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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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Dnmt1 is actively retained in the cytoplasm of early mouse embryos via a sequence in the NH2-terminal, regulatory domain of the enzyme. A–D show the localization of a β-galactosidase fusion protein containing amino acids 1–638 of the oocyte-specific Dnmt1 isoform which is retained in the cytoplasm of two-cell mouse embryos. A shows the DIC image; B shows the DNA stained with Hoechst 33258; C shows the rhodamine detection of the β-galactosidase fusion protein; and D shows the corresponding confocal section clearly showing its cytoplasmic localization. E–H illustrate a cytoplasmic retention deficient β-galactosidase fusion protein containing amino acids 1–259 of the oocyte-specific Dnmt1 isoform, as DIC image (E), DNA staining (F), epifluorescence image of the fusion protein (G), and respective confocal section (H). The dotty cytoplasmic signal visible in the confocal section in H was not reproducible. Fertilized eggs were microinjected with the different fusion constructs schematically shown in I (β-galactosidase was fused at the COOH terminus and it is not shown), processed as described in Fig. 3, and assayed for their ability to be retained in the cytoplasm of two-cell mouse embryos. The previously identified targeting sequence that directs the protein to subnuclear sites of DNA replication (Leonhardt et al. 1992) is indicated in green. The diamond at the NH2 terminus of two fusions signifies the addition of an SV-40 large T antigen NLS. All the constructs listed show a clear nuclear localization in somatic cells (data not shown). The deletion endpoints of the different fusion proteins shown in I define a broad region between amino acids 308 and 854 of the oocyte-specific Dnmt1 isoform (light grey shading) which is necessary and sufficient for cytoplasmic retention (scored as ++) in preimplantation mouse embryos. Within this larger region the most crucial sequence (dark grey shading) lies between amino acids 427 and 638, since deletions in this region are clearly not retained in the cytoplasm (scored as −). Absence of amino acids 308–427 or 638–854 flanking this sequence caused a less efficient cytoplasmic retention (scored as +). Bars, 10 μm.
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Figure 4: Dnmt1 is actively retained in the cytoplasm of early mouse embryos via a sequence in the NH2-terminal, regulatory domain of the enzyme. A–D show the localization of a β-galactosidase fusion protein containing amino acids 1–638 of the oocyte-specific Dnmt1 isoform which is retained in the cytoplasm of two-cell mouse embryos. A shows the DIC image; B shows the DNA stained with Hoechst 33258; C shows the rhodamine detection of the β-galactosidase fusion protein; and D shows the corresponding confocal section clearly showing its cytoplasmic localization. E–H illustrate a cytoplasmic retention deficient β-galactosidase fusion protein containing amino acids 1–259 of the oocyte-specific Dnmt1 isoform, as DIC image (E), DNA staining (F), epifluorescence image of the fusion protein (G), and respective confocal section (H). The dotty cytoplasmic signal visible in the confocal section in H was not reproducible. Fertilized eggs were microinjected with the different fusion constructs schematically shown in I (β-galactosidase was fused at the COOH terminus and it is not shown), processed as described in Fig. 3, and assayed for their ability to be retained in the cytoplasm of two-cell mouse embryos. The previously identified targeting sequence that directs the protein to subnuclear sites of DNA replication (Leonhardt et al. 1992) is indicated in green. The diamond at the NH2 terminus of two fusions signifies the addition of an SV-40 large T antigen NLS. All the constructs listed show a clear nuclear localization in somatic cells (data not shown). The deletion endpoints of the different fusion proteins shown in I define a broad region between amino acids 308 and 854 of the oocyte-specific Dnmt1 isoform (light grey shading) which is necessary and sufficient for cytoplasmic retention (scored as ++) in preimplantation mouse embryos. Within this larger region the most crucial sequence (dark grey shading) lies between amino acids 427 and 638, since deletions in this region are clearly not retained in the cytoplasm (scored as −). Absence of amino acids 308–427 or 638–854 flanking this sequence caused a less efficient cytoplasmic retention (scored as +). Bars, 10 μm.

Mentions: The comparison of the full-length and the truncated fusion protein in Fig. 3 suggests that Dnmt1 contains oocyte-specific retention sequences, since both fusions contain functional NLS. Therefore, we generated a set of deletions to map that putative retention sequence (Fig. 4). All constructs shown in this figure were first tested in somatic cells and showed a clear nuclear localization (data not shown). A deletion series coming from the COOH-terminal end indicated that the first 638 amino acids of the regulatory domain of Dnmt1 are sufficient for retention in the cytoplasm and that the catalytic domain is not required (Fig. 4 D, I). A similar deletion series starting from the NH2 terminus is not possible since the major NLS is located between amino acids 72 and 92. Therefore, internal deletions were done to further narrow down the region involved in cytoplasmic retention. These deletions showed that fusions containing the region from amino acid 308–854 are efficiently retained in the cytoplasm and constructs containing the region from amino acid 427–638 were still retained but less efficiently so that some signal could also be detected in the nucleus (see also Fig. 3 d). We propose that the binding interface mediating the cytoplasmic retention is complex and may involve several stretches of amino acids from different parts of the primary sequence. Deletion of some of these interacting parts may reduce but not totally abolish the affinity for the target(s). Similarly, deletions may affect the three-dimensional structure of the interface and thereby reduce the binding affinity and thus cause some leakage into the nucleus.


