Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development

Abstract

Parental origin-specific DNA methylation regulates the monoallelic expression of the mammalian imprinted genes. The methylation marks or imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear how the methylation imprints are maintained through extensive demethylation in cleavage-stage preimplantation embryos. Previous reports suggested that DNA methyltransferase(s) other than Dnmt1 is involved in the maintenance of the imprints during cleavage. Here we demonstrate, by using conditional knockout mice, that the other known DNA methyltransferases Dnmt3a and Dnmt3b are dispensable for the maintenance of the methylation marks at most imprinted loci. We further demonstrate that a lack of both maternal and zygotic Dnmt1 results in complete demethylation of all imprinted loci examined in blastocysts. Consistent with these results we find that zygotic Dnmt1 is expressed in the preimplantation embryo. Thus, contrary to the previous reports, Dnmt1 alone is sufficient to maintain the methylation marks of the imprinted genes.

Figure 1.

Expression and subcellular localization of Dnmt3a and Dnmt3b in mouse oocytes and preimplantation embryos. (A) Immunostaining of wild-type FG oocytes, MII oocytes, and preimplantation embryos with an anti-Dnmt3a antibody. Dnmt3a signals (green) were detectable in the nucleus of FG oocytes and embryos from the one-cell through to the eight-cell stage. Dnmt3a was diffusely present in the cytoplasm of MII oocytes. Small intense signals represent the nuclei of pole bodies. (B) Absence of detectable Dnmt3a in oocytes and preimplantation embryos from [Dnmt3a^2lox/2lox, Zp3-Cre] females. This confirms the maternal origin of the protein detected in the wild-type embryos. (C) Immunostaining of wild-type oocytes and preimplantation embryos with an anti-Dnmt3b antibody. Dnmt3b (green) was not detectable in oocytes, one-cell embryos, or two-cell embryos and became detectable in the later stages. (D) Zygotic production of Dnmt3b in preimplantation embryos. Dnmt3b was detected in embryos obtained from [Dnmt3b^2lox/2lox, Zp3-Cre] females crossed with wild-type males. (E) Absence of detectable Dnmt3b signals in embryos obtained from [Dnmt3b^2lox/2lox, Zp3-Cre] females crossed with [Dnmt3b^2lox/1lox, Tnap-Cre] males. The cell nucleus was counterstained with propidium iodide (PI) (red).

Figure 2.

DNA methylation status of the imprinted DMRs in Dnmt3a/Dnmt3b double mutants. (A) The mouse crossing scheme for the production of embryos for bisulfite sequencing. Oocyte-specific conditional double knockout females ([Dnmt3a^2lox/2lox, Dnmt3b^2lox/2lox, Zp3-Cre]) were crossed with double heterozygous males ([Dnmt3a^1lox/+, Dnmt3b^1lox/+]), to obtain embryos with four different genotypes. The males used for this cross had a JF1-strain background. Among the embryos obtained from this cross, the [Dnmt3a^1lox/1lox, Dnmt3b^1lox/1lox] embryos completely lacked maternal and zygotic proteins of Dnmt3a and Dnmt3b and were used for the analysis. Strain-specific SNPs were used to determine the parental origin of the DMRs. (B) Methylation status of the paternally methylated DMRs. The paternal alleles of the H19 and Dlk1/Gtl2 DMRs were maintained methylated in [Dnmt3a^1lox/1lox, Dnmt3b^1lox/1lox] embryos at E9.5, whereas the paternal allele of the Rasgrf1 DMR was partially demethylated. (C) Methylation status of the maternally methylated Peg3 DMR. Maternal methylation imprint was not established, due to the lack of Dnmt3a in oocytes, confirming the effective conditional knockout. Open circles and filled circles indicate unmethylated cytosines and methylated cytosines, respectively. (JF1) JF1-derived allele; (dom) domesticus-derived allele.

Figure 3.

Expression and subcellular localization of Dnmt1 in oocytes and preimplantation embryos. (A) Immunostaining of wild-type FG oocytes, MII oocytes, and preimplantation embryos with an anti-Dnmt1 antibody recognizing both Dnmt1o and Dnmt1s. Dnmt1 proteins (green) were mainly detected in the ooplasm and the cytoplasm of preimplantation embryos. Nuclear translocation of the proteins at the eight-cell stage was not observed. (B) Absence of detectable Dnmt1 in oocytes and preimplantation embryos from [Dnmt1^2lox/2lox, Zp3-Cre] females. The cell nucleus was counterstained with PI (red).

Figure 4.

Methylation status of the DMRs in blastocysts lacking both maternal and zygotic Dnmt1. (A) A schematic representation of the flow of the experiment. [Dnmt1^2lox/2lox, Zp3-Cre] females were crossed with Dnmt1^c/+ males, and then the obtained E3.5 blastocysts were genotyped by PCR with primers that specifically amplify the Dnmt1^c allele. DNA from blastocysts of the same genotype (74 blastocysts of Dnmt1^1lox/c and 111 blastocysts of Dnmt1^1lox/+) was pooled and subjected to bisulfite sequencing. (B) Methylation status of the H19, Rasgrf1, Peg3, and Snrpn DMRs in wild-type (top) and mutant blastocysts (middle and bottom). (Middle) Blastocysts lacking maternal Dnmt1 (Dnmt1^1lox/+) showed a partial reduction in methylation at the normally methylated allele of all DMRs. (Bottom) Blastocysts lacking both maternal and zygotic Dnmt1 (Dnmt1^1lox/c) showed near complete loss of methylation at the normally methylated DMR alleles. (JF1) JF1-derived allele; (dom) domesticus-derived allele.

Figure 5.

Detection of zygotic Dnmt1s in preimplantation embryos by immunoblotting. Zygotic Dnmt1s was detected in eightcell embryos and blastocysts obtained from [Dnmt1^2lox/2lox, Zp3-Cre] females. These embryos lacked maternal Dnmt1 (mostly Dnmt1o) and thus allowed the detection of small amounts of zygotic Dnmt1s. Proteins extracted from pools of 250 oocytes or embryos from [Dnmt1^2lox/2lox, Zp3-Cre] females and those extracted from pools of 10 wild-type oocytes or embryos were loaded. Proteins extracted from a wild-type ovary were loaded as a control.