MBD proteins are components of the MeCP1 protein complex

Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. This report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA. Since DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, MBD2 in HeLa cells was found to be associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A. In these experiments MBD2 did not demethylate DNA. These data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes (Ng, 1999).

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, have been found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation (Zhang, 1999).

Histone deacetylation plays an important role in methylated DNA silencing. Recent studies indicated that the methyl-CpG-binding protein, MBD2, is a component of the MeCP1 histone deacetylase complex. Interestingly, MBD2 is able to recruit the nucleosome remodeling and histone deacetylase, NuRD, to methylated DNA in vitro. To understand the relationship between the MeCP1 complex and the NuRD complex, the MeCP1 complex was purified to homogeneity and it was found to contain 10 major polypeptides including MBD2 and all of the known NuRD components. Functional analysis of the purified MeCP1 complex revealed that it preferentially binds, remodels, and deacetylates methylated nucleosomes. Thus, this study defines the MeCP1 complex, and provides biochemical evidence linking nucleosome remodeling and histone deacetylation to methylated gene silencing (Feng, 2001).

Methyl-CpG-binding domain proteins (MBD) mediate functional responses of methylated DNA. MBD2 and MBD3 are components of the MeCP1 protein complex, which contains the Mi-2/NuRD complex and includes 66- and 68-kDa polypeptides. Two highly related 66-kDa proteins have been identified in a yeast two-hybrid screen with MBD2b. Based on the high degree of sequence conservation to the previously identified Xenopus p66 subunit of the Mi-2/NuRD complex, these proteins have been termed hp66alpha and hp66beta. hp66alpha is the human orthologue of Xenopus p66, whereas hp66beta, previously identified as a component of the human MeCP1 complex, is a second member of a p66 gene family. Coprecipitation of hp66alpha and MBD2 demonstrates their in vivo association. Furthermore, confocal microscopy shows a nuclear colocalization of hp66alpha with hp66beta and MBD2 in a speckled pattern. hp66alpha is a potent transcriptional repressor reducing gene activity about 100-fold and is ubiquitously coexpressed with hp66beta in cell lines and in fetal and adult tissues. Direct binding of both p66 family members to MBD2 as well as MBD3 has been demonstrated. Interestingly, hp66alpha, which binds with a higher affinity than hp66beta, interacts via two interaction domains in contrast to a single interaction domain present in hp66beta. These results demonstrate that two highly related mammalian p66 proteins display overlapping functions and are involved in methylation dependent transcriptional repression (Brackertz, 2002).

Methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) are transcriptional repressors that contain methyl-CpG binding domains and are components of a CpG-methylated DNA binding complex named MeCP1. Methyl-CpG-binding protein 3-like 1 (MBD3L1) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG binding domain. MBD3L1 interacts with MBD2 and MBD3 in vitro and in yeast two-hybrid assays. Gel shift experiments with a CpG-methylated DNA probe indicate that recombinant MBD3L1 can supershift an MBD2-methylated DNA complex. In vivo, MBD3L1 associates with and colocalizes with MBD2 but not with MBD3 and is recruited to 5-methylcytosine-rich pericentromeric heterochromatin in mouse cells. In glutathione S-transferase pull-down assays MBD3L1 is found associated with several known components of the MeCP1.NuRD complex, including HDAC1, HDAC2, MTA2, MBD2, RbAp46, and RbAp48, but MBD3 is not found in the MBD3L1-bound fraction. MBD3L1 enhances transcriptional repression of methylated DNA by MBD2. The data are consistent with a role of MBD3L1 as a methylation-dependent transcriptional repressor that may interchange with MBD3 as an MBD2-interacting component of the NuRD complex. MBD3L1 knockout mice were created and were found to be viable and fertile, indicating that MBD3L1 may not be essential or there is functional redundancy (through MBD3) in this pathway. Overall, this study reveals additional complexities in the mechanisms of transcriptional repression by the MBD family proteins (Jiang, 2004).

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription (Jin, 2005).

Miscellaneous MBD protein interactions

A human gene has been identified encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintains its subnuclear localization but fails to recruit MBDin at hypermethylated foci. Functional analyses demonstrate that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids are used in transcriptional assays. Southern blotting and bisulfite analysis shows that transcriptional reactivation occurs without changes of the promoter methylation pattern. These findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation (Lembo, 2003).

