus was individually quantified. The signal intensity of the maternal pronucleus in non-injected zygotes was set as 1. The experiments were repeated for four times. Thirty-four to 45 zygotes were quantified in each experimental group. Error bars represent standard errors.