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First published online 10 November 2004
doi: 10.1242/dev.01531

Development 131, 6033-6039 (2004)
Published by The Company of Biologists 2004
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The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA

Joachim Marhold1, Katja Kramer1, Elisabeth Kremmer2 and Frank Lyko1,*

1 Research Group Epigenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
2 GSF-Forschungszentrum, Institut für Molekulare Immunologie, Marchioninistrasse 25, 81377 München, Germany



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  		Fig. 1. Characterization of the MBD1 allele. (A) Overview indicating the exon-intron structure of the MBD2/3 locus and the position of the P element insertion. (B) Northern analysis of embryonic poly(A)+ RNA from 0- to 6-hour-old embryos. The upper panel shows significant levels of MBD2/3 mRNA in control (yw) embryos, while no signal was detectable in MBD1 mutant embryos. A probe against the elongation factor EF1{alpha}48D was used as a loading control (lower panel). (C) Western analysis of 0-6 hours-old embryonic protein extracts. Nucleosome assembly protein 1 (NAP1) was used as loading control. (D) Confocal sections from embryos double immunostained with antibodies against MBD2/3 (green) and DNA (red). Mutant embryos showed only background levels for MBD2/3, while the DNA signal showed no quantitative differences between mutant and control.

 


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  		Fig. 2. Phenotype of MBD1 mutants. (A) Embryo stained with anti-DNA antibody (red). A significant number of mutant embryos appeared rounder and shorter than control embryos. Scale bar: 50 µm. (B) Percentage of embryos with detectable chromosome segregation defects. The data are derived from the microscopical analysis of 200-300 MBD1 mutant (M) and control (C) embryos, respectively. (C) Chromosome segregation defects in MBD1 mutant embryos. Embryos were collected, stained with an antibody against DNA and screened for abnormal mitotic figures. This revealed multiple chromosomal bridges in MBD1 mutants. Scale bar: 5 µm. (D) Eye phenotype of flies carrying a pUAST-lacZ (control) transgene in the wm4h background. (E) The MBD1 mutation results in a strong dominant suppression of the wm4h phenotype. (F) A similar effect was also observed with an independent MBD2/3 mutant allele (see Materials and methods for details).

 


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  		Fig. 3. Functional interaction between MBD2/3 and MI-2. (A) MBD1/MI-24 compound heterozygotes (left panel) showed significantly smaller and rougher eyes than either parent strain (middle and right panels). (B,C) Analysis of MI-2 protein distribution in control (B) and mutant (C) embryos by double immunostaining with antibodies against MBD2/3 (green) and DNA (red) and subsequent confocal microscopy. Scale bars: 5 µm. (B) MI-2 was ubiquitously distributed in wild type nuclei. (C) MI-2 was displaced from a subset of chromosomal sites in MBD1 embryos.

 


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  		Fig. 4. MBD2/3 colocalizes with a subset of MI-2 proteins. (A) Establishment of a novel MBD2/3-specific antibody. Antibody specificity was confirmed by western analysis of protein extracts from 0- to 6-hour-old embryos (left panel) and confocal analysis of immunostained embryos (right panels). Significant signals for MBD2/3 were detected in control (yw), but not in MBD1 embryos. (B) Confocal sections of embryos double immunostained with antibodies against MBD2/3 (red) and MI-2 (green). MBD2/3 was found to decorate only a limited number of nuclear foci, while MI-2 was broadly distributed over the entire nucleus. Scale bars: 5 µm.

 


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  		Fig. 5. MBD2/3 interacts with CpT/A-methylated oligonucleotides in band shift assays. Drosophila GST-MBD2/3, GST-MBD2/3{Delta} and human GST-MBD2 were analyzed for their ability to bind to radioactively labelled oligonucleotide probes. Binding assays were performed either in the absence (-) or presence (+) of recombinant proteins and competed with a 400-fold excess of the corresponding unlabelled probe (c). (A) Binding assays with a CpG-methylated probe (m) or with the corresponding unmethylated probe (u). (B) Binding assays with a CpT/A-methylated probe (m) or with the corresponding unmethylated probe (u). Arrowheads indicate the position of shifted oligonucleotide probes.

 


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  		Fig. 6. The localization of MBD2/3 depends on DNA methylation. (A) Embryos were demethylated with the DNA methyltransferase inhibitor 5-azacytidine and double immunostained with antibodies against MBD2/3 (green) and DNA (red). This caused a readily detectable displacement of MBD2/3 from DNA. (B) For comparison, pictures from mock-treated embryos are also shown. Scale bars: 25 µm, insets show fourfold magnified details.

 




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