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First published online 27 August 2003
doi: 10.1242/dev.00716

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A Dnmt2-like protein mediates DNA methylation in Drosophila

¹ Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
² Division of Molecular Toxicology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

View larger version (115K): [in a new window]   Fig. 1. Immunological detection of 5-methylcytosine in Drosophila embryos. Embryos were double stained with an antibody that specifically recognizes 5-methylcytosine (5mC, green) and with an antibody that stains DNA (red). The left panel shows images from confocal sections of whole embryos. Scale bars: 50 µm. The right panels show an enlargement from the same embryos. Scale bars in enlarged pictures: 10 µm. (A) Early cleavage stage embryo; (B) cellular blastoderm stage embryo; (C) post-gastrulation stage embryo. (D) Pole cells from blastoderm embryos showed a distinct staining for 5-methylcytosine. (E) The specificity of the 5-methylcytosine antibody was confirmed by slot blot analysis of methylated (Bos taurus, Bt) and unmethylated (S. cerevisiae, Sc) genomic DNA. DNA from 0-6 hour Drosophila embryos (Dm) showed an intermediate staining intensity. Staining with the DNA antibody indicated equal loading of all slots. (F) 5-methylcytosine staining was found to be greatly reduced in Su(var)3-9 mutants (see text for details). (G) Incubation of dechorionated embryos with the DNA methyltransferase inhibitor 5-azacytidine reduced the 5-methylcytosine signal to background levels. The signal for DNA was not affected by the Su(var)3-9 mutation or by 5-azacytidine.

View larger version (91K): [in a new window]   Fig. 2. Expression pattern of Dnmt2. (A) Protein extracts were prepared from the developmental stages indicated and analyzed by western blotting. Significant amounts of Dnmt2 protein were found only in embryos. The position of marker proteins (in kDa) is indicated. A protein extract from adult females overexpressing Dnmt2 was included as an additional control for the specificity of our antibody (right lane). P40 signals are shown to indicate equal loading of all lanes. (B) Subcellular Dnmt2 localization in embryos. Stained embryos were analyzed by confocal microscopy. The protein is green, DNA is coloured red. Scale bars: 10 µm. (C) Depletion of Dnmt2 by RNA interference. Double-stranded RNA was injected into syncytial blastoderm embryos and embryos were stained after a 3 hour staging period. This procedure strongly reduced the amount of Dnmt2 protein.

View larger version (70K): [in a new window]   Fig. 3. Dnmt2 RNA interference results in loss of embryonic DNA methylation. Embryos were injected with Dnmt2 dsRNA and then staged, fixed and double stained with antibodies against 5-methylcytosine (5mC, green) and DNA (red). The left panel shows an overlay of both the 5-methylcytosine and the DNA signal, the right panel shows the 5-methylcytosine signal only. Scale bars: 50 µm. (A) Injection of Dnmt2 dsRNA resulted in a complete loss of nuclear 5-methylcytosine signals. The signal for DNA was not affected. (B) Injection of CG11840 dsRNA revealed a nuclear staining similar to wild-type embryos. (C) The effect of RNA interference on embryonic development. The percentage of hatching first instar larvae (hatch rate) did not differ significantly between embryos injected with Dnmt2 dsRNA (black bar) and embryos injected with injection buffer only (white bar). Embryos injected with CG11840 dsRNA showed detectable embryonic lethality (grey bar). For each experiment, about 300 embryos were analyzed.

View larger version (39K): [in a new window]   Fig. 4. Transgenic overexpression of Dnmt2 causes hypermethylation of the Drosophila genome. (A) Western analysis demonstrates significant levels of Dnmt2 protein in transgenic flies (left lanes). No protein was detectable in controls (right lane). (B) Genomic DNA from transgenic flies was analyzed by capillary electrophoresis. This revealed readily detectable amounts of 5-methylcytosine. (C) Very little 5-methylcytosine was present in matched control samples that did not overexpress Dnmt2. (D) The cytosine methylation level was quantified in several independent measurements from several independent experiments and the average was determined. Standard deviations are indicated by error bars.

View larger version (29K): [in a new window]   Fig. 5. Target sequences of Dnmt2-mediated DNA methylation. Methylated sequences were identified in Drosophila DNA by bisulfite sequencing of random genomic fragments. This revealed that overexpression of Dnmt2 results in specific methylation at CpT and CpA dinucleotides. Methylated cytosine residues are shown in red and their 3'-neighbours are indicated in green.