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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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Tissue specific expression of Dnmt2.(A) Left panel: developmental Western blot of pGeno-Dnmt2-EGFP animals. Whole protein extract from embryonic, larval and adult stages were blotted and probed with anti-Dnmt2. Ponceau staining is shown as a loading control. Right panel: Dnmt2-EGFP is expressed both in the cytoplasm and in nuclei. Fractionation efficiency was confirmed by probing the blot for cytoplasmic 〈-tubulin and nuclear Lamin C. Antibodies against EGFP reveal Dnmt2 in developing embryos (B), third instar larval salivary glands (C), ovaries (D) and testes (E). (F) Dnmt2-EGFP in a developing cyst of the female germline is mostly cytoplasmic. Nuclear staining can be observed in nurse cell nuclei. (G) Dnmt2-EGFP in the male germline is ubiquitous in all cells with the exception of somatic hub cells (marked by Armadillo, red). Scale bars: (B) 100 µm; (C) 10 µm; (D) 50 µm; (E) 100 µm; (F) 20 µm; (G) 10 µm.
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pone-0001414-g002: Tissue specific expression of Dnmt2.(A) Left panel: developmental Western blot of pGeno-Dnmt2-EGFP animals. Whole protein extract from embryonic, larval and adult stages were blotted and probed with anti-Dnmt2. Ponceau staining is shown as a loading control. Right panel: Dnmt2-EGFP is expressed both in the cytoplasm and in nuclei. Fractionation efficiency was confirmed by probing the blot for cytoplasmic 〈-tubulin and nuclear Lamin C. Antibodies against EGFP reveal Dnmt2 in developing embryos (B), third instar larval salivary glands (C), ovaries (D) and testes (E). (F) Dnmt2-EGFP in a developing cyst of the female germline is mostly cytoplasmic. Nuclear staining can be observed in nurse cell nuclei. (G) Dnmt2-EGFP in the male germline is ubiquitous in all cells with the exception of somatic hub cells (marked by Armadillo, red). Scale bars: (B) 100 µm; (C) 10 µm; (D) 50 µm; (E) 100 µm; (F) 20 µm; (G) 10 µm.

Mentions: Since Dnmt2 expression could be detected during all stages of Drosophila development we asked whether Dnmt2 displays a tissue-specific expression. Our affinity purified antibodies against Dnmt2 were not suitable for indirect immunofluoresence experiments, which was most likely due to epitope masking (data not shown). We therefore created several independent transgenic fly lines harbouring a Dnmt2-EGFP fusion gene in the genomic sequence context of the Dnmt2 locus (pGeno-Dnmt2-EGFP). Since Dnmt2 mutant animals are viable and fertile and do not display obvious phenotypes we could not positively test whether the genomic Dnmt2-EGFP contstruct is functional. On the other hand, co-immunoprecipitation experiments using both endogenous Dnmt2 as well as Dnmt2-EGFP constructs revealed that Dnmt2-EGFP is contained in similar protein complexes, suggesting that part of the functionality of Dnmt2 is retained in the fusion protein (unpublished observations). In order to test whether these constructs express tagged Dnmt2 similarly to endogenous Dnmt2 we performed Western blot analysis of protein extracts from various developmental stages and adult tissues using antibodies against Dnmt2. We found that Dnmt2-EGFP was expressed during all stages of development and at levels that were comparable with those of the endogenous Dnmt2 protein (Fig. 2A). Furthermore, biochemical fractionation of embryonic protein extracts and immunolocalization of Dnmt2-EGFP in salivary glands showed that the fusion protein could be imported into nuclei, suggesting that the sub-cellular localization signals were functionally retained (Fig. 2A and C).


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)

Tissue specific expression of Dnmt2.(A) Left panel: developmental Western blot of pGeno-Dnmt2-EGFP animals. Whole protein extract from embryonic, larval and adult stages were blotted and probed with anti-Dnmt2. Ponceau staining is shown as a loading control. Right panel: Dnmt2-EGFP is expressed both in the cytoplasm and in nuclei. Fractionation efficiency was confirmed by probing the blot for cytoplasmic 〈-tubulin and nuclear Lamin C. Antibodies against EGFP reveal Dnmt2 in developing embryos (B), third instar larval salivary glands (C), ovaries (D) and testes (E). (F) Dnmt2-EGFP in a developing cyst of the female germline is mostly cytoplasmic. Nuclear staining can be observed in nurse cell nuclei. (G) Dnmt2-EGFP in the male germline is ubiquitous in all cells with the exception of somatic hub cells (marked by Armadillo, red). Scale bars: (B) 100 µm; (C) 10 µm; (D) 50 µm; (E) 100 µm; (F) 20 µm; (G) 10 µm.
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Related In: Results  -  Collection

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pone-0001414-g002: Tissue specific expression of Dnmt2.(A) Left panel: developmental Western blot of pGeno-Dnmt2-EGFP animals. Whole protein extract from embryonic, larval and adult stages were blotted and probed with anti-Dnmt2. Ponceau staining is shown as a loading control. Right panel: Dnmt2-EGFP is expressed both in the cytoplasm and in nuclei. Fractionation efficiency was confirmed by probing the blot for cytoplasmic 〈-tubulin and nuclear Lamin C. Antibodies against EGFP reveal Dnmt2 in developing embryos (B), third instar larval salivary glands (C), ovaries (D) and testes (E). (F) Dnmt2-EGFP in a developing cyst of the female germline is mostly cytoplasmic. Nuclear staining can be observed in nurse cell nuclei. (G) Dnmt2-EGFP in the male germline is ubiquitous in all cells with the exception of somatic hub cells (marked by Armadillo, red). Scale bars: (B) 100 µm; (C) 10 µm; (D) 50 µm; (E) 100 µm; (F) 20 µm; (G) 10 µm.
Mentions: Since Dnmt2 expression could be detected during all stages of Drosophila development we asked whether Dnmt2 displays a tissue-specific expression. Our affinity purified antibodies against Dnmt2 were not suitable for indirect immunofluoresence experiments, which was most likely due to epitope masking (data not shown). We therefore created several independent transgenic fly lines harbouring a Dnmt2-EGFP fusion gene in the genomic sequence context of the Dnmt2 locus (pGeno-Dnmt2-EGFP). Since Dnmt2 mutant animals are viable and fertile and do not display obvious phenotypes we could not positively test whether the genomic Dnmt2-EGFP contstruct is functional. On the other hand, co-immunoprecipitation experiments using both endogenous Dnmt2 as well as Dnmt2-EGFP constructs revealed that Dnmt2-EGFP is contained in similar protein complexes, suggesting that part of the functionality of Dnmt2 is retained in the fusion protein (unpublished observations). In order to test whether these constructs express tagged Dnmt2 similarly to endogenous Dnmt2 we performed Western blot analysis of protein extracts from various developmental stages and adult tissues using antibodies against Dnmt2. We found that Dnmt2-EGFP was expressed during all stages of development and at levels that were comparable with those of the endogenous Dnmt2 protein (Fig. 2A). Furthermore, biochemical fractionation of embryonic protein extracts and immunolocalization of Dnmt2-EGFP in salivary glands showed that the fusion protein could be imported into nuclei, suggesting that the sub-cellular localization signals were functionally retained (Fig. 2A and C).

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
Related in: MedlinePlus