The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA-protein complexes are uncharacterized. A novel set of RNA-binding proteins has been characterized that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. A subset of the DNMTs and MBD proteins can form RNA-protein complexes. The MBD protein RNA-binding activity has been characterized; it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. It is speculated that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control (Jeffery, 2004).

The induction of Rb gene expression is a key event in the process of myogenic differentiation. Transcription of the Rb gene is repressed by MIZF, an MBD2-binding partner. New roles of MIZF have been identified as a regulator of myogenic differentiation. MIZF mRNA is detected in undifferentiated C2C12 myoblasts but its expression decreases during myogenesis, correlating with an increase in Rb mRNA. To examine the function of MIZF in regulating myogenic differentiation, C2C12 myoblasts were transduced with adenoviral vectors to constitutively produce MIZF at high levels. When switched to differentiation medium, these cells show decreased expression of Rb as well as differentiation markers such as myogenin and Troponin-T, and consequently can not differentiate into multinucleated myotubes. These results suggest that transcriptional repression of Rb by MIZF could be one of the critical determinants in myogenic differentiation (Sekimata, 2004).

Epigenetic regulation of gene expression is critical in the maintenance of cellular homeostasis. Dysregulation of normal epigenetic transcription occurs in abnormal physiological conditions, such as those seen in cancer cells and cells infected with parasites, making the mechanism underlying abnormal epigenetic transcription of great interest. Gene expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by a viral transcriptional stimulator, Tax. A novel mechanism of transcription from the HTLV-1 long terminal repeat (LTR) is reported that is regulated by Tax. Tax is able to activate transcription from the LTR, even when it is heavily methylated. In addition, the methyl-CpG-binding domain 2 (MBD2) protein plays an important role in Tax-mediated transcriptional activation. A physical interaction between Tax and MBD2 is important in enhancing the transcriptional activity of Tax against the methylated LTR. Furthermore, this study identified the formation of a protein complex composed of MBD2 and Tax bound to the methylated LTR. A new model is presented of epigenetic regulation by MBD2 acting in concert with a virally encoded transactivator, Tax. These observations provide insight into the epigenetic regulation of gene expression and the diverse mechanisms of transcriptional regulation using methylated promoters (Ego, 2005).

Transcriptional targets of MBD proteins

DNA methylation is essential for epigenetic gene regulation during development. The cyclic AMP (cAMP)-responsive element (CRE) is found in the promoter of many cAMP-regulated genes and plays important roles in cAMP-regulated gene expression. Methylation occurs on the CRE site and results in transcriptional repression via a direct mechanism, that is, prevention by the methyl group of binding of the cAMP-responsive factor CREB to this site. A recent study indicated that the nucleosome is also important in repressing transcription. In this study, the regulation of transcriptional repression on methylated CRE was investigated. Focus was placed on methyl-CpG binding domain protein 2 (MBD2). MBD2 consists of two forms, MBD2a and MBD2b, the latter lacking the N-terminal extension of MBD2a. Unexpectedly, it was found that MBD2a, but not MBD2b, promotes activation of the unmethylated cAMP-responsive genes. An in vivo binding assay revealed that MBD2a selectively interacts with RNA helicase A (RHA), a component of CREB transcriptional coactivator complexes. MBD2a and RHA cooperatively enhances CREB-dependent gene expression. Interestingly, coimmunoprecipitation assays demonstrate that MBD2a binding to RHA is not associated with histone deacetylase 1. These results indicate a novel role for MBD2a in gene regulation (Fujita, 2003).

Opioid receptors are expressed in a cell type-specific manner. The mouse delta-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells can be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter results in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment forms a MeCP1-like protein complex that contains methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 is decreased when Neuro2A cells are demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells leads to the repression of mDOR promoter activity when the promoter is methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR (Wang, 2003).

The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, pHrD-IRES-Luc (human rRNA promoter-luciferase reporter) was constructed. In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (-9,-102,-347 with respect to the +1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (+152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylation-mediated inhibition of ribosomal RNA gene expression (Ghoshal, 2004).

bMBD2/3 in Bombyx

A cDNA clone encoding methyl DNA binding domain-containing protein (bMBD2/3) was obtained by homology searches using a Bombyx mori fat body cDNA library. The cDNA encoded a polypeptide with 249 amino acids sharing 54% similarity with the methyl DNA binding protein from Drosophila melanogaster. To characterize the biochemical properties of bMBD2/3, the clone was expressed in Escherichia coli as His-tagged protein. The recombinant protein was purified to homogeneity using Ni-NTA superflow resin and heparin agarose. The protein shows specific methyl DNA binding activity and is phosphorylated by protein kinase in vitro. Immunoblotting using the purified antibody indicated that bMBD2/3 is expressed in almost all tissues. Using western blotting analysis, some proteins that interact with bMBD2/3 were identified in the brain. This is the first report that insect MBD is phosphorylated and is present in adult tissues. These results suggest that bMBD2/3 plays important roles in the DNA methylation-specific transcription of Bombyx mori (Uno, 2005).