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

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

Dnmt1 is actively retained in the cytoplasm of early mouse embryos via a sequence in the NH2-terminal, regulatory domain of the enzyme. A–D show the localization of a β-galactosidase fusion protein containing amino acids 1–638 of the oocyte-specific Dnmt1 isoform which is retained in the cytoplasm of two-cell mouse embryos. A shows the DIC image; B shows the DNA stained with Hoechst 33258; C shows the rhodamine detection of the β-galactosidase fusion protein; and D shows the corresponding confocal section clearly showing its cytoplasmic localization. E–H illustrate a cytoplasmic retention deficient β-galactosidase fusion protein containing amino acids 1–259 of the oocyte-specific Dnmt1 isoform, as DIC image (E), DNA staining (F), epifluorescence image of the fusion protein (G), and respective confocal section (H). The dotty cytoplasmic signal visible in the confocal section in H was not reproducible. Fertilized eggs were microinjected with the different fusion constructs schematically shown in I (β-galactosidase was fused at the COOH terminus and it is not shown), processed as described in Fig. 3, and assayed for their ability to be retained in the cytoplasm of two-cell mouse embryos. The previously identified targeting sequence that directs the protein to subnuclear sites of DNA replication (Leonhardt et al. 1992) is indicated in green. The diamond at the NH2 terminus of two fusions signifies the addition of an SV-40 large T antigen NLS. All the constructs listed show a clear nuclear localization in somatic cells (data not shown). The deletion endpoints of the different fusion proteins shown in I define a broad region between amino acids 308 and 854 of the oocyte-specific Dnmt1 isoform (light grey shading) which is necessary and sufficient for cytoplasmic retention (scored as ++) in preimplantation mouse embryos. Within this larger region the most crucial sequence (dark grey shading) lies between amino acids 427 and 638, since deletions in this region are clearly not retained in the cytoplasm (scored as −). Absence of amino acids 308–427 or 638–854 flanking this sequence caused a less efficient cytoplasmic retention (scored as +). Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Dnmt1 is actively retained in the cytoplasm of early mouse embryos via a sequence in the NH2-terminal, regulatory domain of the enzyme. A–D show the localization of a β-galactosidase fusion protein containing amino acids 1–638 of the oocyte-specific Dnmt1 isoform which is retained in the cytoplasm of two-cell mouse embryos. A shows the DIC image; B shows the DNA stained with Hoechst 33258; C shows the rhodamine detection of the β-galactosidase fusion protein; and D shows the corresponding confocal section clearly showing its cytoplasmic localization. E–H illustrate a cytoplasmic retention deficient β-galactosidase fusion protein containing amino acids 1–259 of the oocyte-specific Dnmt1 isoform, as DIC image (E), DNA staining (F), epifluorescence image of the fusion protein (G), and respective confocal section (H). The dotty cytoplasmic signal visible in the confocal section in H was not reproducible. Fertilized eggs were microinjected with the different fusion constructs schematically shown in I (β-galactosidase was fused at the COOH terminus and it is not shown), processed as described in Fig. 3, and assayed for their ability to be retained in the cytoplasm of two-cell mouse embryos. The previously identified targeting sequence that directs the protein to subnuclear sites of DNA replication (Leonhardt et al. 1992) is indicated in green. The diamond at the NH2 terminus of two fusions signifies the addition of an SV-40 large T antigen NLS. All the constructs listed show a clear nuclear localization in somatic cells (data not shown). The deletion endpoints of the different fusion proteins shown in I define a broad region between amino acids 308 and 854 of the oocyte-specific Dnmt1 isoform (light grey shading) which is necessary and sufficient for cytoplasmic retention (scored as ++) in preimplantation mouse embryos. Within this larger region the most crucial sequence (dark grey shading) lies between amino acids 427 and 638, since deletions in this region are clearly not retained in the cytoplasm (scored as −). Absence of amino acids 308–427 or 638–854 flanking this sequence caused a less efficient cytoplasmic retention (scored as +). Bars, 10 μm.
Mentions: The comparison of the full-length and the truncated fusion protein in Fig. 3 suggests that Dnmt1 contains oocyte-specific retention sequences, since both fusions contain functional NLS. Therefore, we generated a set of deletions to map that putative retention sequence (Fig. 4). All constructs shown in this figure were first tested in somatic cells and showed a clear nuclear localization (data not shown). A deletion series coming from the COOH-terminal end indicated that the first 638 amino acids of the regulatory domain of Dnmt1 are sufficient for retention in the cytoplasm and that the catalytic domain is not required (Fig. 4 D, I). A similar deletion series starting from the NH2 terminus is not possible since the major NLS is located between amino acids 72 and 92. Therefore, internal deletions were done to further narrow down the region involved in cytoplasmic retention. These deletions showed that fusions containing the region from amino acid 308–854 are efficiently retained in the cytoplasm and constructs containing the region from amino acid 427–638 were still retained but less efficiently so that some signal could also be detected in the nucleus (see also Fig. 3 d). We propose that the binding interface mediating the cytoplasmic retention is complex and may involve several stretches of amino acids from different parts of the primary sequence. Deletion of some of these interacting parts may reduce but not totally abolish the affinity for the target(s). Similarly, deletions may affect the three-dimensional structure of the interface and thereby reduce the binding affinity and thus cause some leakage into the nucleus.

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
Related in: MedlinePlus