MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells

Abstract

The chicken embryonic ␤-type globin gene, , is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated -globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zincfinger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pulldown assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive -globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active ␤ A -globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent -globin IntroductionMethylation of the 5-position of cytosine residues in DNA has an important role in the regulation of gene expression in higher eukaryotes. The first descriptions of an inverse correlation between DNA methylation and expression of a protein-coding eukaryotic gene were those made for the vertebrate globin genes of the chicken, rabbit, and human. [1][2][3][4] The tremendous interest in DNA methylation seen within the past decade stems from its important role in the silencing of tumor suppressor genes in cancer. 5,6 Despite the prediction of a widespread role for DNA methylation in gene regulation, 7 the expression of only a small number of vertebrate genes has been shown to be tissue-restricted or developmentally restricted by DNA methylation. 8 Studies showing tissue-restricted or developmentally restricted expression in nontransformed, primary cells are even more scarce and are limited to 4 main examples: the Gallus gallus -globin gene, 9,10 the Mus musculus interleukin-4 and interferon-␥ genes, 11 and the Xenopus laevis mesodermal genes. 12 Intense study has been directed toward elucidating the mechanisms through which DNA methylation represses transcription. The discovery of a family of proteins that specifically recognize methylated DNA, the methyl-CpG-binding proteins (MCBPs), 13,14 has led to the general observation of these proteins and their associated corepressor complexes as the mediators of DNA methylation-induced gene silencing in a variety of systems. [15][16][17][18][19] Biochemical studies have shown that the MCBPs are members of distinct and nonoverlapping transcriptional repression complexes. MBD1 was identified as a critical component of an S phase-specific complex that propagates the DNA methylation signal into a dimethylation of lysine 9 of histone H3 (H3-K9-Me 2 ) signal during DNA replication. 17 MBD2 is the methyl-CpG-binding component of the MeCP1 transcriptional repression complex. 20 MBD3 is a core component of the NuRD transcriptional repression complex that can be recruited by ...

MBD2 and Multiple Domains of CHD4 Are Required for Transcriptional Repression by Mi-2/NuRD Complexes

“…Expression of transcriptional activators in the appropriate developmental context leads to promoter binding and eviction of the NuRD complex, followed by DNA demethylation and transcriptional activation mediated by the remodeling activities of activating CRCs such as SWI/SNF. NuRD-mediated repression of the rho-globin gene in primary erythroid cells is also dependent on MBD2, supporting our model and providing evidence that the significance of our findings is not limited to the mb-1 promoter (19).…”

Identification of a PRMT5-dependent repressor complex linked to silencing of human fetal globin gene expression

“…34 MBD2 has also been linked to silencing of the chicken embryonic genes, where it binds in a repressor complex containing NuRD components. 35 The identification of both MBD2 and MBD3 in the ␥-globin gene PRMT5-dependent repressor complex is in contrast to the PRMT5-containing complex that assembles on the P14 ARF and P16 INK4a CpG islands, which contains only MBD2. 23 Unlike our findings, recruitment of PRMT5 in that context is dependent on DNA methylation, although a direct link between histone and DNA methyltransferases was not established.…”

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

“…The NuRD complex has chromosome remodeling and histone deacetylase activity (15,78). Mi2 coprecipitates with the MBD2 methylCpG binding protein complex in a repressed chicken ␤-like globin gene locus, suggesting that repression is linked to a closed chromatin structure and increased methylation of the genes (34). The recruitment of the repressive NuRD complex to the Ϫ566 silencer region of the ␥-globin genes may change the acetylation/methylation pattern of the ␥-globin genes, leading to a transcriptionally repressed state.…”

“…Nuclear extracts were prepared from K562 and MEL cell lines as described elsewhere (see materials and methods in the supplemental material) (34). Western blotting was performed according to standard procedures (see materials and methods in the supplemental material) (5).…”