Northern analysis was used to ascertain the expression patterns of the two MBD-like isoforms during Drosophila development. Two transcripts, corresponding to the splice variants of MBD-like, are present in early embryos. In 0-2h-old embryos, only the longer mRNA encoding MBD-like is detectable. The abundance of this mRNA declines precipitously after 12 h of embryonic development and is undetectable in larval stages and 1- to 5-day-old adult males, but is present in adult females of a similar age, possibly due to maternal mRNA in the ovary. Protein expression patterns were also examined. Similarly, only MBD-like was observed in 0 to 2-h embryos while both MBD-like and MBD-likedelta were present in 3 to 12-h embryos. In the final embryonic stage and in the first larval stage, only the MBD-likedelta isoform was present. Neither protein isoform was detected in the remaining larval stages or in 1- to 5-day-old-adults, despite the presence of mRNA. The inability to detect MBD-likedelta protein in later developmental stages is probably a reflection of the low concentration of MBD-likedelta relative to total protein during these developmental stages. Immunostaining of salivary gland cells shows that MBD-likedelta is expressed in third instar larvae (Ballestar, 2001).

The Drosophila gene dMBD2/3 encodes a protein with significant homologies to the mammalian methyl-DNA binding proteins MBD2 and MBD3. These proteins are essential components of chromatin complexes involved in epigenetic gene regulation. Because the available in vitro data on dMBD2/3 are conflicting, an in vivo characterization of dMBD2/3 was undertaken. Expression of two isoforms specifically was detected during embryonic development. Staining of whole embryos combined with high-resolution confocal microscopy revealed a highly regulated spatial distribution. During the syncytial blastoderm stage, dMBD2/3 forms speckles that localized to the cytoplasm. Shortly after, during the cellular blastoderm stage, the protein enters the nucleus and forms bright foci that associate with DNA. This rapid transition coincides with the activation of the embryonic genome. A similar observation was made during activation of the spermatocyte genome; dMBD2/3 forms distinct foci associated with the activated Y chromosome. The results indicate that dMBD2/3 forms specialized nuclear compartments to keep certain genes epigenetically silenced during genome activation (Marhold, 2002).

Effects of Mutation or Deletion

To address the function of MBD2/3 in vivo, an insertion mutant was identified and characterized that contains an EP element in the 5'-coding sequence of the MBD2/3 gene, 54 bp downstream of the initiation codon. This mutant allele was designated MBD1. In order to characterize this mutation, mRNA was isolated from homozygous MBD1 embryos and it was analyzed for the presence of MBD2/3 transcripts by Northern blotting. This revealed that MBD2/3 expression was reduced to background levels. Consistently, Western blotting failed to detect any MBD2/3 protein in extracts from homozygous mutant embryos. Last, homozygous MBD1 embryos were stained with a polyclonal MBD2/3-specific antibody and no signals above the background level were observed. These results strongly suggest that the MBD1 mutation represents a null allele (Marhold, 2004b).

Homozygous mutant flies were viable and fertile. This indicates that MBD2/3 is not essential for Drosophila development. However, a more detailed analysis of mutant embryos by immunofluorescence microscopy revealed that a significant fraction (>20%) appeared smaller and more rounded than matched controls. However, this phenotype did not seem to have a significant effect on embryonic viability. The effect of the MBD1 mutation on the organization of embryonic DNA was analyzed. To this end, homozygous mutant and control embryos were collected, immunostained with an antibody against DNA and analyzed by confocal microscopy. This revealed chromosome segregation defects in 37% of MBD1 embryos, but only in 1% of control embryos. More specifically, a high number of chromosome bridges and lagging anaphase chromosomes were observed, indicating a potential role of MBD2/3 in the stability of pericentric heterochromatin. This prompted an investigation of the effect of the mutation on the expression of a variegating pericentric white gene. To this end, the MBD1 mutation was introduced into the wm4h background. These experiments revealed a strong dominant suppression of white variegation, which can be seen by an uniform red eye colour and the loss of variegating spots in the eyes of the adult progeny. The same effect was also observed with an independent mutant MBD2/3 allele that contains a P-element insertion 300 bp upstream from the transcriptional start site. This confirmed the specificity of the observation and is consistent with a role of MBD2/3 in chromatin regulation (Marhold, 2004b).


Reference names in red indicate recommended papers.

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MBD-like: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 1 June 2005

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