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The Drosophila cytosine-5 methyltransferase Dnmt2 is associated with the nuclear matrix and can access DNA during mitosis.

Schaefer M, Steringer JP, Lyko F - PLoS ONE (2008)

Bottom Line: Dnmt2 localization is highly dynamic during the cell cycle.Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions.Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.

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Dnmt2 localization to mitotic nuclei is dynamic and microtubuli-dependent.(A) Nuclear division cycle 10 embryos expressing pUbq-Dnmt2-EGFP and His2Av-mRFP1 were mounted on an open coverslip and viewed by dual wavelength time-lapse LSCM. Selected frames are shown for Dnmt2-EGFP (gray, upper panel) and as merged images (lower panel) with Dnmt2-EGFP in green and His2A-mRFP1 in red. A movie of the sequence is available as supporting material. (B) 1–3 h old embryos expressing pGeno-Dnmt2-EGFP were collected and fixed immediately (left panel), treated with colcemide (middle panel) or with taxol (right panel) in order to disrupt or stabilize microtubuli, respectively. Metaphase chromosomes are shown with Dnmt2 (gray), DNA (red) and 〈-tubulin (green). While Dnmt2-EGFP is maintained around metaphase plates in taxol-treated embryos, the protein is delocalized in colcemide-treated embryos. Scale bars: (A) 10 µm; (B) 5 µm.
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pone-0001414-g005: Dnmt2 localization to mitotic nuclei is dynamic and microtubuli-dependent.(A) Nuclear division cycle 10 embryos expressing pUbq-Dnmt2-EGFP and His2Av-mRFP1 were mounted on an open coverslip and viewed by dual wavelength time-lapse LSCM. Selected frames are shown for Dnmt2-EGFP (gray, upper panel) and as merged images (lower panel) with Dnmt2-EGFP in green and His2A-mRFP1 in red. A movie of the sequence is available as supporting material. (B) 1–3 h old embryos expressing pGeno-Dnmt2-EGFP were collected and fixed immediately (left panel), treated with colcemide (middle panel) or with taxol (right panel) in order to disrupt or stabilize microtubuli, respectively. Metaphase chromosomes are shown with Dnmt2 (gray), DNA (red) and 〈-tubulin (green). While Dnmt2-EGFP is maintained around metaphase plates in taxol-treated embryos, the protein is delocalized in colcemide-treated embryos. Scale bars: (A) 10 µm; (B) 5 µm.

Mentions: Imaging Dnmt2-EGFP through consecutive cleavage cycles revealed that the fusion protein is distributed diffusely during S-phase but rapidly localizes to prophase nuclei at the onset of mitosis. During meta- and anaphase Dnmt2-EGFP formed spindle-like structures and re-localized to midbody structures during karyokinesis. During the following S-phase Dnmt2-EGFP was diffusely distributed throughout the syncitial cytoplasm until the start of the next mitotic phase. (Fig. 5A and movie S1).


The Drosophila cytosine-5 methyltransferase Dnmt2 is associated with the nuclear matrix and can access DNA during mitosis.

Schaefer M, Steringer JP, Lyko F - PLoS ONE (2008)

Dnmt2 localization to mitotic nuclei is dynamic and microtubuli-dependent.(A) Nuclear division cycle 10 embryos expressing pUbq-Dnmt2-EGFP and His2Av-mRFP1 were mounted on an open coverslip and viewed by dual wavelength time-lapse LSCM. Selected frames are shown for Dnmt2-EGFP (gray, upper panel) and as merged images (lower panel) with Dnmt2-EGFP in green and His2A-mRFP1 in red. A movie of the sequence is available as supporting material. (B) 1–3 h old embryos expressing pGeno-Dnmt2-EGFP were collected and fixed immediately (left panel), treated with colcemide (middle panel) or with taxol (right panel) in order to disrupt or stabilize microtubuli, respectively. Metaphase chromosomes are shown with Dnmt2 (gray), DNA (red) and 〈-tubulin (green). While Dnmt2-EGFP is maintained around metaphase plates in taxol-treated embryos, the protein is delocalized in colcemide-treated embryos. Scale bars: (A) 10 µm; (B) 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169302&req=5

pone-0001414-g005: Dnmt2 localization to mitotic nuclei is dynamic and microtubuli-dependent.(A) Nuclear division cycle 10 embryos expressing pUbq-Dnmt2-EGFP and His2Av-mRFP1 were mounted on an open coverslip and viewed by dual wavelength time-lapse LSCM. Selected frames are shown for Dnmt2-EGFP (gray, upper panel) and as merged images (lower panel) with Dnmt2-EGFP in green and His2A-mRFP1 in red. A movie of the sequence is available as supporting material. (B) 1–3 h old embryos expressing pGeno-Dnmt2-EGFP were collected and fixed immediately (left panel), treated with colcemide (middle panel) or with taxol (right panel) in order to disrupt or stabilize microtubuli, respectively. Metaphase chromosomes are shown with Dnmt2 (gray), DNA (red) and 〈-tubulin (green). While Dnmt2-EGFP is maintained around metaphase plates in taxol-treated embryos, the protein is delocalized in colcemide-treated embryos. Scale bars: (A) 10 µm; (B) 5 µm.
Mentions: Imaging Dnmt2-EGFP through consecutive cleavage cycles revealed that the fusion protein is distributed diffusely during S-phase but rapidly localizes to prophase nuclei at the onset of mitosis. During meta- and anaphase Dnmt2-EGFP formed spindle-like structures and re-localized to midbody structures during karyokinesis. During the following S-phase Dnmt2-EGFP was diffusely distributed throughout the syncitial cytoplasm until the start of the next mitotic phase. (Fig. 5A and movie S1).

Bottom Line: Dnmt2 localization is highly dynamic during the cell cycle.Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions.Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
Cytosine-5 methyltransferases of the Dnmt2 family are highly conserved in evolution and their biological function is being studied in several organisms. Although all structural DNA methyltransferase motifs are present in Dnmt2, these enzymes show a strong tRNA methyltransferase activity. In line with an enzymatic activity towards substrates other than DNA, Dnmt2 has been described to localize to the cytoplasm. Using molecular and biochemical approaches we show here that Dnmt2 is both a cytoplasmic and a nuclear protein. Sub-cellular fractionation shows that a significant amount of Dnmt2 is bound to the nuclear matrix. Sub-cellular localization analysis reveals that Dnmt2 proteins are enriched in actively dividing cells. Dnmt2 localization is highly dynamic during the cell cycle. Using live imaging we observed that Dnmt2-EGFP enters prophase nuclei and shows a spindle-like localization pattern during mitotic divisions. Additional experiments suggest that this localization is microtubule dependent and that Dnmt2 can access DNA during mitotic cell divisions. Our results represent the first comprehensive characterization of Dnmt2 proteins on the cellular level and have important implications for our understanding of the molecular activities of Dnmt2.

Show MeSH