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).
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).
Ballestar, E., Pile, L. A., Wassarman, D. A., Wolffe, A. P. and Wade, P. A. (2001). A Drosophila MBD family member is a transcriptional co-repressor associated with specific genes. Eur. J. Biochem. 268: 5397-540. 11606202
Bird, A. P. and Wolffe, A. P. (1999). Methylation-induced repression-belts, braces, and chromatin. Cell 99: 451-454. 10589672
Brackertz, M., Boeke, J., Zhang, R. and Renkawitz, R. (2002). Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3. J. Biol. Chem. 277(43): 40958-66. 12183469
Ego, T., Tanaka, Y. and Shimotohno, K. (2005). Interaction of HTLV-1 Tax and methyl-CpG-binding domain 2 positively regulates the gene expression from the hypermethylated LTR. Oncogene 24(11): 1914-234. 15674330
Feng, Q. and Zhang, Y. (2001). The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes. Genes Dev. 15: 827-8324. 11297506
Fujita, H., Fujii, R., Aratani, S., Amano, T., Fukamizu, A. and Nakajima, T. (2003). Antithetic effects of MBD2a on gene regulation. Mol. Cell. Biol. 23(8): 2645-57. 12665568
Fuks, F., Hurd, P. J., Wolf, D., Nan, X., Bird, A. P. and Kouzarides, T. (2003). The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J. Biol. Chem. 278: 4035-4040. 12427740
Ghoshal, K., et al. (2004). Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J. Biol. Chem. 279(8): 6783-93. 14610093
Gutierrez, A. and Sommer, R. J. (2004). Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae. Nucleic Acids Res. 32(21): 6388-96. 15576683
Hendrich, B. and Bird, A. (1998). Identification and characterization of a family of mammalian methyl-CpG binding proteins. Mol. Cell. Biol. 18: 6538-6547. 9774669
Hendrich, B., Guy, J., Ramsahoye, B., Wilson, V. A. and Bird, A. (2001). Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes Dev. 15: 710-723. 11274056
Hendrich, B. and Tweedie, S. (2003). The methyl-CpG binding domain and the evolving role of DNA methylation in animals. Trends Genet. 19: 269-277. 12711219
Hutchins, A. S., et al. (2002). Gene silencing quantitatively controls the function of a developmental trans-activator. Mol. Cell 10(1): 81-91. 12150909
Iwano, H., Nakamura, M. and Tajima, S. (2004). Xenopus MBD3 plays a crucial role in an early stage of development. Dev. Biol. 268(2): 416-28. 15063177
Jeffery, L. and Nakielny, S. (2004). Components of the DNA methylation system of chromatin control are RNA-binding proteins. J. Biol. Chem. 279(47): 49479-87. 15342650
Jiang, C. L., Jin, S. G. and Pfeifer, G. P. (2004). MBD3L1 is a transcriptional repressor that interacts with methyl-CpG-binding protein 2 (MBD2) and components of the NuRD complex. J. Biol. Chem. 279(50): 52456-64. 15456747
Jin, S. G., Jiang, C. L., Rauch, T., Li, H. and Pfeifer, G. P. (2005). MBD3L2 interacts with MBD3 and components of the NuRD complex and can oppose MBD2-MeCP1-mediated methylation silencing. J. Biol. Chem. 280(13): 12700-9. 15701600
Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J. and Wolffe, A. P. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19: 187-191. 9620779
Kunert, N., Marhold, J., Stanke, J., Stach, D. and Lyko, F. (2003). A Dnmt2-like protein mediates DNA methylation in Drosophila. Development 130: 5083-5090. 12944428
Lembo, F., et al. (2003). MBDin, a novel MBD2-interacting protein, relieves MBD2 repression potential and reactivates transcription from methylated promoters. Mol. Cell. Biol. 23(5): 1656-65. 12588985
Lyko, F., Ramsahoye, B. H. and Jaenisch, R. (2000). DNA methylation in Drosophila melanogaster. Nature 408: 538-540. 11117732
Marhold, J., Zbylut, M., Lankenau, D. H., Li, M., Gerlich, D., Ballesar, E., Mechler, B. M. and Lyko, F. (2002). Stage-specific chromosomal association of Drosophila dMBD2/3 during genome activation. Chromosoma 111: 13-21. 12068919
Marhold, J., Brehm, A. and Kramer, K. (2004a). The Drosophila methyl-DNA binding protein MBD2/3 interacts with the NuRD complex via p55 and MI-2. BMC Mol Biol. 5(1): 20. 15516265
Marhold, J., Kramer, K., Kremmer, E. and Lyko, F. (2004b). The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA. Development 131: 6033-6039. 15537686
Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N. and Bird, A. (1998). Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393: 386-389. 9620804
Ng, H. H., Zhang, Y., Hendrich, B., Johnson, C. A., Turner, B. M., Erdjument-Bromage, H., Tempst, P., Reinberg, D. and Bird, A. (1999). MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat. Genet. 23: 58-61. 10471499
Roder, K., Hung, M. S., Lee, T. L., Lin, T. Y., Xiao, H., Isobe, K. I., Juang, J. L. and Shen, C. J. (2000). Transcriptional repression by Drosophila methyl-CpG-binding proteins. Mol. Cell. Biol. 20: 7401-7409. 10982856
Sansom, O. J., Berger, J., Bishop, S. M., Hendrich, B., Bird, A. and Clarke, A. R. (2003). Deficiency of Mbd2 suppresses intestinal tumorigenesis. Nat. Genet. 34(2): 145-7. 12730693
Santos, F., Hendrich, B., Reik, W. and Dean, W. (2002). Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241(1): 172-82. 11784103
Sekimata, M. and Homma, Y. (2004). Regulation of Rb gene expression by an MBD2-interacting zinc finger protein MIZF during myogenic differentiation. Biochem. Biophys. Res. Commun. 325(3): 653-9. 15541338
Tweedie, S., Ng, H. H., Barlow, A. L., Turner, B. M., Hendrich, B. and Bird, A. (1999). Vestiges of a DNA methylation system in Drosophila melanogaster? Nat. Genet. 23: 389-390. 10581020
Uno, T., et al. (2005). Expression, purification, and characterization of methyl DNA binding protein from Bombyx mori Journal of Insect Science 5: 8. Full Text
Wade, P. A., Gegonne, A., Jones, P. L., Ballestar, E., Aubry, F. and Wolffe, A. P. (1999). Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nat. Genet. 23: 62-66. 10471500
Wang, G., Wei, L. N. and Loh, H. H. (2003). Transcriptional regulation of mouse delta-opioid receptor gene by CpG methylation: involvement of Sp3 and a methyl-CpG-binding protein, MBD2, in transcriptional repression of mouse delta-opioid receptor gene in Neuro2A cells. J. Biol. Chem. 278(42): 40550-6. Epub 2003 Jul 30. 12890683
Weissmann, F., Muyrers-Chen, I., Musch, T., Stach, D., Wiessler, M., Paro, R. and Lyko, F. (2003). DNA hypermethylation in Drosophila melanogaster causes irregular chromosome condensation and dysregulation of epigenetic histone modifications. Mol. Cell. Biol. 23: 2577-2586. 12640138
Zhang, Y., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Bird, A. and Reinberg, D. (1999). Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev. 13: 1924-1935. 10444591
date revised: 1 June 2005
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