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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 has at least three independent nuclear localization signals. (A) Diagram outlining the structure of the oocyte-specific murine Dnmt1 protein and indicating the different parts of the Dnmt1 present in fusions with the β-galactosidase epitope (for simplicity, the latter is not depicted). The different fusion constructs were expressed in murine myoblast or fibroblast cells, formalin fixed, stained with anti–β-galactosidase monoclonal antibody, and screened for their nuclear or cytoplasmic localization. B shows a representative example of a cytoplasmic fusion protein containing amino acids 1-72; 92-139 of Dnmt1 and C shows a nuclear fusion protein containing amino acids 1-72; 92-259 of Dnmt1, including only the second NLS. The analysis of all listed constructs lead to the identification of three regions (highlighted by grey shading) that contain independently functional NLS. Sequences representing possible NLS within each shaded area are specified below. The black box indicates the location of the cysteine-rich region that had been shown to bind zinc ions (Bestor 1992). Bar, 10 μm.
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Figure 2: Dnmt1 has at least three independent nuclear localization signals. (A) Diagram outlining the structure of the oocyte-specific murine Dnmt1 protein and indicating the different parts of the Dnmt1 present in fusions with the β-galactosidase epitope (for simplicity, the latter is not depicted). The different fusion constructs were expressed in murine myoblast or fibroblast cells, formalin fixed, stained with anti–β-galactosidase monoclonal antibody, and screened for their nuclear or cytoplasmic localization. B shows a representative example of a cytoplasmic fusion protein containing amino acids 1-72; 92-139 of Dnmt1 and C shows a nuclear fusion protein containing amino acids 1-72; 92-259 of Dnmt1, including only the second NLS. The analysis of all listed constructs lead to the identification of three regions (highlighted by grey shading) that contain independently functional NLS. Sequences representing possible NLS within each shaded area are specified below. The black box indicates the location of the cysteine-rich region that had been shown to bind zinc ions (Bestor 1992). Bar, 10 μm.

Mentions: Therefore, we expressed this shorter isoform in different somatic cell lines including COS1, mouse fibroblasts, and myoblast cells. For visualization by immunofluorescence, the oocyte-specific isoform was fused with an unrelated β-galactosidase epitope. In all tested somatic cell lines the fusion protein showed a clear nuclear localization like the longer somatic isoform (data not shown). To further study the regulation of the nuclear uptake of Dnmt1, we generated a set of deletion mutants and determined their subcellular localization (Fig. 2). A series of deletions showed that the first 139 amino acids of the oocyte-specific form are sufficient for nuclear uptake of the β-galactosidase fusion in somatic cells. Deletion of one candidate NLS (KKRR from position 72–92) prevented nuclear uptake. This region clearly contains a functional NLS since this sequence alone allows nuclear uptake of the fusion protein (Fig. 2). The regulatory domain of Dnmt1, however, contains further stretches of basic amino acids that could serve as NLS. Further deletion constructs identified at least two independent additional NLS (positions 140–259 and 511–638). These results clearly show that the oocyte-specific Dnmt1 form enters the nucleus of somatic cells and contains at least three independent NLS. Though these three regions function as NLS, a set of internal deletions (from positions 310–637, 310–511, 511–637, and 636–972) that retain all three NLS or at least the two first NLS show cytoplasmic localization of the fusion protein in somatic cells (Fig. 2). Similar deletions constructs (not fused to β-galactosidase) expressed in mammalian cells are enzymatically inactive (Margot, J.B., A.M. Aguirre-Arteta, V. Di Giacco, S. Pradhan, R. Roberts, M.C. Cardoso, and H. Leonhardt, manuscript submitted for publication) suggesting an important role of this region in the proper folding of Dnmt1, rather than a specific effect on nuclear localization.


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

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

Dnmt1 has at least three independent nuclear localization signals. (A) Diagram outlining the structure of the oocyte-specific murine Dnmt1 protein and indicating the different parts of the Dnmt1 present in fusions with the β-galactosidase epitope (for simplicity, the latter is not depicted). The different fusion constructs were expressed in murine myoblast or fibroblast cells, formalin fixed, stained with anti–β-galactosidase monoclonal antibody, and screened for their nuclear or cytoplasmic localization. B shows a representative example of a cytoplasmic fusion protein containing amino acids 1-72; 92-139 of Dnmt1 and C shows a nuclear fusion protein containing amino acids 1-72; 92-259 of Dnmt1, including only the second NLS. The analysis of all listed constructs lead to the identification of three regions (highlighted by grey shading) that contain independently functional NLS. Sequences representing possible NLS within each shaded area are specified below. The black box indicates the location of the cysteine-rich region that had been shown to bind zinc ions (Bestor 1992). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Dnmt1 has at least three independent nuclear localization signals. (A) Diagram outlining the structure of the oocyte-specific murine Dnmt1 protein and indicating the different parts of the Dnmt1 present in fusions with the β-galactosidase epitope (for simplicity, the latter is not depicted). The different fusion constructs were expressed in murine myoblast or fibroblast cells, formalin fixed, stained with anti–β-galactosidase monoclonal antibody, and screened for their nuclear or cytoplasmic localization. B shows a representative example of a cytoplasmic fusion protein containing amino acids 1-72; 92-139 of Dnmt1 and C shows a nuclear fusion protein containing amino acids 1-72; 92-259 of Dnmt1, including only the second NLS. The analysis of all listed constructs lead to the identification of three regions (highlighted by grey shading) that contain independently functional NLS. Sequences representing possible NLS within each shaded area are specified below. The black box indicates the location of the cysteine-rich region that had been shown to bind zinc ions (Bestor 1992). Bar, 10 μm.
Mentions: Therefore, we expressed this shorter isoform in different somatic cell lines including COS1, mouse fibroblasts, and myoblast cells. For visualization by immunofluorescence, the oocyte-specific isoform was fused with an unrelated β-galactosidase epitope. In all tested somatic cell lines the fusion protein showed a clear nuclear localization like the longer somatic isoform (data not shown). To further study the regulation of the nuclear uptake of Dnmt1, we generated a set of deletion mutants and determined their subcellular localization (Fig. 2). A series of deletions showed that the first 139 amino acids of the oocyte-specific form are sufficient for nuclear uptake of the β-galactosidase fusion in somatic cells. Deletion of one candidate NLS (KKRR from position 72–92) prevented nuclear uptake. This region clearly contains a functional NLS since this sequence alone allows nuclear uptake of the fusion protein (Fig. 2). The regulatory domain of Dnmt1, however, contains further stretches of basic amino acids that could serve as NLS. Further deletion constructs identified at least two independent additional NLS (positions 140–259 and 511–638). These results clearly show that the oocyte-specific Dnmt1 form enters the nucleus of somatic cells and contains at least three independent NLS. Though these three regions function as NLS, a set of internal deletions (from positions 310–637, 310–511, 511–637, and 636–972) that retain all three NLS or at least the two first NLS show cytoplasmic localization of the fusion protein in somatic cells (Fig. 2). Similar deletions constructs (not fused to β-galactosidase) expressed in mammalian cells are enzymatically inactive (Margot, J.B., A.M. Aguirre-Arteta, V. Di Giacco, S. Pradhan, R. Roberts, M.C. Cardoso, and H. Leonhardt, manuscript submitted for publication) suggesting an important role of this region in the proper folding of Dnmt1, rather than a specific effect on nuclear localization.

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