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DNA methyltransferase is actively retained in the cytoplasm during early development.

Cardoso MC, Leonhardt H - J. Cell Biol. (1999)

Bottom Line: The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1).We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development.Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Franz Volhard Clinic, 13125 Berlin, Germany.

ABSTRACT
The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

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Cytoplasmic retention of Dnmt1 is independent of protein size. Two β-galactosidase fusion constructs were generated, one containing amino acids 1–854 of Dnmt1 (depicted in the diagram, Fig. 4 I) and the other containing amino acids 1–796 of human DNA ligase I (Cardoso et al. 1997). The size of both fusion proteins is between 160–170 kD. Both plasmids were transfected into mouse fibroblasts and microinjected into fertilized mouse eggs. After a 1-d incubation, localization of the fusion proteins was assayed by immunofluorescence staining with anti–β-galactosidase monoclonal antibody. A shows a confocal section through the middle of a mouse embryo expressing the Dnmt1 fusion which is mostly cytoplasmic. B shows the DNA ligase I fusion expressed in mouse embryos, which clearly has an exclusive nuclear localization. C and D illustrate the localization of the same proteins as in A and B, respectively, in the nuclei of tissue culture cells, in these images undergoing S-phase as seen by the ring and dot-shaped pattern of subnuclear foci (Leonhardt et al. 1992; Cardoso et al. 1997). Bar 10 μm.
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Figure 5: Cytoplasmic retention of Dnmt1 is independent of protein size. Two β-galactosidase fusion constructs were generated, one containing amino acids 1–854 of Dnmt1 (depicted in the diagram, Fig. 4 I) and the other containing amino acids 1–796 of human DNA ligase I (Cardoso et al. 1997). The size of both fusion proteins is between 160–170 kD. Both plasmids were transfected into mouse fibroblasts and microinjected into fertilized mouse eggs. After a 1-d incubation, localization of the fusion proteins was assayed by immunofluorescence staining with anti–β-galactosidase monoclonal antibody. A shows a confocal section through the middle of a mouse embryo expressing the Dnmt1 fusion which is mostly cytoplasmic. B shows the DNA ligase I fusion expressed in mouse embryos, which clearly has an exclusive nuclear localization. C and D illustrate the localization of the same proteins as in A and B, respectively, in the nuclei of tissue culture cells, in these images undergoing S-phase as seen by the ring and dot-shaped pattern of subnuclear foci (Leonhardt et al. 1992; Cardoso et al. 1997). Bar 10 μm.

Mentions: All tested Dnmt1 fusion constructs are relatively large, ranging in size from 100 kD to ∼250 kD. Although this size range does not make any difference for nuclear uptake in somatic cells, we generated a similar fusion construct of equal size using the DNA ligase I, which is involved in DNA replication and is like Dnmt1 targeted to nuclear replication foci (Cardoso et al. 1997). Fig. 5 shows the side-by-side comparison of both the Dnmt1 and the DNA ligase I construct. In somatic cells, both fusion constructs are targeted to nuclear replication foci, but only the DNA ligase fusion protein enters the nucleus of early mouse embryos. These results clearly show that the observed cytoplasmic retention of fusion proteins is specific for the Dnmt1 protein and is developmentally controlled. Moreover, the retention mechanism is active also in early preimplantation embryos and not only in oocytes, where the maternal stock of Dnmt1 is accumulated.


DNA methyltransferase is actively retained in the cytoplasm during early development.

Cardoso MC, Leonhardt H - J. Cell Biol. (1999)

Cytoplasmic retention of Dnmt1 is independent of protein size. Two β-galactosidase fusion constructs were generated, one containing amino acids 1–854 of Dnmt1 (depicted in the diagram, Fig. 4 I) and the other containing amino acids 1–796 of human DNA ligase I (Cardoso et al. 1997). The size of both fusion proteins is between 160–170 kD. Both plasmids were transfected into mouse fibroblasts and microinjected into fertilized mouse eggs. After a 1-d incubation, localization of the fusion proteins was assayed by immunofluorescence staining with anti–β-galactosidase monoclonal antibody. A shows a confocal section through the middle of a mouse embryo expressing the Dnmt1 fusion which is mostly cytoplasmic. B shows the DNA ligase I fusion expressed in mouse embryos, which clearly has an exclusive nuclear localization. C and D illustrate the localization of the same proteins as in A and B, respectively, in the nuclei of tissue culture cells, in these images undergoing S-phase as seen by the ring and dot-shaped pattern of subnuclear foci (Leonhardt et al. 1992; Cardoso et al. 1997). Bar 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164986&req=5

Figure 5: Cytoplasmic retention of Dnmt1 is independent of protein size. Two β-galactosidase fusion constructs were generated, one containing amino acids 1–854 of Dnmt1 (depicted in the diagram, Fig. 4 I) and the other containing amino acids 1–796 of human DNA ligase I (Cardoso et al. 1997). The size of both fusion proteins is between 160–170 kD. Both plasmids were transfected into mouse fibroblasts and microinjected into fertilized mouse eggs. After a 1-d incubation, localization of the fusion proteins was assayed by immunofluorescence staining with anti–β-galactosidase monoclonal antibody. A shows a confocal section through the middle of a mouse embryo expressing the Dnmt1 fusion which is mostly cytoplasmic. B shows the DNA ligase I fusion expressed in mouse embryos, which clearly has an exclusive nuclear localization. C and D illustrate the localization of the same proteins as in A and B, respectively, in the nuclei of tissue culture cells, in these images undergoing S-phase as seen by the ring and dot-shaped pattern of subnuclear foci (Leonhardt et al. 1992; Cardoso et al. 1997). Bar 10 μm.
Mentions: All tested Dnmt1 fusion constructs are relatively large, ranging in size from 100 kD to ∼250 kD. Although this size range does not make any difference for nuclear uptake in somatic cells, we generated a similar fusion construct of equal size using the DNA ligase I, which is involved in DNA replication and is like Dnmt1 targeted to nuclear replication foci (Cardoso et al. 1997). Fig. 5 shows the side-by-side comparison of both the Dnmt1 and the DNA ligase I construct. In somatic cells, both fusion constructs are targeted to nuclear replication foci, but only the DNA ligase fusion protein enters the nucleus of early mouse embryos. These results clearly show that the observed cytoplasmic retention of fusion proteins is specific for the Dnmt1 protein and is developmentally controlled. Moreover, the retention mechanism is active also in early preimplantation embryos and not only in oocytes, where the maternal stock of Dnmt1 is accumulated.

Bottom Line: The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1).We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development.Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Franz Volhard Clinic, 13125 Berlin, Germany.

ABSTRACT
The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

Show MeSH