MBD proteins in C. elegans

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode C. elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster and the absence of a Hedgehog signaling system. The recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C. elegans is described. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C. elegans 200-300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P. pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. It is speculated that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C. elegans (Gutierrez, 2004).

Mutation of vertebrate MBD proteins

MBD2 and MBD3 are closely related proteins with consensus methyl-CpG binding domains. MBD2 is a transcriptional repressor that specifically binds to methylated DNA and is a component of the MeCP1 protein complex. In contrast, MBD3 fails to bind methylated DNA in murine cells, and is a component of the Mi-2/NuRD corepressor complex. It has been shown by gene targeting that the two proteins are not functionally redundant in mice, since Mbd3-/- mice die during early embryogenesis, whereas Mbd2-/- mice are viable and fertile. Maternal behavior of Mbd2-/- mice is however defective and, at the molecular level, Mbd2-/- mice lack a component of MeCP1. Mbd2-mutant cells fail to fully silence transcription from exogenous methylated templates, but inappropriate activation of endogenous imprinted genes or retroviral sequences was not detected. Despite their differences, Mbd3 and Mbd2 interact genetically suggesting a functional relationship. Genetic and biochemical data together favor the view that MBD3 is a key component of the Mi-2/NuRD corepressor complex, whereas MBD2 may be one of several factors that can recruit this complex to DNA (Hendrich, 2001).

How a single cell gives rise to progeny with differing fates remains poorly understood. Cells have been examined lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity (Hutchins, 2002).

Dynamic epigenetic modification of the genome occurs during early development of the mouse. Active demethylation of the paternal genome occurs in the zygote, followed by passive demethylation during cleavage stages, and de novo methylation, which is thought to happen after implantation. These processes were investigated by using indirect immunofluorescence with an antibody to 5-methyl cytosine. In contrast to previous work, demethylation of the male pronucleus was shown to be completed within 4 h of fertilisation. This activity is intricately linked with and not separable from pronucleus formation. In conditions permissive for polyspermy, up to five male pronuclei undergo demethylation in the same oocyte. Paternal demethylation in fertilised oocytes deficient for MBD2, the only candidate demethylase, occurs normally. Passive loss of methylation occurs in a stepwise fashion up to the morulae stage without any evidence of spatial compartmentalisation. De novo methylation is observed specifically in the inner cell mass (ICM) but not in the trophectoderm of the blastocyst and hence may have an important role in early lineage specification. This is the first complete and detailed analysis of the epigenetic reprogramming cycle during preimplantation development. The three phases of methylation reprogramming may have roles in imprinting, the control of gene expression, and the establishment of nuclear totipotency (Santos, 2002).

Gene silencing through de novo methylation of CpG island promoters contributes to cancer. Mbd2, which recruits co-repressor complexes to methylated DNA, is essential for efficient tumorigenesis in the mouse intestine. Since Mbd2-deficient mice are viable and fertile, their resistance to intestinal cancer may be of therapeutic relevance (Sansom, 2003).

DNA methylation plays a crucial role in gene silencing via recruitment of the proteins that specifically recognize methyl-CpG. Two splicing isoforms of MBD3, xMBD3 and xMBD3LF, are the major methyl-CpG binding proteins in Xenopus eggs and early stage embryos. They were highly expressed in the eyes and central nerve system of tadpoles. Inhibition of the expression of xMBD3 by antisense oligonucleotides severely affects embryogenesis. Low-dose injection of antisense oligonucleotides specifically affected eye formation. An identical phenotype is observed on the forced expression of xMBD3 mutated in the methyl-CpG binding domain (MBD) and xMBD3LF, those of which lack methylated DNA binding activity. In contrast, the eye-defective phenotype is not induced on the injection of truncated forms of mutant xMBD3 or xMBD3LF that contain MBD. It is proposed that MBD3, distinct from the case in mouse, plays a crucial role in the recognition of methylated genes as an intrinsic component of the complex to guide the corepressor complex during an early stage of Xenopus embryogenesis (Iwano, 2004).

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

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